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| <?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">01498</article-id><article-id pub-id-type="doi">10.7554/eLife.01498</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in <italic>Caenorhabditis elegans</italic> neurons</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-7588"><name><surname>Richardson</surname><given-names>Claire E</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10301"><name><surname>Spilker</surname><given-names>Kerri A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7797"><name><surname>Cueva</surname><given-names>Juan G</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7798"><name><surname>Perrino</surname><given-names>John</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7799"><name><surname>Goodman</surname><given-names>Miriam B</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1880"><name><surname>Shen</surname><given-names>Kang</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff4"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biology</institution>, <institution>Stanford University</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Molecular and Cellular Physiology</institution>, <institution>Stanford University</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Cell Sciences Imaging Facility</institution>, <institution>Stanford University</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution>Howard Hughes Medical Institute, Stanford University</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Hobert</surname><given-names>Oliver</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>kangshen@stanford.edu</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Metabolic Engineering and Systems Biology, Biogen Indec, Cambridge, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>25</day><month>02</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01498</elocation-id><history><date date-type="received"><day>10</day><month>09</month><year>2013</year></date><date date-type="accepted"><day>10</day><month>01</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Richardson et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Richardson 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="elife01498.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="article-reference" xlink:href="10.7554/eLife.01637"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01498.001</object-id><p>In neuronal processes, microtubules (MTs) provide structural support and serve as tracks for molecular motors. While it is known that neuronal MTs are more stable than MTs in non-neuronal cells, the molecular mechanisms underlying this stability are not fully understood. In this study, we used live fluorescence microscopy to show that the <italic>C. elegans</italic> CAMSAP protein PTRN-1 localizes to puncta along neuronal processes, stabilizes MT foci, and promotes MT polymerization in neurites. Electron microscopy revealed that <italic>ptrn-1</italic> null mutants have fewer MTs and abnormal MT organization in the PLM neