Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00744</article-id><article-id pub-id-type="doi">10.7554/eLife.00744</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>Intraflagellar transport drives flagellar surface motility</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-3552"><name><surname>Shih</surname><given-names>Sheng Min</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3553"><name><surname>Engel</surname><given-names>Benjamin D</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-4907"><name><surname>Kocabas</surname><given-names>Fatih</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3555"><name><surname>Bilyard</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3556"><name><surname>Gennerich</surname><given-names>Arne</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3557"><name><surname>Marshall</surname><given-names>Wallace F</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-3258"><name><surname>Yildiz</surname><given-names>Ahmet</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff5"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Physics</institution>, <institution>University of California, Berkeley</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Biochemistry</institution>, <institution>University of California, San Francisco</institution>, <addr-line><named-content content-type="city">San Francisco</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Internal Medicine</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Anatomy and Structural Biology</institution>, <institution>Albert Einstein College of Medicine</institution>, <addr-line><named-content content-type="city">Bronx</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Department of Molecular Cell Biology</institution>, <institution>University of California, Berkeley</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pfeffer</surname><given-names>Suzanne</given-names></name><role>Reviewing editor</role><aff><institution>Stanford University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>yildiz@berkeley.edu</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Texas Institute of Biotechnology, North American College, Houston, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>11</day><month>06</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00744</elocation-id><history><date date-type="received"><day>14</day><month>03</month><year>2013</year></date><date date-type="accepted"><day>30</day><month>04</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Shih et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Shih et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00744.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00744.001</object-id><p>The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used <italic>Chlamydomonas</italic> flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca<sup>2+</sup>-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of <italic>Chlamydomonas</italic> gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.001">http://dx.doi.org/10.7554/eLife.00744.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00744.002</object-id><title>eLife digest</title><p>Cilia and flagella protrude like bristles from the cell surface. They share the same basic ‘9+2’ axoneme structure, being made up of nine microtubule doublets that surround a central pair of singlet microtubules. Flagella are generally involved in cell propulsion, whereas motile cilia help to move fluids over cell surfaces.</p><p>Maintaining cilia and flagella is a challenge for cells, which must find a way to send new proteins all the way along the axoneme to the site of assembly at the flagellar tip. Cells achieve this via a process called intraflagellar transport, in which proteins are carried back and forth by kinesin and dynein motors along the axonemal doublet microtubules. Intraflagellar transport has been proposed to influence other functions of cilia and flagella, including the propulsion of cells over surfaces. However, the details of these interactions are unclear.</p><p>Through a combination of biophysical and microscopy approaches, Shih et al. describe the mechanism that the green alga <italic>Chalmydomonas</italic> uses to power flagellar gliding over surfaces. By tracking single fluorescently tagged molecules, Shih et al. observed that flagellar membrane glycoproteins are carried along the axoneme by the intraflagellar transport machinery. During transport, flagellar membrane glycoproteins transiently adhere to the surface, and dynein motors that were previously engaged in carrying these glycoproteins now transmit force that moves the axonemal microtubules. This process, which is dependent on the concentration of calcium ions in the extracellular environment, generates the force that propels the alga's flagella along the surface.</p><p>Gliding motility is thought to have been one of the initial driving forces for the evolution of cilia and flagella. How the intricate mechanism of flagellar beat motility could have evolved has been the subject of much discussion, as it would require the flagellum to have evolved first. In demonstrating that gliding motility is powered by the same intraflagellar transport mechanism that is required for flagellar assembly, Shih et al. provide strong evidence for the evolution of primitive flagella before the evolution of flagellar beating. Furthermore, since algal flagella have essentially the same structure as the cilia of human cells, these findings could ultimately aid in the development of treatments for diseases that result from defects in intraflagellar transport, including polycystic kidney disease and retinal degeneration.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.002">http://dx.doi.org/10.7554/eLife.00744.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>intraflagellar transport</kwd><kwd>gliding motility</kwd><kwd>Chlamydomonas</kwd><kwd>dynein</kwd><kwd>kinesin</kwd><kwd>single molecule</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM094522</award-id><principal-award-recipient><name><surname>Yildiz</surname><given-names>Ahmet</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM097017</award-id><principal-award-recipient><name><surname>Marshall</surname><given-names>Wallace F</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>MCB-1055017</award-id><principal-award-recipient><name><surname>Yildiz</surname><given-names>Ahmet</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Burroughs Welcome Foundation</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Yildiz</surname><given-names>Ahmet</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Hellman Faculty Fund</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Yildiz</surname><given-names>Ahmet</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The transport of proteins along flagellar microtubules generates the force that enables flagella to propel cells across surfaces.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Cilia and flagella are microtubule-based organelles that power the locomotion of many organisms, generate fluid flow over multiciliated surfaces, and mediate cell signaling (<xref ref-type="bibr" rid="bib36">Liem et al., 2012</xref>). In order to assemble and maintain cilia, ciliary proteins are transported from cytoplasm to the tip by IFT along axonemes (<xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>). In IFT, linear arrays of multiprotein complexes (IFT trains) are transported by kinesin-2 and dynein-1b in anterograde and retrograde directions, respectively (<xref ref-type="bibr" rid="bib11">Cole et al., 1998</xref>; <xref ref-type="bibr" rid="bib45">Porter et al., 1999</xref>). IFT is a universal mechanism for nearly all eukaryotic cilia and flagella, and defects in this process are linked to a wide range of human diseases, including polycystic kidney disease, retinal degeneration (<xref ref-type="bibr" rid="bib47">Rosenbaum and Witman, 2002</xref>; <xref ref-type="bibr" rid="bib27">Ishikawa and Marshall, 2011</xref>), and Bardet-Biedl syndrome (<xref ref-type="bibr" rid="bib39">Ou et al., 2005</xref>; <xref ref-type="bibr" rid="bib33">Lechtreck et al., 2009</xref>, <xref ref-type="bibr" rid="bib32">2013</xref>; <xref ref-type="bibr" rid="bib52">Wei et al., 2012</xref>).</p><p>Several studies have suggested that IFT not only transports material between the cell body and the flagellar tip, but also interacts dynamically with the flagellar membrane (<xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>) to regulate diverse ciliary functions including motility, mating, sensing extracellular signals and influencing developmental decisions (<xref ref-type="bibr" rid="bib26">Huangfu et al., 2003</xref>; <xref ref-type="bibr" rid="bib49">Snell et al., 2004</xref>; <xref ref-type="bibr" rid="bib43">Pedersen and Rosenbaum, 2008</xref>; <xref ref-type="bibr" rid="bib27">Ishikawa and Marshall, 2011</xref>). However, it has remained unclear how transport of IFT trains underneath the flagellar membrane transmits force to components at the exterior of the flagellar membrane.</p><p>In order to investigate interactions between IFT and the ciliary surface, we used <italic>Chlamydomonas reinhardtii</italic> gliding motility as a model system. In <italic>Chlamydomonas</italic>, the flagellar surface is highly dynamic; polystyrene microspheres and other inanimate small objects adhere to and are moved bidirectionally along the flagellar surface, and cells glide over solid surfaces via adhesion of their flagella (<xref ref-type="bibr" rid="bib35">Lewin, 1952</xref>; <xref ref-type="bibr" rid="bib1">Bloodgood, 1981</xref>). Gliding motility is central to understanding the function and evolution of cilia, as it may have existed in early cilia before the establishment of axonemal beating. There are indications that gliding and FMG1-B motility are driven by the same process (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>), because they move at comparable speeds and both require ligation of the major flagellar surface protein, FMG1-B, into large clusters (<xref ref-type="bibr" rid="bib9">Bloodgood and Workman, 1984</xref>), accompanied by a Ca<sup>2+</sup>-dependent signaling pathway (<xref ref-type="bibr" rid="bib5">Bloodgood and Salomonsky, 1994</xref>). Microsphere movement is considered a more easily assayed and quantitated surrogate for the force transduction system that drives whole cell gliding motility. However, gliding is driven by the pulling motion of the leading flagellum, whereas FMG1-B movement is bidirectional (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>), implying that the two motilities could employ different motors.</p><p>It has been proposed that IFT provides the force for gliding motility (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). IFT trains make multiple connections with the flagellar membrane (<xref ref-type="bibr" rid="bib44">Pigino et al., 2009</xref>) and carry several ciliary and flagellar membrane proteins (<xref ref-type="bibr" rid="bib46">Qin et al., 2005</xref>; <xref ref-type="bibr" rid="bib25">Huang et al., 2007</xref>; <xref ref-type="bibr" rid="bib33">Lechtreck et al., 2009</xref>, <xref ref-type="bibr" rid="bib32">2013</xref>). Inactivation of kinesin-2 in the temperature-sensitive (<italic>ts</italic>) mutant <italic>fla10</italic><sup><italic>ts</italic></sup> stops both IFT and gliding motility (<xref ref-type="bibr" rid="bib29">Kozminski et al., 1995</xref>). While these results suggest that kinesin-2 serves as the anterograde motor responsible for both microsphere movement and gliding motility (<xref ref-type="bibr" rid="bib29">Kozminski et al., 1995</xref>; <xref ref-type="bibr" rid="bib31">Laib et al., 2009</xref>), the retrograde motor for these motilities has not been clearly identified. Mutations in the LC8 subunit of dynein do not abolish FMG1-B movement (<xref ref-type="bibr" rid="bib42">Pazour et al., 1998</xref>), and other flagellar motors, such as the minus-end directed kinesin KCBP (<xref ref-type="bibr" rid="bib14">Dymek et al., 2006</xref>), have been proposed to drive FSM (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>).</p><p>Several studies have raised arguments against this model. IFT motility differs significantly from FSM in that trains move faster and more processively along the length of the flagellum (<xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>; <xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). FSM requires micromolar levels of free calcium, whereas IFT is Ca<sup>2+</sup>-independent (<xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>; <xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). Sexual agglutinins were observed to migrate from the cell body into flagella in the absence of IFT (<xref ref-type="bibr" rid="bib40">Pan and Snell, 2002</xref>). Therefore, evidence supporting the role of IFT in gliding motility is indirect and the precise functions of IFT, molecular motors, FMG1-B and Ca<sup>2+</sup> in FSM remain unclear.</p></sec><sec id="s2" sec-type="results"><title>Results</title><p>To dissect the mechanism of FSM, we directly observed IFT, FMG1-B and gliding motilities using single-molecule imaging techniques. We monitored the movement of individual IFT trains by using total internal reflection fluorescence (TIRF) illumination to image paralyzed-flagella (<italic>pf</italic>) mutant cells that had adhered to the glass surface with both flagella. To establish a link between FMG1-B transport and IFT, we simultaneously tracked the movement of FMG1-B antibody-coated fluorescent beads (200 nm diameter, dark red) and IFT27-GFP in the <italic>pf18</italic> strain. The beads performed short processive runs with reversals of direction whereas IFT trains moved in a regular and unidirectional manner. Multicolor kymography analysis shows that the beads transiently dissociated from one IFT train, diffused for a period of time and then bound to another IFT train (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, <xref ref-type="other" rid="video1">Video 1</xref>) similar to the movement of extraflagellar particles observed along the flagellar membrane (<xref ref-type="bibr" rid="bib12">Dentler, 2005</xref>). Diffraction-limited images of beads and IFT trains were fitted to a two-dimensional Gaussian to achieve a higher localization precision (<xref ref-type="bibr" rid="bib53">Yildiz et al., 2003</xref>). <xref ref-type="fig" rid="fig1">Figure 1B</xref> shows the colocalization of a membrane-attached bead with an individual IFT train as it moves unidirectionally for >500 nm. The movements of the bead and the colocalized IFT trains correlate strongly (>0.99) with each other during the processive run (N = 30, see <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref> for more examples), excluding the possibility that microspheres coincidentally moved together with IFT trains. We measured the distance between microspheres and the nearest IFT trains moving in the same direction for both the experimental data and randomly generated kymographs. The Kolmogorov-Smirnov test (p=7 × 10<sup>−9</sup>, <xref ref-type="fig" rid="fig1">Figure 1C</xref>) demonstrates that IFT trains colocalize with FMG1-B along the flagellar membrane (<xref ref-type="fig" rid="fig1">Figure 1D</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.003</object-id><label>Figure 1.</label><caption><title>IFT transports FMG1-B.</title><p>(<bold>A</bold>) (Top) Simultaneous tracking of anti-FMG1-B beads (red) and IFT27-GFP (green). (Bottom) Kymographs show that bead motility colocalizes with IFT trains during processive runs. Between the runs, the bead transiently attaches to and detaches from IFT trains. The white arrows indicate the IFT trains transporting the bead. (<bold>B</bold>) Two-dimensional Gaussian fitting of the bead and IFT trains show that bead motility correlates strongly (>0.99) with the movement of individual anterograde (green shaded region) and retrograde (blue shaded region) IFT trains. The bead moves at similar speeds to IFT trains in both anterograde and retrograde directions. (<bold>C</bold>) Comparison of distances from beads to the closest IFT train moving in the same direction (grey bars) to the predicted distribution without correlation (null hypothesis, black line). Kolmogorov-Smirnov statistics indicate that bead and IFT train positions strongly correlate with each other. (<bold>D</bold>) A model for IFT particles transporting FMG1-B. The bead is attached to FMG1-B in the flagellar membrane through antibody linkages (not to scale).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.003">http://dx.doi.org/10.7554/eLife.00744.003</ext-link></p></caption><graphic xlink:href="elife00744f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Additional examples of simultaneous tracking of bead motility and IFT.</title><p>(<bold>A–C</bold>) Three additional examples for colocalization and correlated movement of IFT trains (IFT27 GFP) and beads. (<bold>D</bold>) Two-dimensional Gaussian fitting reveals the position of the bead and IFT individual trains as a function of time. Bead motility correlates strongly with the movement of individual IFT trains.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.004">http://dx.doi.org/10.7554/eLife.00744.004</ext-link></p></caption><graphic xlink:href="elife00744fs001"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife00744v001.avi"><object-id pub-id-type="doi">10.7554/eLife.00744.005</object-id><label>Video 1.</label><caption><title>Simultaneous imaging of bead motility and IFT.</title><p>The left channel shows fluorescent beads coated with anti-FMG1-B. One bead displays bidirectional movement along the flagellum. The middle channel shows the movement of IFT trains within the IFT27-GFP <italic>pf18</italic> cell. The right channel is the superimposed image of the two channels (IFT: green, bead: red). Movement of the bead colocalizes with that of individual IFT trains. The data was collected at 5 frames/s. The size of a single channel is 6.8 × 19.1 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.005">http://dx.doi.org/10.7554/eLife.00744.005</ext-link></p></caption></media></p><p>We next investigated whether IFT plays a direct role in gliding motility by monitoring individual IFT trains in IFT20-GFP and IFT27-GFP <italic>pf18</italic> cells as they glided on coverslips. A small fraction of IFT trains displayed rare pauses (0.125 s<sup>−1</sup> per cell, N<sub>cells</sub> = 23, N<sub>pauses</sub> = 247) as they moved either in the retrograde or anterograde direction. Remarkably, the pausing of IFT trains was required for the initiation of whole-cell movements (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1 and 2</xref>). We did not observe the start of a gliding event without a paused IFT train. We were able to determine the directionality of 60% of the paused IFT trains, and all of the trains correspondent to the initiation of gliding motility stopped moving during retrograde transport (N = 148). To rule out the possibility that IFT pausing events and initiation of gliding motility are simply coincidental, we quantified the lag time between the pausing of the last retrograde IFT train and the initiation of gliding motility. The average lag time was 0.48 ± 0.05 s. In comparison, we observed individual IFT pausing events (including both anterograde and retrograde pauses) every 8.25 ± 0.96 s per cell (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref>). The Student’s <italic>t</italic>-test excludes the null hypothesis that the timing of retrograde IFT pausing and gliding motility are independent of each other (p<0.0001). Anterograde trains also displayed rare pauses (∼0.02 s<sup>−1</sup> per cell, N = 27), but we never observed pausing of an anterograde train before the initiation of gliding motility.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.006</object-id><label>Figure 2.</label><caption><title>Dynein-1b drives gliding motility.</title><p>(<bold>A</bold>) (Left) Kymograph of a gliding IFT20-GFP cell. A single retrograde IFT train transiently pauses (red arrow) and initiates the gliding movement of the cell toward the paused train. (Right) A schematic representing the timing and trajectory of the paused IFT train (red curve) in the gliding cell shown on the left. (<bold>B</bold>) (Left) Kymograph of an IFT27-GFP cell, pseudo-colored to show the corresponding velocity of each IFT train. Multiple IFT trains (red arrows) pause (green color) prior to gliding motility. The cell glides until it reaches the paused IFT trains. (Right) A schematic representing the timing and trajectories of the paused IFT trains (red curves) in the gliding cell shown on the left. (<bold>C</bold>) Gliding of uniflagellate cells under bright field illumination. A uniflagellate <italic>pf18</italic> cell glides unidirectionally toward its flagellum. A uniflagellate <italic>dhc1b-3</italic><sup><italic>ts</italic></sup> cell displays bidirectional gliding at the restrictive temperature. Red and blue arrowheads represent forward (flagellum in the lead) and backward (cell body in the lead) gliding directions. (<bold>D</bold>) All of the uniflagellate <italic>pf18</italic> cells glided with the flagellum in the lead. Heat inactivation of dynein-1b in <italic>dhc1b-3</italic><sup><italic>ts</italic></sup> cells at 37°C resulted in a 46% reduction in gliding frequency (N = 35) and led to bidirectional gliding motility. 8% of the cells glided with the flagellum leading the cell body (0.34 ± 0.09 µm/s, mean ± SEM), while 23% glided with the cell body leading the flagellum (0.47 ± 0.16 µm/s). 23% of the cells displayed saltatory gliding movement. (<bold>E</bold>) Uniflagellate cells glided ∼20% faster than biflagellate cells. Inhibition of dynein-1b (<italic>dhc1b-3</italic><sup><italic>ts</italic></sup>) resulted in a fivefold decrease in gliding speed, whereas inhibition of kinesin-2 (<italic>pf1 fla10-1</italic><sup><italic>ts</italic></sup>) led to a twofold speed increase (mean ± SEM). In temperature-insensitive paralyzed (<italic>pf18</italic>) and WT cells, changes in gliding speed between permissive and restrictive temperatures were negligible. (<bold>F</bold>) A model for gliding motility. Retrograde IFT trains adhere to the glass surface through FMG1-B, and the surface-tethered dynein motors pull the cell body through microtubules toward the flagellar tip.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.006">http://dx.doi.org/10.7554/eLife.00744.006</ext-link></p></caption><graphic xlink:href="elife00744f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Three additional examples for kymographs of gliding IFT27-GFP cells.</title><p>(<bold>A–C</bold>) Multiple retrograde IFT trains pause in the leading flagellum prior to gliding motility.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.007">http://dx.doi.org/10.7554/eLife.00744.007</ext-link></p></caption><graphic xlink:href="elife00744fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.008</object-id><label>Figure 2—figure supplement 2.</label><caption><title>The procedure for Fourier space direction analysis.</title><p>(<bold>A</bold>) The original kymograph of an IFT27-GFP cell during gliding motility. (<bold>B</bold>) Magnitude of the fast Fourier transform of the kymograph. (<bold>C</bold>) Mask with colors assigned to different slopes. (<bold>D</bold>) The pattern in Fourier space was color-coded using the mask. Green and red colors represent anterograde and retrograde IFT, blue represents paused IFT trains. Only the magnitude is shown. (<bold>E</bold>) Inverse Fourier transform of the colored Fourier pattern in (<bold>C</bold>) shows the kymograph with trajectories colored by slope.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.008">http://dx.doi.org/10.7554/eLife.00744.008</ext-link></p></caption><graphic xlink:href="elife00744fs003"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.009</object-id><label>Figure 2—figure supplement 3.</label><caption><title>Lag time between IFT pausing and initiation of gliding motility.</title><p>(<bold>A</bold>) A kymograph of IFT trains in a uniflagellate gliding cell. (<bold>B</bold>) A schematic representing the timing and trajectories of paused IFT trains in the gliding cell shown in (<bold>A</bold>). Arrows show the time interval between the pausing of the retrograde IFT train and the start of gliding motility. (<bold>C</bold>) The histogram shows the time between the start of gliding motility and the pausing time of the last retrograde IFT train in the leading flagellum. The histogram was fitted to a single exponential decay (blue curve). Average lag time is 0.48 ± 0.05 s (N = 36), which is significantly shorter than the average time between individual IFT pausing events (8 s).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.009">http://dx.doi.org/10.7554/eLife.00744.009</ext-link></p></caption><graphic xlink:href="elife00744fs004"/></fig><fig id="fig2s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.010</object-id><label>Figure 2—figure supplement 4.</label><caption><title>Three examples for kymograph of gliding uniflagellate IFT27-GFP cells.</title><p>(<bold>A</bold>)<bold>–</bold>(<bold>C)</bold> Multiple retrograde IFT trains pause prior to gliding motility.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.010">http://dx.doi.org/10.7554/eLife.00744.010</ext-link></p></caption><graphic xlink:href="elife00744fs005"/></fig></fig-group></p><p>In 24% of all cases (N = 50), a single paused train appeared sufficient to pull the entire cell (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="other" rid="video2">Video 2</xref>). In other cases, multiple trains paused before the start of gliding motility (<xref ref-type="fig" rid="fig2">Figure 2B</xref>, <xref ref-type="other" rid="video2">Video 2</xref>). Pauses occurred in the leading flagellum, and cells moved in the opposite direction relative to the transport trajectory of the paused trains. In uniflagellate IFT27-GFP <italic>pf18</italic> cells, we also always observed pausing of single or multiple IFT trains before the initiation of gliding motility (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4</xref>). Gliding motility either stopped when paused IFT trains resumed movement (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), or continued until the cell bodies reached the paused trains (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). We never observed the cell body gliding further than the position of the paused IFT train(s).<media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife00744v002.avi"><object-id pub-id-type="doi">10.7554/eLife.00744.011</object-id><label>Video 2.</label><caption><title>Pausing of IFT trains during gliding motility.</title><p><bold>A</bold>, Gliding motility of an IFT20-GFP cell on a glass surface. A single IFT train tethered to the surface (arrow) immediately preceding the initiation of gliding motility. The size of the window is 24.1 × 24.9 µm. The data was collected at 5 frames/s. <bold>B</bold>, Gliding motility of an IFT27-GFP cell on a glass surface. Multiple IFT trains became surface-tethered (arrows), providing force for gliding motility. The size of the window is 24.5 × 24.9 µm. The data was collected at 5 frames/s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.011">http://dx.doi.org/10.7554/eLife.00744.011</ext-link></p></caption></media></p><p>We found compelling evidence that dynein-1b is the primary motor responsible for gliding motility. First, uniflagellate <italic>pf18</italic> cells always glided with the flagellum leading the cell body at 1.49 ± 0.10 µm/s (mean ± SEM) (<xref ref-type="fig" rid="fig2">Figure 2C–E</xref>, <xref ref-type="other" rid="video3">Video 3</xref>) (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>), suggesting that gliding motility is driven by pulling forces generated through a minus end directed microtubule motor. In contrast to <italic>pf18</italic> cells, uniflagellate dynein-1b <italic>ts</italic> cells (<italic>dhc1b-3</italic><sup><italic>ts</italic></sup>) (<xref ref-type="bibr" rid="bib16">Engel et al., 2012</xref>) were unable to display robust gliding at restrictive temperatures (<xref ref-type="fig" rid="fig2">Figure 2C</xref>, <xref ref-type="other" rid="video3">Video 3</xref>). We observed only short-range (<500 nm) gliding-like motion in 54% of these cells, compared to robust unidirectional gliding motility observed over 5 µm in <italic>pf18</italic> cells. These short-range motions were bidirectional (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) and significantly slower (forward: 0.35 ± 0.09 µm/s, backward: 0.47 ± 0.16 µm/s) than the gliding speed of <italic>pf18</italic> cells (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Second, inactivation of dynein-1b in biflagellate <italic>dhc1b-3</italic><sup><italic>ts</italic></sup> cells decreased the fraction of gliding cells from 100% to 52% and reduced the gliding speed from 0.86 ± 0.08 µm/s to 0.18 ± 0.03 µm/s. In contrast, inactivation of kinesin-2 in <italic>fla10</italic><sup><italic>ts</italic></sup> cells resulted in a twofold increase in gliding speed (<xref ref-type="fig" rid="fig2">Figure 2E</xref>), indicating that kinesin plays an inhibitory role during dynein-1b driven gliding motility. These results agree with our observations that the pausing of anterograde trains does not initiate gliding motility.<media content-type="glencoe play-in-place height-250 width-310" id="video3" mime-subtype="avi" mimetype="video" xlink:href="elife00744v003.avi"><object-id pub-id-type="doi">10.7554/eLife.00744.012</object-id><label>Video 3.</label><caption><title>Gliding motility of uniflagellate WT and <italic>dhc1b-3</italic> cells.</title><p>Uniflagellate WT and <italic>dhc1b-3 Chlamydomonas</italic> cells glide with the flagellum in the lead. The size of the whole window is 47.0 × 11.9 µm. The data was collected at 10 frames/s under a bright-field microscope.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.012">http://dx.doi.org/10.7554/eLife.00744.012</ext-link></p></caption></media></p><p>To further test the role of dynein-1b in gliding motility, we treated the cells with a small molecule inhibitor of dynein, ciliobrevin D (<xref ref-type="bibr" rid="bib20">Firestone et al., 2012</xref>). We varied ciliobrevin D concentration between 0–150 µM and monitored IFT in cells adhered both of their flagella to surface 2 min after drug treatment (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>, <xref ref-type="other" rid="video4">Video 4</xref>). Both anterograde and retrograde IFT train frequencies dropped with increasing concentrations of ciliobrevin D (<xref ref-type="fig" rid="fig3">Figure 3B</xref>), and at >100 µM ciliobrevin D we observed accumulation of IFT trains at the flagellar tip (see example kymograph in <xref ref-type="fig" rid="fig3">Figure 3A</xref>). At 150 µM ciliobrevin D, retrograde IFT frequency was reduced by 92% compared to the 70% decrease observed after 6 hr of heat inactivation of <italic>dhc1b-3</italic><sup><italic>ts</italic></sup> cells (<xref ref-type="bibr" rid="bib16">Engel et al., 2012</xref>). The velocities of retrograde and anterograde trains also decreased by 60% and 36%, respectively (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). Importantly, inhibition of dynein-1b resulted in a significant reduction in the speed (79%) and frequency (79%) of gliding motility (N = 50, <xref ref-type="fig" rid="fig3">Figure 3D</xref>). These results further support our conclusion that dynein-1b motors produce the force for gliding motility.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.013</object-id><label>Figure 3.</label><caption><title>Ciliobrevin D inhibits dynein-1b and stops gliding motility.</title><p>(<bold>A</bold>) Kymographs of IFT20-GFP cells treated with varying concentrations of ciliobrevin D. Images were acquired 5 min after addition of ciliobrevin D. (<bold>B</bold> and <bold>C</bold>) Frequency and speed of retrograde and anterograde IFT trains at different ciliobrevin D concentrations. The frequency of retrograde IFT was reduced by 92% at 150 µM ciliobrevin D. (<bold>D</bold>) The speed and fraction of gliding cells decreased by 79% at 150 µM ciliobrevin D.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.013">http://dx.doi.org/10.7554/eLife.00744.013</ext-link></p></caption><graphic xlink:href="elife00744f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.014</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Additional example for kymographs of IFT20-GFP cells treated with varying concentrations of ciliobrevin D.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.014">http://dx.doi.org/10.7554/eLife.00744.014</ext-link></p></caption><graphic xlink:href="elife00744fs006"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video4" mime-subtype="avi" mimetype="video" xlink:href="elife00744v004.avi"><object-id pub-id-type="doi">10.7554/eLife.00744.015</object-id><label>Video 4.</label><caption><title>IFT motility at different concentrations of ciliobrevin D.</title><p>IFT27-GFP <italic>pf18</italic> cells were immobilized on a glass coverslip. 0–150 µM ciliobrevin D was added to the cell culture and the movies were recorded at 10 frames/s within 2–10 min of drug treatment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.015">http://dx.doi.org/10.7554/eLife.00744.015</ext-link></p></caption></media></p><p>Based on these results, we propose a model to describe the functions of IFT motors, IFT trains and FMG1-B transport in gliding motility (<xref ref-type="fig" rid="fig2">Figure 2F</xref>). Surface adhesion of the FMG1-B cargo through its large extracellular carbohydrate domain (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>) stops the retrograde IFT train. Dynein motors previously engaged in transporting the paused IFT train exert force towards the microtubule minus end, causing the whole cell to move toward the plus-end flagellar tip. Thus, gliding motility works similarly to microtubule gliding assays, in which surface-immobilized dyneins glide microtubules with their plus-end tips in the lead.</p><p>To investigate how cells reverse gliding direction, we developed a kymography method for monitoring IFT trains as the cells reorient their flagella during gliding (<xref ref-type="fig" rid="fig4">Figure 4A–B</xref>, <xref ref-type="other" rid="video5">Video 5</xref>). During 60% of reversals (N = 40), the cell lifted one of its flagella and the paused IFT trains on the surface-adhered flagellum drove the motility (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). In other cases, single or multiple paused IFT trains accumulated in one flagellum, and the cell body glided toward this cluster until the paused trains either detached from the glass surface or reached the flagellar base. We also observed cases with paused IFT trains in both flagella where the cell remained immotile, likely due to the balance of forces between bound dynein-1b motors (<xref ref-type="fig" rid="fig4">Figure 4C–D</xref>). There were no indications of coordination of the pausing events between the two flagella. Different modes of reversals in gliding motility may allow cells to search through the environment by a random walk when they adhere both of their flagella, and to move in unidirectional manner by lifting one of the flagella.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.016</object-id><label>Figure 4.</label><caption><title>Mechanisms of reversal in gliding motility.</title><p>(<bold>A</bold>) The average intensity of all the frames in the first 60 s of <xref ref-type="other" rid="video5">Video 5</xref> of an IFT27-GFP <italic>pf18</italic> cell, showing the path of the gliding flagella. (<bold>B</bold>) To monitor IFT trains while the cells alter their flagellar orientation in gliding motility, different curves were plotted before and after cells reoriented their flagella (red dotted lines). Each curve shares at least one common point with previous and subsequent curves. Intermediary frames were analyzed by interpolation of the assigned curves, and the intensity along the line in each frame was stacked according to the fixed point (yellow dot). (<bold>C</bold>) A kymograph generated by this method reveals multiple ways that the cell can control gliding direction. (i) When the cell lifts one flagellum (red arrow), the cell body moves toward the surface-attached flagellum. When both flagella are attached to the surface, gliding direction is determined by the balance of forces exerted by surface-tethered IFT trains. (ii) The cell glides toward the flagellum with more paused IFT particles (yellow arrow). (iii) When there are equal numbers of surface-tethered IFT particles in both flagella (cyan arrow), opposite forces cancel out and the cell remains stationary. (<bold>D</bold>) A schematic model representing the three different modes (i, ii, iii) of gliding direction reversal observed in (<bold>C</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.016">http://dx.doi.org/10.7554/eLife.00744.016</ext-link></p></caption><graphic xlink:href="elife00744f004"/></fig><media content-type="glencoe play-in-place height-250 width-310" id="video5" mime-subtype="avi" mimetype="video" xlink:href="elife00744v005.avi"><object-id pub-id-type="doi">10.7554/eLife.00744.017</object-id><label>Video 5.</label><caption><title>Reversal of direction in gliding motility.</title><p>Gliding motility of an IFT27-GFP cell on a glass surface. The cell changes the direction and speed of gliding motility either by raising one of its flagella or by the pausing of one or more IFT trains relative to the glass surface. The data was collected at 10 frames/s. The size of the window is 33.9 × 23.7 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.017">http://dx.doi.org/10.7554/eLife.00744.017</ext-link></p></caption></media></p><p>Gliding motility in <italic>C. reinhardtii</italic> is controlled by a Ca<sup>2+</sup>-calmodulin regulated kinase and requires micromolar levels of Ca<sup>2+</sup> in the media (<xref ref-type="bibr" rid="bib7">Bloodgood and Spano, 2002</xref>). We tested whether the pausing of IFT trains depends on Ca<sup>2+</sup> (<xref ref-type="fig" rid="fig5">Figure 5A–B</xref>) by analyzing the kymographs of IFT at different Ca<sup>2+</sup> concentrations. To quantify pausing in flagella, we used Fourier space direction analysis (FSDA, see <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>) to separate the traces of paused IFT trains and moving IFT trains into two kymographs. Kymographs of paused IFT trains in <xref ref-type="fig" rid="fig5">Figure 5A,B</xref> show that pausing along the length of the flagellum was significantly reduced in Ca<sup>2+</sup>-deprived cells (see additional examples in <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). After Ca<sup>2+</sup> depletion, IFT trains rarely paused in the middle regions of flagella and accumulated at the base (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, middle). <xref ref-type="fig" rid="fig5">Figure 5C</xref> plots the total fluorescence intensity of paused IFT trains (N<sub>cells</sub> = 30 for each case) along the lengths of flagella at different Ca<sup>2+</sup> levels. The frequency of pausing in Ca<sup>2+</sup>-deprived cells was significantly lower than IFT pausing in cells at a normal Ca<sup>2+</sup> concentration. In regular TAP media (free [Ca<sup>2+</sup>] = 0.34 mM), 95% of all surface-adhered cells displayed gliding motility and individual pausing events were observed every ∼8 s per flagellum, on average. In contrast, both the fraction of gliding cells and IFT pausing frequency were reduced by ∼10-fold at <1 µM Ca<sup>2+</sup> (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Next, we compared the IFT pausing frequencies in gliding and non-gliding cells at different Ca<sup>2+</sup> levels. The pausing frequency in non-gliding cells was low and independent of Ca<sup>2+</sup> concentration. In contrast, the pausing frequency in gliding cells was high at normal Ca<sup>2+</sup> levels and gradually declined to match that of non-gliding cells as the Ca<sup>2+</sup> concentration decreased below 1 nM (<xref ref-type="fig" rid="fig5">Figure 5E</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.018</object-id><label>Figure 5.</label><caption><title>Ca<sup>2+</sup> is required for the pausing of IFT trains at flagellar adhesion sites.</title><p>(<bold>A</bold>) (Left) Kymograph of an IFT27-GFP cell adhering both of its flagella in the presence of 0.34 mM free Ca<sup>2+</sup>. Pausing (middle) and moving (right) IFT trains were split into separate kymographs by FSDA analysis. IFT trains pause frequently along the length of the flagellum and drive gliding motility. (<bold>B</bold>) When cells are deprived of free Ca<sup>2+</sup>, immotile IFT trains accumulate near the cell body and do not display frequent pauses between the flagellar base and the tip. (<bold>C</bold>) The average fluorescence intensity of ‘pausing IFT trains’ in kymographs relative to the length of the flagellum (N = 6 cells for each condition). Cells in 0.34 mM Ca<sup>2+</sup> show robust pausing uniformly along the length of the flagellum. In contrast, Ca<sup>2+</sup>-deprived cells show significantly reduced pausing events. Background fluorescence was excluded from the analysis. (<bold>D</bold>) IFT pausing frequency and the fraction of gliding cells as a function of free Ca<sup>2+</sup> concentration. The free Ca<sup>2+</sup> concentration was controlled through Ca<sup>2+</sup>-EGTA buffering. (<bold>E</bold>) IFT pausing frequency of gliding and non-gliding cells as a function of free Ca<sup>2+</sup>. At 0.34 mM Ca<sup>2+</sup>, the IFT pausing frequency in gliding cells is very high compared to non-gliding cells. In Ca<sup>2+</sup>-deprived cells, the pausing frequency in gliding cells is reduced to the residual level of pausing events observed in non-gliding cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.018">http://dx.doi.org/10.7554/eLife.00744.018</ext-link></p></caption><graphic xlink:href="elife00744f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.019</object-id><label>Figure 5—figure supplement 1.</label><caption><title>The analysis of IFT pausing in the presence and absence of free Ca2+ in media.</title><p>(<bold>A</bold>) Two additional examples for kymographs of IFT27-GFP cells adhering both flagella to the surface in the presence of 0.34 mM free Ca<sup>2+</sup>. Pausing (middle) and moving (right) IFT trains were split to separate kymographs by FSDA analysis. IFT trains pause frequently along the length of flagellum and drive gliding motility. (<bold>B</bold>) Two additional examples for kymographs of IFT27-GFP cells adhering both flagella to the surface when cells are deprived of free Ca<sup>2+</sup>. IFT trains accumulate near the cell body and do not display frequent pauses between the flagellar base and the tip.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.019">http://dx.doi.org/10.7554/eLife.00744.019</ext-link></p></caption><graphic xlink:href="elife00744fs007"/></fig></fig-group></p><p>In Ca<sup>2+</sup>-deprived cells, the beads freely diffused on the flagellar membrane but did not move processively by IFT trains (not shown). Thus, we propose that Ca<sup>2+</sup> is required for attachment of FMG1-B to IFT trains, not for the activation of a motor protein that provides force for gliding, as previously suggested (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). As the flagellar membrane is enriched with a PKD2-like Ca<sup>2+</sup> channel (<xref ref-type="bibr" rid="bib41">Pazour et al., 2005</xref>; <xref ref-type="bibr" rid="bib25">Huang et al., 2007</xref>), Ca<sup>2+</sup> signaling at flagellar adhesion sites may play a role in controlling the directionality and timing of gliding movement.</p><p>To measure the forces exerted by motors bound to individual IFT trains, we tracked the movement of FMG1-B-antibody coated bead motility using an optical trap. To rule out the possibility that loads exerted by the trap might disrupt the linkage between IFT and FMG1-B, we performed TIRF imaging of IFT27-GFP and optical trapping of beads simultaneously (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). We observed that IFT trains remained colocalized with trapped beads when subjected to external forces (<xref ref-type="fig" rid="fig6">Figure 6B</xref>; see <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> for additional examples). The average offset between the positions of the trapped beads and colocalized IFT trains was 280 ± 10 nm (N = 11). This is due to the fact that the beads (920 nm diameter) and IFT trains (200–1000 nm in length) (<xref ref-type="bibr" rid="bib44">Pigino et al., 2009</xref>) are large objects relative to the resolution of conventional fluorescence imaging (approximately 200–250 nm) and the forces that stretch the bead-FMG1-B-IFT linkage displace the center of the bead from the IFT train (see the schematics in <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). The trap measurements correspond to forces generated by IFT motors and provide direct evidence that IFT transports FMG1-B. Since beads are outside the cell, but are physically linked to the action of IFT trains, the assay combines the advantages of precise in vitro trapping with the ability to manipulate IFT motility under load.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.020</object-id><label>Figure 6.</label><caption><title>Stall force measurements on single IFT trains.</title><p>(<bold>A</bold>) Schematic representation of combined optical trapping of bead motility and fluorescent tracking of IFT. (<bold>B</bold>) Simultaneous tracking of IFT27-GFP and bead motility. At t = 3 s, the microscope stage was moved to bring the flagellum underneath the trapped bead. An IFT train stalls at the trap position (t = 5–8 s) and stays adhered to the bead until the bead escapes the trap (t = 10 s). The CCD camera is toggled between fluorescence and bright field imaging to monitor IFT trains as well as the bead when it is out of the detection range. (<bold>C</bold>) Displacement records of bead motility show successive runs including stalling (inset) and releasing events. (<bold>D</bold>) The peak force distributions and statistics (mean ± SEM) of stalling and releasing events in <italic>pf18</italic> cells. IFT particles exert 25–30 pN of peak forces with slightly less force produced in the anterograde direction.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.020">http://dx.doi.org/10.7554/eLife.00744.020</ext-link></p></caption><graphic xlink:href="elife00744f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.021</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Three additional examples of simultaneous bead trapping and IFT27-GFP fluorescence tracking.</title><p>(<bold>A</bold>) A single anterograde IFT train stalls directly underneath the trapped bead for 20 s. (<bold>B</bold> and <bold>C</bold>) The bead shows multiple runs and escapes the trap. The optical trapping beam was on throughout the duration of the kymograph.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.021">http://dx.doi.org/10.7554/eLife.00744.021</ext-link></p></caption><graphic xlink:href="elife00744fs008"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.022</object-id><label>Figure 6—figure supplement 2.</label><caption><title>The offset between the bead position and IFT trains.</title><p>(<bold>A</bold>) Schematic for an in vitro fixed-trap assay with a single motor bound to a microtubule. (<bold>B</bold>) Schematic for the optical trap assay of <italic>Chlamydomonas</italic> bead motility (roughly to scale). In both (<bold>A</bold> and <bold>B</bold>), there is a large offset between the bead center and the motor/cargo. The size of the offset depends on tether length, the bead diameter and the length of IFT train. (<bold>C</bold>) Histogram for the separation between the bead center and IFT position in the combined optical trap and fluorescence experiment. We observe up to 500 nm offset between an IFT train (∼400 nm in length) and the polystyrene bead (900 nm in diameter). The average separation between the bead and IFT is 280 nm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.022">http://dx.doi.org/10.7554/eLife.00744.022</ext-link></p></caption><graphic xlink:href="elife00744fs009"/></fig><fig id="fig6s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.023</object-id><label>Figure 6—figure supplement 3.</label><caption><title>Additional examples for bead motility under fixed trap.</title><p>(Top) Displacement records of bead motility in <italic>fla10</italic><sup><italic>ts</italic></sup> cells show successive runs, stalling and releasing events along the anterograde direction at permissive temperatures. (Bottom) The bead motility in <italic>fla10</italic><sup><italic>ts</italic></sup> cells displays runs, stalling and releasing events mostly along the retrograde direction at restrictive temperatures.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.023">http://dx.doi.org/10.7554/eLife.00744.023</ext-link></p></caption><graphic xlink:href="elife00744fs010"/></fig><fig id="fig6s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.024</object-id><label>Figure 6—figure supplement 4.</label><caption><title>The effect of antibody concentration on peak forces measurements.</title><p>(<bold>A</bold>) The fraction of beads moving along the flagellar surface as a function of the antibody concentration on the bead surface. (<bold>B</bold>) The fraction of stall, release and escape events along the anterograde and retrograde directions in optical trapping assays as a function of antibody concentration used to coat the bead surface. (<bold>C</bold>) Histograms of peak forces measured in the optical trap assay along the anterograde and retrograde directions in WT cells. 1 µm diameter polystyrene beads were coated with 0.05, 0.5 and 10 mg/ml FMG1-B antibody.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.024">http://dx.doi.org/10.7554/eLife.00744.024</ext-link></p></caption><graphic xlink:href="elife00744fs011"/></fig></fig-group></p><p>In a fixed trap assay, ∼50% of processive bead movements terminated with a stall before returning to the trap center (<xref ref-type="fig" rid="fig6">Figure 6C</xref>, <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>). We did not observe a significant difference between the force values of stall and release events. This suggests that motors attached to an IFT train may not be able to reach their maximum stall force, defined as the stall force of a single motor multiplied by the number of bound motors (<xref ref-type="bibr" rid="bib48">Shubeita et al., 2008</xref>). Histograms of peak forces (<xref ref-type="fig" rid="fig6">Figure 6D</xref>) show that anterograde and retrograde IFT trains moved against 21.4 ± 0.7 pN and 25.2 ± 1.3 pN (SEM), respectively. Forces exerted on IFT trains well exceed the force-generation capability of a single motor. Previous studies demonstrated that multiple motors produce larger forces, move with higher velocities under load and carry cargos further than a single kinesin or dynein motor (<xref ref-type="bibr" rid="bib37">Mallik et al., 2005</xref>; <xref ref-type="bibr" rid="bib50">Vershinin et al., 2007</xref>). It remains controversial whether motor stall forces are additive at low motor copy numbers (<xref ref-type="bibr" rid="bib50">Vershinin et al., 2007</xref>) or whether multiple motors tend to transport their cargo using only one load-bearing motor at a time (<xref ref-type="bibr" rid="bib28">Jamison et al., 2010</xref>). Therefore, we believe that peak forces in our experiment represent a lower boundary for estimates of motor copy number. Assuming that IFT motors produce 6–7 pN forces (<xref ref-type="bibr" rid="bib21">Gennerich et al., 2007</xref>; <xref ref-type="bibr" rid="bib10">Brunnbauer et al., 2010</xref>), we estimate that at least four motors transport IFT trains at a time, in agreement with previously reported values (<xref ref-type="bibr" rid="bib18">Engel et al., 2009b</xref>). It is possible that multiple motor engagements enhance the run length of individual IFT trains, allowing them to traverse the length of a flagellum. In addition, the fast transport of IFT trains in a viscous cellular environment and IFT’s role in FSM may require forces larger than single motor stall forces.</p><p>The measured forces in both directions only marginally changed (<20%) under different bead antibody-coating conditions (<xref ref-type="fig" rid="fig6s4">Figure 6—figure supplement 4)</xref>. This result argues against the possibility that antibody-coating of the beads leads to the crosslinking of more than one IFT trains, which would be expected to multiply the average peak force. 20% of retrograde runs escaped the trap by producing more than 80 pN of force, presumably via clustering of FMG1-B (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>), while <1% escape events were observed in the anterograde direction.</p><p>We next investigated why the speed of gliding motility (1.49 ± 0.10 µm/s, SEM) is significantly slower than that of retrograde IFT (2.96 ± 0.14 µm/s), even though both processes are powered by dynein-1b motors actively transporting IFT trains. IFT trains that carry 1-μm beads moved ∼30% slower than IFT trains that were not associated with the beads (two-tailed Student’s <italic>t</italic>-test, p = 2.1 × 10<sup>−9</sup> (anterograde) and 1.8 × 10<sup>−11</sup> (retrograde), <xref ref-type="fig" rid="fig7">Figure 7A</xref>). We reasoned that the viscous drag of the membrane may slow down IFT trains transporting FMG1-B clusters and led to the observed differences in speed between IFT and FSM. To estimate the drag constant of the bead-IFT complex, we analyzed individual stalling events in the optical trap assay (<xref ref-type="fig" rid="fig7">Figure 7B</xref>) and measured the recoil time of the bead after a stall (<xref ref-type="fig" rid="fig7">Figure 7C</xref>). The average drag constant of the bead-IFT complex was found to be 8.0 ± 2.8 pN s/μm (SEM, N = 30).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.025</object-id><label>Figure 7.</label><caption><title>Viscous drag of the membrane slows down the motility of IFT trains that carry beads.</title><p>(<bold>A</bold>) The average speed of FMG1-B antibody-coated beads and IFT trains. Coating of the coverslip surface with 0.7 mg/ml polylysine led to a ∼20% reduction in the speed of IFT motility (two tailed <italic>t</italic>-test, p=5.2 × 10<sup>−14</sup> [anterograde] and 5.6 × 10<sup>−6</sup> [retrograde]). IFT trains that carry 0.2-μm beads move at 10–30% slower speeds than IFT trains that are not associated with the beads (p=1.1 × 10<sup>−2</sup> [anterograde] and 7.2 × 10<sup>−5</sup> [retrograde]). IFT trains that carry 0.2-μm beads move at similar speeds to beads (p=0.4 [anterograde] and 0.6 [retrograde]) (error bars represent SEM). (<bold>B</bold>) A typical example for stalling of bead motility by the optical trap. The bead recovers slowly to the trap center after a stall (rectangular box). (<bold>C</bold>) The recoiling of a trapped bead to the trap center after a stall was fitted to single exponential decay. The mean drag constant of the bead-FMG1-B-IFT complex is 8.0 ± 0.28 pN s/µm. (<bold>D</bold>) The IFT-bead complex experiences large drag forces as the IFT train moves several micrometers per second inside the flagellum (not to scale).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.025">http://dx.doi.org/10.7554/eLife.00744.025</ext-link></p></caption><graphic xlink:href="elife00744f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00744.026</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Measurement of viscous drag on bead movement along the surface of the flagellar membrane.</title><p>(<bold>A</bold>) A polystyrene bead was trapped by the laser beam and brought on top of a surface-immobilized flagellum. After the bead was bound to FMG-1B, the trap was oscillated 500 nm back and forth along the flagellar surface in a square wave pattern. Trap stiffness was set to 0.07 pN/nm. Due to the viscous drag of the membrane, the bead displays a relaxation curve when it follows the trap. (<bold>B</bold>) The recoiling of a trapped bead to the trap center after a stall was fitted to single exponential decay. The mean drag constant of the bead-FMG1-B-IFT complex is 8.4 ± 4.2 pN.s/µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.026">http://dx.doi.org/10.7554/eLife.00744.026</ext-link></p></caption><graphic xlink:href="elife00744fs012"/></fig></fig-group></p><p>We hypothesized that the large drag constants we measured were due to the interaction between the bead and the membrane. Our results rule out the possibility that microtubule motors step backwards during the recoiling of the bead, as the average bead velocity was 20 µm/s (an order of magnitude faster than that of dynein and kinesin) and there were no detectable backward steps. To rule out other possible interactions between IFT trains and axonemes, we oscillated individual beads on a flagellar membrane surface in a square wave pattern and measured the recoiling time when the beads are decoupled from IFT (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). The average viscous drag constant was 8.4 ± 4.2 pN.s/µm, which is similar to the drag that beads experience when they interact with IFT trains. Anterograde and retrograde trains moving at 2–3 µm/s would experience 16–24 pN resistive forces, comparable to the total motor force exerted on a single IFT train (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). Therefore, IFT is subjected to a high drag force when it carries a large bead along the flagellar surface, which leads to the reduction of transport velocity.</p><p>We next investigated how kinesin-2 activity reduces the speed of gliding motility (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). This could theoretically be explained by a tug-of-war between active kinesin and dynein motors that are both present on the same IFT train. Alternatively, kinesin-2 and dynein-1b motors may be exclusively active on anterograde and retrograde cargos, respectively, and pausing of anterograde trains could produce forces that oppose the gliding forces of paused retrograde trains. To distinguish between tug-of-war and coordinated transport mechanisms, we examined how inhibition of one class of motors affected the forces exerted on IFT trains traveling in the opposite direction (<xref ref-type="bibr" rid="bib31">Laib et al., 2009</xref>). At permissive temperatures, the peak forces of IFT in <italic>fla10-1</italic><sup><italic>ts</italic></sup> and <italic>dhc1b-3</italic><sup><italic>ts</italic></sup> were in close agreement with wild-type (WT) cells. Heat-inactivation of kinesin-2 reduced the frequency of anterograde runs by 66%, but did not alter the peak forces on retrograde runs (<xref ref-type="bibr" rid="bib31">Laib et al., 2009</xref>) (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). Similarly, heat inactivation of dynein-1b significantly reduced the ratio of retrograde to anterograde transport events (0.15), but had minimal effect on the peak forces of anterograde runs (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). These results exclude a tug-of-war mechanism in IFT, which would lead to an increase of force from motors walking in one direction upon inactivation of the motors walking in the opposite direction. Instead, only one type of a motor remains active on IFT trains at a time, which is consistent with the result that retrograde speed of <italic>fla10-1</italic><sup><italic>ts</italic></sup> does not change after kinesin-2 is inactivated. We propose that kinesin-2 on paused anterograde trains slows down gliding motility by exerting forces in the opposite direction to that of dynein-1b on paused retrograde trains.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.00744.027</object-id><label>Figure 8.</label><caption><title>Force measurements on temperature-sensitive mutants.</title><p>(<bold>A</bold>) Peak force histograms for IFT movement in <italic>pf1 fla10</italic><sup><italic>ts</italic></sup> cells at 22°C and 34°C. Stalling events are less common than the release of the bead. The frequency of anterograde IFT trains is reduced after switching the temperature to 34°C. Forces along the retrograde direction remain unaltered after the inhibition of kinesin-2 (mean ± SEM). (<bold>B</bold>) Peak force histograms for IFT in <italic>dhc1b-3</italic><sup><italic>ts</italic></sup>. The frequency and force production of retrograde IFT trains are reduced after switching the temperature to 34°C. Forces along the anterograde direction remain unaltered after the inhibition of dynein-1b (mean ± SEM).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00744.027">http://dx.doi.org/10.7554/eLife.00744.027</ext-link></p></caption><graphic xlink:href="elife00744f008"/></fig></p></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>We present direct evidence for the mechanism of IFT-mediated cell-surface interactions using <italic>Chlamydomonas</italic> gliding motility and FMG1-B transport as a model system. In <italic>Chlamydomonas,</italic> IFT trains carry FMG1-B as a cargo. The interaction between IFT trains and FMG1-B clusters is transient, as FMG1-B boards moving trains but usually unloads and diffuses away before the trains arrive at the flagellar base or tip. During gliding motility, IFT trains pause due to attachment of the FMG1-B cargo to the surface, and the dynein-1b motors engaged to the paused IFT trains generate pulling forces along the microtubule long axis. While the majority of organismal motilities rely on the actin cytoskeleton or axonemal beating, gliding motility in <italic>C. reinhardtii</italic> is distinct in that it is powered by intracellular transport machinery along microtubules. By providing surface adhesion points, FMG1-B performs an analogous function to integrins in mammalian cell motility (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>; <xref ref-type="bibr" rid="bib34">Lecuit et al., 2011</xref>).</p><p>Our results also demonstrate how different types of flagellar motility in <italic>Chlamydomonas</italic> have distinct characteristics despite being driven by the same forces. First, we showed that viscous drag of the membrane slows down the motility of IFT trains that carry FMG1-B-conjugated beads, causing gliding motility and FMG1-B transport to proceed slower than IFT. Second, previous work showed that IFT is unaffected by changes in environmental Ca<sup>2+</sup> concentration (<xref ref-type="bibr" rid="bib4">Bloodgood and Salomonsky, 1990</xref>; <xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>), whereas gliding motility is Ca<sup>2+</sup>-dependent (<xref ref-type="bibr" rid="bib4">Bloodgood and Salomonsky, 1990</xref>; <xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>). This raised the possibility that Ca<sup>2+</sup> may be required to activate a specific motor protein that provides force for gliding (<xref ref-type="bibr" rid="bib3">Bloodgood, 2009</xref>). Our results are inconsistent with this hypothesis. We found that the presence of free Ca<sup>2+</sup> leads to frequent pauses in IFT motility at flagellar adhesion sites. In the absence of Ca<sup>2+</sup>, IFT moves unidirectionally without interruptions, suggesting that attachment of FMG1-B to IFT trains is regulated by a Ca<sup>2+</sup>-dependent signaling pathway. The signaling pathway responsible for linking FMG1-B to IFT remains unclear. There is evidence that surface adhesion or crosslinking of FMG1-B into large clusters induces dephosphorylation of a transmembrane protein that co-immunoprecipitates with FMG1-B (<xref ref-type="bibr" rid="bib5">Bloodgood and Salomonsky, 1994</xref>, <xref ref-type="bibr" rid="bib6">1998</xref>). The complex may be regulated by a calcium-calmodulin dependent gliding associated kinase (GAK), which is required for the gliding motility (<xref ref-type="bibr" rid="bib7">Bloodgood and Spano, 2002</xref>). Further investigation is required to identify the rest of the signaling pathway regulating IFT-flagellar surface interactions.</p><p>Our force measurements show a number of similarities and differences with earlier published work. In a previous trapping assay, beads nonspecifically adsorbed to the flagellar membrane were found to exert 60 pN average peak forces (<xref ref-type="bibr" rid="bib31">Laib et al., 2009</xref>). In contrast, our measured peaked forces are significantly lower (20–30 pN) than these values. Laib et al. could not determine whether their force measurements reflect the forces produced by motors attached to IFT trains or by motors that are directly bound to FMG1-B clusters. Our experiments directly link optical trap recordings of bead motility to forces generated by flagellar motors attached to IFT trains (<xref ref-type="fig" rid="fig1">Figure 1</xref>), enabling us to propose physical models for the coordination of IFT and its role in FSM. In agreement with Laib et al., we observe reciprocal coordination of motors during anterograde transport. Additionally, our measurements of dynein-1b inactivation verify that motors do not engage in a tug-of-war while travelling in the retrograde direction.</p><p>The mechanism we described for IFT pausing and force generation has broad implications for the traffic of ciliary sensory proteins as well as cell signaling at ciliary membrane adhesion points (<xref ref-type="bibr" rid="bib51">Wang et al., 2006</xref>). For example, IFT is required for signal transduction during the mating of ciliated organisms. The initial interaction between gametes of two mating types in <italic>Chlamydomonas</italic> can occur at any point along their flagellar membranes, but the tips of their flagella must be aligned and locked before activating gametic fusion (<xref ref-type="bibr" rid="bib24">Homan et al., 1987</xref>). Since IFT trains transport sexual agglutinins within the flagellar membrane of gametic cells (<xref ref-type="bibr" rid="bib19">Ferris et al., 2005</xref>), adhesion between sexual agglutinins of opposite cell types may stall retrograde IFT movement. As a result, forces produced by IFT trains would move the flagellar contact points until a balance of forces is achieved when the flagella are properly aligned with respect to each other. Indeed, microspheres have been observed to move and accumulate at the flagellar tips during mating (<xref ref-type="bibr" rid="bib23">Hoffman and Goodenough, 1980</xref>). We propose that the absence of retrograde bead movement may be related to the formation of adhesion contacts between the flagella and pausing of retrograde IFT trains.</p><p>Cilia in the retina, liver, and kidney cells were recently observed to make direct physical contacts, which may serve as ‘bridges’ for signaling networks between many cells (<xref ref-type="bibr" rid="bib38">Ott et al., 2012</xref>). These contacts are tight adhesions between the ciliary membranes, mediated by N-linked glycoproteins. It is possible that IFT exerts force on cell-cell adhesion sites and determines the positioning of these adhesion sites by moving them along the length of the cilium. Ca<sup>2+</sup> signaling at flagellar adhesion sites may play a major role in regulating attachment to IFT trains and controlling the direction of force generation. We believe that the assay we developed will be a starting point for deciphering the role of IFT in the signaling, mating and development of ciliated cells.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Strains and culture conditions</title><p>Vegetative <italic>C. reinhardtii</italic> cells were grown in Tris-acetate-phosphate (TAP) media in an illuminated plant growth chamber at 22°C. WT mt+ (cc125), <italic>pf18</italic> mt− (cc1297), and <italic>pf18</italic> mt+ (cc1036) strains were obtained from the <italic>Chlamydomonas</italic> Resource Center. The IFT20-GFP ΔIFT20 strain was provided by K Lechtreck and G Witman. The IFT27-GFP mt+ strain and <italic>pf1 fla10-1</italic><sup><italic>ts</italic></sup> were provided by J Rosenbaum. IFT27-GFP <italic>pf18</italic> strain was generated through crosses (<xref ref-type="bibr" rid="bib18">Engel et al., 2009b</xref>). <italic>pf1 fla10-1</italic><sup><italic>ts</italic></sup> cells were incubated at 37°C for 15 min for heat inactivation of kinesin-2, and the movies were recorded within 30 min at 37°C, before the complete impairment of IFT and the start of flagellar resorption (<xref ref-type="bibr" rid="bib30">Kozminski et al., 1993</xref>). In the <italic>dhc1b</italic>-3<sup>ts</sup> mt- mutant strain (cc4422) (<xref ref-type="bibr" rid="bib16">Engel et al., 2012</xref>), dynein-1b is inhibited after 6 hr incubation at 37°C. Paralyzed flagella (<italic>pf)</italic> strains and paralyzing compounds (<xref ref-type="bibr" rid="bib15">Engel et al., 2011</xref>) were utilized to impair swimming motility. Ciliobrevin D was dissolved in DMSO and then added to the <italic>pf18</italic> cell culture to inhibit dynein activity. IFT and gliding motility were recorded simultaneously 2 min after addition of 0–150 µM ciliobrevin D to the flow chamber.</p></sec><sec id="s4-2"><title>Single-molecule fluorescence imaging</title><p>The IFT and FSM imaging assays were performed with an objective-type TIRF microscope (<xref ref-type="bibr" rid="bib13">DeWitt et al., 2012</xref>). The GFP signal was recorded by an electron multiplied charge-coupled device (EM-CCD) camera, with an effective pixel size of 106 nm. IFT27-GFP <italic>pf18</italic> and IFT20-GFP ΔIFT20 strains were used for tracking individual IFT trains (<xref ref-type="bibr" rid="bib17">Engel et al., 2009a</xref>). The sample chamber was pre-treated with 0.7 mg/ml poly-L-lysine to adhere flagella on a glass surface. 15 µl cells in TAP media were applied to cover glass and inverted onto slides. Gliding motility assays were performed without polylysine treatment and cells were imaged under bright-field illumination. <italic>ts</italic> mutants were assayed at permissive (22°C) and restrictive (37°C) conditions by controlling the temperature with an objective heater (Bioptechs). Because paralyzed cells glide 30–40% faster than non-paralyzed cells (<xref ref-type="bibr" rid="bib2">Bloodgood, 1995</xref>), gliding speeds of <italic>ts</italic> mutants of kinesin-2 (<italic>pf1 fla10-1</italic><sup><italic>ts</italic></sup>) and dynein-1b (<italic>dhc1b-3</italic><sup><italic>ts</italic></sup>) were compared to those of both <italic>pf18</italic> and WT cells at permissive (22°C) and restrictive (37°C) temperatures.</p></sec><sec id="s4-3"><title>Bead-motility imaging</title><p>To visualize bead movement along the flagellar surface, 200 nm carboxyl-modified nile-red fluorescent beads (Invitrogen) were coated with anti-FMG1-B antibody (<xref ref-type="bibr" rid="bib8">Bloodgood et al., 1986</xref>) by EDC-NHS cross linking. 1 ml <italic>pf18</italic> cell culture was spun down at 400<italic>g</italic> for 1 min and resuspended in 100 µl fivefold diluted TAP media. 1 µl bead stock solution (25 mg/ml) was then added to 20 µl of resuspended cell culture and the mixture was incubated in ice for 10 min, in order to slow down IFT movement for bead attachment. 1 µl of 20 mM CaCl<sub>2</sub> was then added, the cell culture was incubated at room temperature for 10 min for bead movement to recover. To immobilize cells on a glass surface, the cover glasses (18 × 18 mm) were pre-treated with 0.7 mg/ml poly-L-lysine (MW = 150–300 kDa; Sigma) for 5 min. The nile-red bead fluorescence was recorded with Andor iXon 128 × 128 EM-CCD at 400 µs per frame. To prevent deflagellation of cells under intense (100 mW) laser illumination, only the flagellar regions of immobilized cells were excited. Flagellar membrane adsorption of carboxylated 100 nm-beads was significantly reduced without antibody crosslinking (∼50-fold), and only two bead motility events were observed in 500 cells.</p></sec><sec id="s4-4"><title>Multicolor-tracking assays</title><p>To simultaneously monitor the movement of IFT trains and FMG1-B proteins, IFT27-GFP <italic>pf18</italic> cells were incubated with anti-FMG1-B-coated dark-red beads. The GFP and dark-red bead fluorescence were simultaneously recorded at 200 ms per frame. The image was split into two fluorescent channels, which were registered to a sub-pixel accuracy (<xref ref-type="bibr" rid="bib13">DeWitt et al., 2012</xref>) with respect to each other, prior to live-cell imaging. The crosstalk between the two fluorescent channels was below 0.1%.</p><p>Various concentrations of EGTA were added to IFT27-GFP <italic>pf18</italic> cells grown in TAP media (contains 0.34 mM of Ca<sup>2+</sup>) and the movies were recorded within 15 min after EGTA addition. The concentration of free Ca<sup>2+</sup> in the assay buffer as a function of added EGTA was calculated from the Chelator program (<ext-link ext-link-type="uri" xlink:href="http://maxchelator.stanford.edu">http://maxchelator.stanford.edu</ext-link>). Only the cells with fully grown flagella (8–12 µm in length) were analyzed. Immotile IFT trains from the beginning to the end of the image acquisition were excluded from data analysis. The pausing frequency was calculated from dividing the total number of pausing events by the image acquisition time.</p></sec><sec id="s4-5"><title>Optical trapping assays</title><p>Force measurements on bead motility were carried out with a custom-built optical trap microscope (<xref ref-type="bibr" rid="bib21">Gennerich et al., 2007</xref>), with single molecule fluorescence detection ability. A 1064 nm laser (Coherent, Compass) was mounted on an inverted microscope equipped with a 100× 1.49 NA oil immersion objective (Nikon). The trapping beam was steered by a computer-controlled acousto-optic deflector (AA Electronics) at 20 kHz. Trap stiffness was calibrated for each bead from the amplitude of its thermal diffusion. The beads were trapped by a 400 mW 1064 nm laser beam to achieve an average spring constant of ∼0.4 pN/nm. The bead displacement was detected by a quadrant photodiode (QPD) and recorded at 2 kHz. Experiments were carried out with the trap position fixed. Stall forces were defined as the magnitude of the opposing force that reduces the mean velocity of the cargo to 0 for more than 100 ms. Returning events with stalling periods shorter than 100 ms at peak forces were scored as releases. Movements of beads beyond the linear range of the detector (±200 nm, ±80 pN) were scored as escapes. The trapping assays at restrictive conditions were performed at 34°C. Optical trap microscope was equipped with a 488 nm laser beam with near-TIRF excitation to track individual trains in IFT27-GFP cells. Bright-field illumination was turned on for ∼1 s at every 1–2 min to monitor the bead position along the flagella and to synchronize the PSD signal and CCD image.</p><p>Carboxylated latex beads (0.92 µm diameter, Invitrogen) were coated with 0.05 mg/ml anti-carbohydrate mouse monoclonal antibody to FMG1-B for flagellar membrane attachment. Cover glasses were pre-treated with 0.7 mg/ml polylysine for 5 min. 10 µl <italic>pf18</italic> cell culture was flowed into a flow chamber and incubated for 30 s to allow cells to attach to the cover glass. 10 µl solution including 0.2× TAP, 1 mM CaCl<sub>2</sub>, 0.3 mg/ml BSA, and 0.1 g/l beads was then flowed into the chamber to replace the buffer. Trapped beads were positioned over surface immobilized flagella (<xref ref-type="bibr" rid="bib22">Guilford and Bloodgood, 2013</xref>). The fraction of moving beads was measured by resting beads on the flagellar surface for 1 min.</p><p>Viscous drag constant between the bead and the flagellar membrane was measured by oscillating a flagellar surface attached bead ±500 nm along the length of flagellum in a square wave pattern at 0.2 Hz. The connection between the bead and FMG-1B was verified by moving the trap away from the flagellum. If the bead displayed active movement powered by IFT trains during bead oscillation measurements, the bead was lifted for 0.5–1.0 μm to dissociate it away from IFT and then brought back to flagella.</p></sec><sec id="s4-6"><title>Data analysis</title><p>Speeds were measured from linear fits to the displacement traces observed in kymographs. In order to distinguish between IFT trains moving in anterograde and retrograde directions and IFT trains pausing over a period of time, we assigned different colors to individual IFT trains as a function of their velocity by Fourier space directional analysis (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). The image was then transformed back into real space to recover the kymographs. To perform IFT kymography analysis during gliding motility, we separately determined the position of IFT trains relative to the cell body, and the position of the cell body relative to the glass surface (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Correlation between the movements of beads and IFT trains was demonstrated by rejecting the null hypothesis (IFT and bead movement was uncorrelated) using Kolmogorov-Smirnov statistics.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank RD Vale and RA Bloodgood for evaluation of the manuscript, and M Porter, K Lechtreck, G Witman and J Rosenbaum for providing <italic>C. reinhardtii</italic> mutant strains and antibodies.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SMS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>BDE, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>FK, Acquisition of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>AG, Acquisition of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>TB, Conception and design, Acquisition of data</p></fn><fn fn-type="con" id="con6"><p>WFM, Conception and design, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>AY, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name></person-group><year>1981</year><article-title>Flagella-dependent gliding motility in Chlamydomonas</article-title><source>Protoplasma</source><volume>106</volume><fpage>183</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1007/BF01275550</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name></person-group><year>1995</year><article-title>Flagellar surface motility: gliding and microsphere movements</article-title><source>Methods Cell Biol</source><volume>47</volume><fpage>273</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/S0091-679X(08)60820-1</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name></person-group><year>2009</year><article-title>The <italic>Chlamydomonas</italic> flagellar membrane and its dynamic properties</article-title><person-group person-group-type="editor"><name><surname>Witman</surname><given-names>G</given-names></name><name><surname>Harris</surname><given-names>E</given-names></name></person-group><source>The <italic>Chlamydomonas</italic> sourcebook: cell motility and behavior</source><volume>Vol 3</volume><edition>2nd Ed.</edition><publisher-loc>San Diego</publisher-loc><publisher-name>Academic Press</publisher-name><fpage>309</fpage><lpage>68</lpage></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Salomonsky</surname><given-names>NL</given-names></name></person-group><year>1990</year><article-title>Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane</article-title><source>J Cell Sci</source><volume>96</volume><issue>Pt 1</issue><fpage>27</fpage><lpage>33</lpage></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Salomonsky</surname><given-names>NL</given-names></name></person-group><year>1994</year><article-title>The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein</article-title><source>J Cell Biol</source><volume>127</volume><fpage>803</fpage><lpage>11</lpage><pub-id pub-id-type="doi">10.1083/jcb.127.3.803</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Salomonsky</surname><given-names>NL</given-names></name></person-group><year>1998</year><article-title>Microsphere attachment induces glycoprotein redistribution and transmembrane signaling in the Chlamydomonas flagellum</article-title><source>Protoplasma</source><volume>202</volume><fpage>76</fpage><lpage>83</lpage><pub-id pub-id-type="doi">10.1007/BF01280876</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Spano</surname><given-names>AJ</given-names></name></person-group><year>2002</year><article-title>Gliding motility in <italic>Chlamydomonas reinhardtii</italic> requires a calcium dependent protein kinase</article-title><source>Mol Biol Cell</source><volume>13</volume><fpage>191a</fpage></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Woodward</surname><given-names>MP</given-names></name><name><surname>Salomonsky</surname><given-names>NL</given-names></name></person-group><year>1986</year><article-title>Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A</article-title><source>J Cell Biol</source><volume>102</volume><fpage>1797</fpage><lpage>812</lpage><pub-id pub-id-type="doi">10.1083/jcb.102.5.1797</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Workman</surname><given-names>LJ</given-names></name></person-group><year>1984</year><article-title>A flagellar surface glycoprotein mediating cell-substrate interaction in Chlamydomonas</article-title><source>Cell Motil</source><volume>4</volume><fpage>77</fpage><lpage>87</lpage><pub-id pub-id-type="doi">10.1002/cm.970040202</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brunnbauer</surname><given-names>M</given-names></name><name><surname>Mueller-Planitz</surname><given-names>F</given-names></name><name><surname>Kosem</surname><given-names>S</given-names></name><name><surname>Ho</surname><given-names>TH</given-names></name><name><surname>Dombi</surname><given-names>R</given-names></name><name><surname>Gebhardt</surname><given-names>JC</given-names></name><etal/></person-group><year>2010</year><article-title>Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner</article-title><source>Proc Natl Acad Sci USA</source><volume>107</volume><fpage>10460</fpage><lpage>5</lpage><pub-id pub-id-type="doi">10.1073/pnas.1005177107</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cole</surname><given-names>DG</given-names></name><name><surname>Diener</surname><given-names>DR</given-names></name><name><surname>Himelblau</surname><given-names>AL</given-names></name><name><surname>Beech</surname><given-names>PL</given-names></name><name><surname>Fuster</surname><given-names>JC</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>1998</year><article-title>Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in <italic>Caenorhabditis elegans</italic> sensory neurons</article-title><source>J Cell Biol</source><volume>141</volume><fpage>993</fpage><lpage>1008</lpage><pub-id pub-id-type="doi">10.1083/jcb.141.4.993</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dentler</surname><given-names>W</given-names></name></person-group><year>2005</year><article-title>Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella</article-title><source>J Cell Biol</source><volume>170</volume><fpage>649</fpage><lpage>59</lpage><pub-id pub-id-type="doi">10.1083/jcb.200412021</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>DeWitt</surname><given-names>MA</given-names></name><name><surname>Chang</surname><given-names>AY</given-names></name><name><surname>Combs</surname><given-names>PA</given-names></name><name><surname>Yildiz</surname><given-names>A</given-names></name></person-group><year>2012</year><article-title>Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains</article-title><source>Science</source><volume>335</volume><fpage>221</fpage><lpage>5</lpage><pub-id pub-id-type="doi">10.1126/science.1215804</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dymek</surname><given-names>EE</given-names></name><name><surname>Goduti</surname><given-names>D</given-names></name><name><surname>Kramer</surname><given-names>T</given-names></name><name><surname>Smith</surname><given-names>EF</given-names></name></person-group><year>2006</year><article-title>A kinesin-like calmodulin-binding protein in Chlamydomonas: evidence for a role in cell division and flagellar functions</article-title><source>J Cell Sci</source><volume>119</volume><fpage>3107</fpage><lpage>16</lpage><pub-id pub-id-type="doi">10.1242/jcs.03028</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Engel</surname><given-names>BD</given-names></name><name><surname>Ishikawa</surname><given-names>H</given-names></name><name><surname>Feldman</surname><given-names>JL</given-names></name><name><surname>Wilson</surname><given-names>CW</given-names></name><name><surname>Chuang</surname><given-names>PT</given-names></name><name><surname>Snedecor</surname><given-names>J</given-names></name><etal/></person-group><year>2011</year><article-title>A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow</article-title><source>Cytoskeleton (Hoboken)</source><volume>68</volume><fpage>188</fpage><lpage>203</lpage><pub-id pub-id-type="doi">10.1002/cm.20504</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Engel</surname><given-names>BD</given-names></name><name><surname>Ishikawa</surname><given-names>H</given-names></name><name><surname>Wemmer</surname><given-names>KA</given-names></name><name><surname>Geimer</surname><given-names>S</given-names></name><name><surname>Wakabayashi</surname><given-names>K</given-names></name><name><surname>Hirono</surname><given-names>M</given-names></name><etal/></person-group><year>2012</year><article-title>The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function</article-title><source>J Cell Biol</source><volume>199</volume><fpage>151</fpage><lpage>67</lpage><pub-id pub-id-type="doi">10.1083/jcb.201206068</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Engel</surname><given-names>BD</given-names></name><name><surname>Lechtreck</surname><given-names>KF</given-names></name><name><surname>Sakai</surname><given-names>T</given-names></name><name><surname>Ikebe</surname><given-names>M</given-names></name><name><surname>Witman</surname><given-names>GB</given-names></name><name><surname>Marshall</surname><given-names>WF</given-names></name></person-group><year>2009a</year><article-title>Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella</article-title><source>Methods Cell Biol</source><volume>93</volume><fpage>157</fpage><lpage>77</lpage><pub-id pub-id-type="doi">10.1016/S0091-679X(08)93009-0</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Engel</surname><given-names>BD</given-names></name><name><surname>Ludington</surname><given-names>WB</given-names></name><name><surname>Marshall</surname><given-names>WF</given-names></name></person-group><year>2009b</year><article-title>Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model</article-title><source>J Cell Biol</source><volume>187</volume><fpage>81</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1083/jcb.200812084</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ferris</surname><given-names>PJ</given-names></name><name><surname>Waffenschmidt</surname><given-names>S</given-names></name><name><surname>Umen</surname><given-names>JG</given-names></name><name><surname>Lin</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>JH</given-names></name><name><surname>Ishida</surname><given-names>K</given-names></name><etal/></person-group><year>2005</year><article-title>Plus and minus sexual agglutinins from <italic>Chlamydomonas reinhardtii</italic></article-title><source>Plant Cell</source><volume>17</volume><fpage>597</fpage><lpage>615</lpage><pub-id pub-id-type="doi">10.1105/tpc.104.028035</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Firestone</surname><given-names>AJ</given-names></name><name><surname>Weinger</surname><given-names>JS</given-names></name><name><surname>Maldonado</surname><given-names>M</given-names></name><name><surname>Barlan</surname><given-names>K</given-names></name><name><surname>Langston</surname><given-names>LD</given-names></name><name><surname>O’Donnell</surname><given-names>M</given-names></name><etal/></person-group><year>2012</year><article-title>Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein</article-title><source>Nature</source><volume>484</volume><fpage>125</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1038/nature10936</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gennerich</surname><given-names>A</given-names></name><name><surname>Carter</surname><given-names>AP</given-names></name><name><surname>Reck-Peterson</surname><given-names>SL</given-names></name><name><surname>Vale</surname><given-names>RD</given-names></name></person-group><year>2007</year><article-title>Force-induced bidirectional stepping of cytoplasmic dynein</article-title><source>Cell</source><volume>131</volume><fpage>952</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2007.10.016</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Guilford</surname><given-names>WH</given-names></name><name><surname>Bloodgood</surname><given-names>RA</given-names></name></person-group><year>2013</year><article-title>Chapter five - laser trap measurements of flagellar membrane motility</article-title><person-group person-group-type="editor"><name><surname>Wallace</surname><given-names>FM</given-names></name></person-group><source>Methods in enzymology</source><publisher-name>Academic Press</publisher-name><fpage>85</fpage><lpage>107</lpage></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoffman</surname><given-names>JL</given-names></name><name><surname>Goodenough</surname><given-names>UW</given-names></name></person-group><year>1980</year><article-title>Experimental dissection of flagellar surface motility in Chlamydomonas</article-title><source>J Cell Biol</source><volume>86</volume><fpage>656</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1083/jcb.86.2.656</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Homan</surname><given-names>W</given-names></name><name><surname>Sigon</surname><given-names>C</given-names></name><name><surname>van den Briel</surname><given-names>W</given-names></name><name><surname>Wagter</surname><given-names>R</given-names></name><name><surname>de Nobel</surname><given-names>H</given-names></name><name><surname>Mesland</surname><given-names>D</given-names></name><etal/></person-group><year>1987</year><article-title>Transport of membrane receptors and the mechanics of sexual cell fusion in <italic>Chlamydomonas eugametos</italic></article-title><source>FEBS Lett</source><volume>215</volume><fpage>323</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1016/0014-5793(87)80170-9</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huang</surname><given-names>K</given-names></name><name><surname>Diener</surname><given-names>DR</given-names></name><name><surname>Mitchell</surname><given-names>A</given-names></name><name><surname>Pazour</surname><given-names>GJ</given-names></name><name><surname>Witman</surname><given-names>GB</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>2007</year><article-title>Function and dynamics of PKD2 in <italic>Chlamydomonas reinhardtii</italic> flagella</article-title><source>J Cell Biol</source><volume>179</volume><fpage>501</fpage><lpage>14</lpage><pub-id pub-id-type="doi">10.1083/jcb.200704069</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huangfu</surname><given-names>D</given-names></name><name><surname>Liu</surname><given-names>A</given-names></name><name><surname>Rakeman</surname><given-names>AS</given-names></name><name><surname>Murcia</surname><given-names>NS</given-names></name><name><surname>Niswander</surname><given-names>L</given-names></name><name><surname>Anderson</surname><given-names>KV</given-names></name></person-group><year>2003</year><article-title>Hedgehog signalling in the mouse requires intraflagellar transport proteins</article-title><source>Nature</source><volume>426</volume><fpage>83</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/nature02061</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ishikawa</surname><given-names>H</given-names></name><name><surname>Marshall</surname><given-names>WF</given-names></name></person-group><year>2011</year><article-title>Ciliogenesis: building the cell’s antenna</article-title><source>Nat Rev Mol Cell Biol</source><volume>12</volume><fpage>222</fpage><lpage>34</lpage><pub-id pub-id-type="doi">10.1038/nrm3085</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jamison</surname><given-names>DK</given-names></name><name><surname>Driver</surname><given-names>JW</given-names></name><name><surname>Rogers</surname><given-names>AR</given-names></name><name><surname>Constantinou</surname><given-names>PE</given-names></name><name><surname>Diehl</surname><given-names>MR</given-names></name></person-group><year>2010</year><article-title>Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport</article-title><source>Biophys J</source><volume>99</volume><fpage>2967</fpage><lpage>77</lpage><pub-id pub-id-type="doi">10.1016/j.bpj.2010.08.025</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kozminski</surname><given-names>KG</given-names></name><name><surname>Beech</surname><given-names>PL</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>1995</year><article-title>The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane</article-title><source>J Cell Biol</source><volume>131</volume><fpage>1517</fpage><lpage>27</lpage><pub-id pub-id-type="doi">10.1083/jcb.131.6.1517</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kozminski</surname><given-names>KG</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name><name><surname>Forscher</surname><given-names>P</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>1993</year><article-title>A motility in the eukaryotic flagellum unrelated to flagellar beating</article-title><source>Proc Natl Acad Sci USA</source><volume>90</volume><fpage>5519</fpage><lpage>23</lpage><pub-id pub-id-type="doi">10.1073/pnas.90.12.5519</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Laib</surname><given-names>JA</given-names></name><name><surname>Marin</surname><given-names>JA</given-names></name><name><surname>Bloodgood</surname><given-names>RA</given-names></name><name><surname>Guilford</surname><given-names>WH</given-names></name></person-group><year>2009</year><article-title>The reciprocal coordination and mechanics of molecular motors in living cells</article-title><source>Proc Natl Acad Sci USA</source><volume>106</volume><fpage>3190</fpage><lpage>5</lpage><pub-id pub-id-type="doi">10.1073/pnas.0809849106</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lechtreck</surname><given-names>KF</given-names></name><name><surname>Brown</surname><given-names>JM</given-names></name><name><surname>Sampaio</surname><given-names>JL</given-names></name><name><surname>Craft</surname><given-names>JM</given-names></name><name><surname>Shevchenko</surname><given-names>A</given-names></name><name><surname>Evans</surname><given-names>JE</given-names></name><etal/></person-group><year>2013</year><article-title>Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase</article-title><source>J Cell Biol</source><volume>201</volume><fpage>249</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1083/jcb.201207139</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lechtreck</surname><given-names>KF</given-names></name><name><surname>Johnson</surname><given-names>EC</given-names></name><name><surname>Sakai</surname><given-names>T</given-names></name><name><surname>Cochran</surname><given-names>D</given-names></name><name><surname>Ballif</surname><given-names>BA</given-names></name><name><surname>Rush</surname><given-names>J</given-names></name><etal/></person-group><year>2009</year><article-title>The <italic>Chlamydomonas reinhardtii</italic> BBSome is an IFT cargo required for export of specific signaling proteins from flagella</article-title><source>J Cell Biol</source><volume>187</volume><fpage>1117</fpage><lpage>32</lpage><pub-id pub-id-type="doi">10.1083/jcb.200909183</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lecuit</surname><given-names>T</given-names></name><name><surname>Lenne</surname><given-names>PF</given-names></name><name><surname>Munro</surname><given-names>E</given-names></name></person-group><year>2011</year><article-title>Force generation, transmission, and integration during cell and tissue morphogenesis</article-title><source>Annu Rev Cell Dev Biol</source><volume>27</volume><fpage>157</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1146/annurev-cellbio-100109-104027</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lewin</surname><given-names>RA</given-names></name></person-group><year>1952</year><article-title>Studies on the flagella of Algae. I. General observations on <italic>Chlamydomonas Moewusii</italic> Gerloff</article-title><source>Biol Bull</source><volume>103</volume><fpage>74</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.2307/1538407</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liem</surname><given-names>KF</given-names><suffix>Jnr.</suffix></name><name><surname>Ashe</surname><given-names>A</given-names></name><name><surname>He</surname><given-names>M</given-names></name><name><surname>Satir</surname><given-names>P</given-names></name><name><surname>Moran</surname><given-names>J</given-names></name><name><surname>Beier</surname><given-names>D</given-names></name><etal/></person-group><year>2012</year><article-title>The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking</article-title><source>J Cell Biol</source><volume>197</volume><fpage>789</fpage><lpage>800</lpage><pub-id pub-id-type="doi">10.1083/jcb.201110049</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mallik</surname><given-names>R</given-names></name><name><surname>Petrov</surname><given-names>D</given-names></name><name><surname>Lex</surname><given-names>SA</given-names></name><name><surname>King</surname><given-names>SJ</given-names></name><name><surname>Gross</surname><given-names>SP</given-names></name></person-group><year>2005</year><article-title>Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications</article-title><source>Curr Biol</source><volume>15</volume><fpage>2075</fpage><lpage>85</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2005.10.039</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ott</surname><given-names>C</given-names></name><name><surname>Elia</surname><given-names>N</given-names></name><name><surname>Jeong</surname><given-names>SY</given-names></name><name><surname>Insinna</surname><given-names>C</given-names></name><name><surname>Sengupta</surname><given-names>P</given-names></name><name><surname>Lippincott-Schwartz</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Primary cilia utilize glycoprotein-dependent adhesion mechanisms to stabilize long-lasting cilia-cilia contacts</article-title><source>Cilia</source><volume>1</volume><fpage>3</fpage><pub-id pub-id-type="doi">10.1186/2046-2530-1-3</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ou</surname><given-names>G</given-names></name><name><surname>Blacque</surname><given-names>OE</given-names></name><name><surname>Snow</surname><given-names>JJ</given-names></name><name><surname>Leroux</surname><given-names>MR</given-names></name><name><surname>Scholey</surname><given-names>JM</given-names></name></person-group><year>2005</year><article-title>Functional coordination of intraflagellar transport motors</article-title><source>Nature</source><volume>436</volume><fpage>583</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/nature03818</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pan</surname><given-names>J</given-names></name><name><surname>Snell</surname><given-names>WJ</given-names></name></person-group><year>2002</year><article-title>Kinesin-II is required for flagellar sensory transduction during fertilization in Chlamydomonas</article-title><source>Mol Biol Cell</source><volume>13</volume><fpage>1417</fpage><lpage>26</lpage><pub-id pub-id-type="doi">10.1091/mbc.01-11-0531</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pazour</surname><given-names>GJ</given-names></name><name><surname>Agrin</surname><given-names>N</given-names></name><name><surname>Leszyk</surname><given-names>J</given-names></name><name><surname>Witman</surname><given-names>GB</given-names></name></person-group><year>2005</year><article-title>Proteomic analysis of a eukaryotic cilium</article-title><source>J Cell Biol</source><volume>170</volume><fpage>103</fpage><lpage>13</lpage><pub-id pub-id-type="doi">10.1083/jcb.200504008</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pazour</surname><given-names>GJ</given-names></name><name><surname>Wilkerson</surname><given-names>CG</given-names></name><name><surname>Witman</surname><given-names>GB</given-names></name></person-group><year>1998</year><article-title>A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT)</article-title><source>J Cell Biol</source><volume>141</volume><fpage>979</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1083/jcb.141.4.979</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pedersen</surname><given-names>LB</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>2008</year><article-title>Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling</article-title><source>Curr Top Dev Biol</source><volume>85</volume><fpage>23</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1016/S0070-2153(08)00802-8</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pigino</surname><given-names>G</given-names></name><name><surname>Geimer</surname><given-names>S</given-names></name><name><surname>Lanzavecchia</surname><given-names>S</given-names></name><name><surname>Paccagnini</surname><given-names>E</given-names></name><name><surname>Cantele</surname><given-names>F</given-names></name><name><surname>Diener</surname><given-names>DR</given-names></name><etal/></person-group><year>2009</year><article-title>Electron-tomographic analysis of intraflagellar transport particle trains in situ</article-title><source>J Cell Biol</source><volume>187</volume><fpage>135</fpage><lpage>48</lpage><pub-id pub-id-type="doi">10.1083/jcb.200905103</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Porter</surname><given-names>ME</given-names></name><name><surname>Bower</surname><given-names>R</given-names></name><name><surname>Knott</surname><given-names>JA</given-names></name><name><surname>Byrd</surname><given-names>P</given-names></name><name><surname>Dentler</surname><given-names>W</given-names></name></person-group><year>1999</year><article-title>Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas</article-title><source>Mol Biol Cell</source><volume>10</volume><fpage>693</fpage><lpage>712</lpage></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qin</surname><given-names>H</given-names></name><name><surname>Burnette</surname><given-names>DT</given-names></name><name><surname>Bae</surname><given-names>YK</given-names></name><name><surname>Forscher</surname><given-names>P</given-names></name><name><surname>Barr</surname><given-names>MM</given-names></name><name><surname>Rosenbaum</surname><given-names>JL</given-names></name></person-group><year>2005</year><article-title>Intraflagellar transport is required for the vectorial movement of TRPV channels in the ciliary membrane</article-title><source>Curr Biol</source><volume>15</volume><fpage>1695</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2005.08.047</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rosenbaum</surname><given-names>JL</given-names></name><name><surname>Witman</surname><given-names>GB</given-names></name></person-group><year>2002</year><article-title>Intraflagellar transport</article-title><source>Nat Rev Mol Cell Biol</source><volume>3</volume><fpage>813</fpage><lpage>25</lpage><pub-id pub-id-type="doi">10.1038/nrm952</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shubeita</surname><given-names>GT</given-names></name><name><surname>Tran</surname><given-names>SL</given-names></name><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Vershinin</surname><given-names>M</given-names></name><name><surname>Cermelli</surname><given-names>S</given-names></name><name><surname>Cotton</surname><given-names>SL</given-names></name><etal/></person-group><year>2008</year><article-title>Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets</article-title><source>Cell</source><volume>135</volume><fpage>1098</fpage><lpage>107</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2008.10.021</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snell</surname><given-names>WJ</given-names></name><name><surname>Pan</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Q</given-names></name></person-group><year>2004</year><article-title>Cilia and flagella revealed: from flagellar assembly in Chlamydomonas to human obesity disorders</article-title><source>Cell</source><volume>117</volume><fpage>693</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2004.05.019</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vershinin</surname><given-names>M</given-names></name><name><surname>Carter</surname><given-names>BC</given-names></name><name><surname>Razafsky</surname><given-names>DS</given-names></name><name><surname>King</surname><given-names>SJ</given-names></name><name><surname>Gross</surname><given-names>SP</given-names></name></person-group><year>2007</year><article-title>Multiple-motor based transport and its regulation by Tau</article-title><source>Proc Natl Acad Sci USA</source><volume>104</volume><fpage>87</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1073/pnas.0607919104</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Pan</surname><given-names>J</given-names></name><name><surname>Snell</surname><given-names>WJ</given-names></name></person-group><year>2006</year><article-title>Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas</article-title><source>Cell</source><volume>125</volume><fpage>549</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2006.02.044</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wei</surname><given-names>Q</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Ling</surname><given-names>K</given-names></name><name><surname>Hu</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>The BBSome controls IFT assembly and turnaround in cilia</article-title><source>Nat Cell Biol</source><volume>14</volume><fpage>950</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/ncb2560</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yildiz</surname><given-names>A</given-names></name><name><surname>Forkey</surname><given-names>JN</given-names></name><name><surname>McKinney</surname><given-names>SA</given-names></name><name><surname>Ha</surname><given-names>T</given-names></name><name><surname>Goldman</surname><given-names>YE</given-names></name><name><surname>Selvin</surname><given-names>PR</given-names></name></person-group><year>2003</year><article-title>Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization</article-title><source>Science</source><volume>300</volume><fpage>2061</fpage><lpage>5</lpage><pub-id pub-id-type="doi">10.1126/science.1084398</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00744.028</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pfeffer</surname><given-names>Suzanne</given-names></name><role>Reviewing editor</role><aff><institution>Stanford University</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://www.elifesciences.org/the-journal/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Intraflagellar Transport Drives Flagellar Surface Motility” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission want to reveal their identity: Suzanne Pfeffer (Reviewing editor), and William Hancock and Robert Bloodgood (peer reviewers).</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. All the comments can likely be addressed by clarification rather than any further experimental work.</p><p>1) Please clarify the viscosity measurement. This measurement is important because it implies that there are 16–24 pN viscous drag loads on the train-glycoprotein complexes, which is a significant insight. The observation is that when trapped beads attached to moving trains stall and then release, the bead is pulled back to the center of the trap relatively slowly (if it was simply a bead in solution, the bead drag forces are such that it would return within 1ms presumably). From the exponential fit and known trap stiffness they calculate the drag coefficient. One question is, what is the mode of failure? Is it motors detaching from the microtubules or could it be glycoprotein detaching from the train? (It seems that the simultaneous imaging says that the fluorescent train comes back with the bead, but we were not able to find this.) If the train is moving back, then the assumption is that the motors don't interact with the filament at all, but dynein has been shown to backstep and it's hard to rule out other motor-microtubule interactions, so the observed viscosity may not be just from the membrane. A possible experiment would be to step or oscillate a bead bound to transmembrane glycoprotein (but not train) and pull out viscosity that way.</p><p>2) The diagram of the cell body in <xref ref-type="fig" rid="fig3">Figure 3</xref> was really helpful for orienting the transport directions. Can such a diagram be added to the other figures as well to orient the reader?</p><p>3) It is quite difficult to see the pausing in <xref ref-type="fig" rid="fig2">Figure 2A/B</xref>. In the figure supplement there are some clearer examples, but the yellow highlighting in 2a makes it quite difficult to make out the pauses. The pausing is abundantly clear and quite convincing in <xref ref-type="other" rid="video2">Video 2</xref>. If the trains of interest were tracked and their position highlighted in a side-by-side Figure in 2a or possibly overlaid, that might help the reader see the paused trains. <xref ref-type="fig" rid="fig2">Figure 2C</xref> is difficult to understand. The odd coloring of the background is distracting, the red and blue arrows in the right panel are too small to see, and it's not described well. Perhaps thresholding to clean it up would help.</p><p>4) For <xref ref-type="fig" rid="fig2s2">Figure 2–figure supplement 2B</xref>, specify this is magnitude of FFT (if this is correct). Figure 2–figure supplement 5 was very informative. Can it be moved to the main text. Also, the i) and ii) notations on the kymograph aren't referenced in the legend or the adjacent cartoon, where they apply.</p><p>5) “The data indicate that multiple motors attached to an IFT train may not be able to reach their maximum stall force”: this statement is not well supported by the data.</p><p>6) <xref ref-type="fig" rid="fig5">Figure 5B</xref> is confusing. The bead seems to move anterograde and then retrograde and then escape, but just before escape it seems to be settling to its original position. Is the position actually the QPD signal and not the real position in those cases around +/- 200 nm because that is the extent of the linear range of the QPD? Please clarify.</p><p>It is interesting that for wt the gliding speed is independent of temperature (22 vs 37) – shouldn’t motor speed vary with temperature?</p><p>7) <xref ref-type="other" rid="video1">Video 1</xref>: why does the level of red fluorescence appear to vary so much from one microsphere to another in the field of view?</p><p>8) The caption to <xref ref-type="fig" rid="fig5">Figure 5</xref> gives the incorrect impression that the typical method of reversal of the direction of gliding motility of a <italic>Chlamydomonas</italic> involves the lifting of one flagellum off the substrate. While this can be sometimes observed, the vast majority of cases of reversals of direction are accomplished while both flagellar remain rigid and in contact with the substrate (albeit to different degrees).</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00744.029</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Please clarify the viscosity measurement. This measurement is important because it implies that there are 16–24 pN viscous drag loads on the train-glycoprotein complexes, which is a significant insight. The observation is that when trapped beads attached to moving trains stall and then release, the bead is pulled back to the center of the trap relatively slowly (if it was simply a bead in solution, the bead drag forces are such that it would return within 1ms presumably). From the exponential fit and known trap stiffness they calculate the drag coefficient. One question is, what is the mode of failure? Is it motors detaching from the microtubules or could it be glycoprotein detaching from the train? (It seems that the simultaneous imaging says that the fluorescent train comes back with the bead, but we were not able to find this.) If the train is moving back, then the assumption is that the motors don’t interact with the filament at all, but dynein has been shown to backstep and it’s hard to rule out other motor-microtubule interactions, so the observed viscosity may not be just from the membrane. A possible experiment would be to step or oscillate a bead bound to transmembrane glycoprotein (but not train) and pull out viscosity that way</italic>.</p><p>If dynein steps backward during the recoiling of the bead, we would expect to observe backward steps and the velocity of the bead to be similar to that of dynein. However, the average velocity of the bead is 20 µm/s (an order of magnitude faster than that of dynein 1b) and there are no backward steps detectable, which rule out the possibility of dynein backtracking.</p><p>To rule out the possibility that our optical trapping measures glycoprotein rupture forces from IFT trains, we performed simultaneous optical trapping of the bead and fluorescence tracking of IFT trains. <xref ref-type="fig" rid="fig6">Figure 6B</xref> shows that IFT train underneath the bead (red dashed curve) recoils with the IFT train. <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1B and C</xref> also show that the bead makes multiple runs and recoils back to the trap center as an IFT train remains underneath the trapped bead, before the bead escapes the trap. The data indicate that we can measure the force production of the same IFT train multiple runs without rupturing the glycoprotein/bead linkage.</p><p>We performed the experiment the reviewers suggested. We oscillated a bead on a flagellar membrane surface in a square wave pattern and measured the recoiling time of the bead. The average viscous drag coefficient was 8.4 ± 4.2 pN.s/µm, similar to the drag that beads experience when they interact with IFT trains (8.0 ± 2.8 pN.s/μm). We added a short discussion about these results in the main text and showed the results in <xref ref-type="fig" rid="fig7s1">Figure 7–figure supplement 1</xref>.</p><p><italic>2) The diagram of the cell body in</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref> <italic>was really helpful for orienting the transport directions. Can such a diagram be added to the other figures as well to orient the reader</italic>?</p><p>We have added a diagram of a <italic>Chlamydomonas</italic> cell adhered to surface with both of its flagella to kymographs to other figures.</p><p><italic>3) It is quite difficult to see the pausing in</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2A/B</italic></xref><italic>. In the figure supplement there are some clearer examples, but the yellow highlighting in 2a makes it quite difficult to make out the pauses. The pausing is abundantly clear and quite convincing in</italic> <xref ref-type="other" rid="video2"><italic>Video 2</italic></xref><italic>. If the trains of interest were tracked and their position highlighted in a side-by-side Figure in 2a or possibly overlaid, that might help the reader see the paused trains.</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2C</italic></xref> <italic>is difficult to understand. The odd coloring of the background is distracting, the red and blue arrows in the right panel are too small to see, and it’s not described well. Perhaps thresholding to clean it up would help</italic>.</p><p>We thank the reviewers for their suggestions. In the revised manuscript, we included diagrams next to kymographs to highlight the paused IFT trains and gliding motility. Coloring in <xref ref-type="fig" rid="fig2">Figure 2C</xref> is removed, which makes red and blue arrows easier to see. Arrows are made bigger and extra description for these arrows are added in the figure legend.</p><p><italic>4) For</italic> <xref ref-type="fig" rid="fig2s2"><italic>Figure 2–figure supplement 2B</italic></xref><italic>, specify this is magnitude of FFT (if this is correct)</italic>.</p><p>Yes. We specified the intensity as the magnitude of FFT in the figure legend.</p><p><italic>Figure 2–figure supplement 5 was very informative. Can it be moved to the main text. Also, the i) and ii) notations on the kymograph aren’t referenced in the legend or the adjacent cartoon, where they apply</italic>.</p><p>We moved the figure to the main text and referred to the notations (shown in C) in D and the figure legend.</p><p><italic>5) “The data indicate that multiple motors attached to an IFT train may not be able to reach their maximum stall force”: this statement is not well supported by the data</italic>.</p><p>This is one of the debated issues in multiple motors field. We clarified this statement by indicating the origin of discussion in the main text as follows: “Previous studies showed that multiple motors produce larger forces, move with higher velocities under load and carry cargos further than a single kinesin or dynein motor (<xref ref-type="bibr" rid="bib37">Mallik et al., 2005</xref>; <xref ref-type="bibr" rid="bib50">Vershinin et al., 2007</xref>). It remains controversial whether motor stall forces are additive at low motor copy numbers (<xref ref-type="bibr" rid="bib50">Vershinin et al., 2007</xref>) or whether multiple motors tend to transport their cargo using only one load-bearing motor at a time (<xref ref-type="bibr" rid="bib28">Jamison et al., 2010</xref>). Our data show that the average forces exerted on IFT trains well exceed the force-generation capability of a single motor. [We did not observe a significant difference between the force values of stall and release events. The data indicate that multiple motors attached to an IFT train may not be able to reach their maximum stall force, defined as the stall force of a single motor multiplied by the number of bound motors (<xref ref-type="bibr" rid="bib48">Shubeita et al., 2008</xref>).]”</p><p><italic>6)</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5B</italic></xref> <italic>is confusing. The bead seems to move anterograde and then retrograde and then escape, but just before escape it seems to be settling to its original position. Is the position actually the QPD signal and not the real position in those cases around +/- 200 nm because that is the extent of the linear range of the QPD? Please clarify</italic>.</p><p>We chose this example in which the bead exceeded the linear range of the position sensitive detector (PSD) and escaped the trap. Additional examples with the beads stall/release within the linear range of PSD are provided in the <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>. In those cases, the movements of IFT trains are within 100 nm and can barely be observed in the fluorescence channel. The reviewers are right. The linear range of the PSD is roughly ±150 nm. As the bead moves further away from the trap center, the position calculated from the PSD signal decreases. To make this point clear, we are attaching a typical PSD response as a function of true distance between the bead and the trap center (<xref ref-type="fig" rid="fig9">Author response image 1</xref>, below).<fig id="fig9" position="float"><label>Author response image 1</label><caption><p>PSD response curve as a function of bead-trap separation. The linear range of the PSD is ±150 nm. PSD signal decreases as the bead separation exceeds 200–300 nm.</p></caption><graphic xlink:href="elife00744f009"/></fig></p><p>We also show the rest of the bead movement trace, which shows the reduction in QPD signal, when the bead moves towards the extent of the linear range of the PSD (<xref ref-type="fig" rid="fig10">Author response image 2</xref>, below).<fig id="fig10" position="float"><label>Author response image 2</label><caption><p>Simultaneous tracking of IFT27-GFP and bead motility. At t = 3 s, the microscope stage was moved to bring the flagellum underneath the trapped bead. An IFT train stalls at the trap position (t = 5–8 s) and stays adhered to the bead until the bead escapes the trap (t = 10 s). PSD signal shows a plateau and then decreases as the bead moves further away from the trap center, as shown in <xref ref-type="fig" rid="fig9">Author response image 1</xref>.</p></caption><graphic xlink:href="elife00744f010"/></fig></p><p><italic>It is interesting that for wt the gliding speed is independent of temperature (22 vs 37) – shouldn’t motor speed vary with temperature</italic>?</p><p>In vitro motility assays showed that the velocity of conventional kinesin motors increase significantly when the temperature is shifted from 22°C to 37°C in accordance with the Arrhenius rate law, but the force generation remain largely constant within a wide range of temperatures (e.g., Kawaguchi et al., BBRC, 2001). However, temperature dependence of <italic>Chlamydomonas</italic> dynein-1b and kinesin-2 velocity has not been explored in vitro. Most relevant studies were performed in in live <italic>Chlamydomonas</italic> cells, which showed that IFT speed does not vary between 22°C and 32°C (Lomini et al., JCB 2001). It remains unclear whether the speed of IFT motors is independent of temperature, or the gliding speed is limited by the net force production on paused IFT trains, which is temperature independent.</p><p><italic>7)</italic> <xref ref-type="other" rid="video1"><italic>Video 1</italic></xref><italic>: why does the level of red fluorescence appear to vary so much from one microsphere to another in the field of view</italic>?</p><p>First, small fraction of the 200 nm beads forms aggregates, and those aggregates tend to attach to the glass surface. These unusually bright beads were not included in the analysis of the data. Second, the sample is illuminated with TIRF, in which the illumination intensity decays exponentially by the z position of the object. The beads positioned on top of the flagella appear dimmer than the ones nonspecifically adhered to the glass surface.</p><p><italic>8) The caption to</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref> <italic>gives the incorrect impression that the typical method of reversal of the direction of gliding motility of a Chlamydomonas involves the lifting of one flagellum off the substrate. While this can be sometimes observed, the vast majority of cases of reversals of direction are accomplished while both flagellar remain rigid and in contact with the substrate (albeit to different degrees)</italic>.</p><p>We actually observed 60% of the time the cell lifted one flagellum to glide toward the other direction. We reckon that there can be several reasons why our observations are different from the previously published results. First, we use TIRF illumination, which is highly sensitive to the distance to the surface compared to brightfield imaging. Second, in certain cases, the cell only needs to lift a part of its flagellum to release the surface-attached IFT trains. We categorize these events as the cell lifts its flagellum to reverse gliding direction. We are not sure how these events were classified in previous studies. Third, different cell strains seem to have different tendency to lift their flagella. For example, <italic>pf18</italic> strain tends to lift one of its flagella during gliding less frequently. In our experiments, we used IFT27-GFP strain, which is different from the strains that are previously used for this experiment.</p></body></sub-article></article> |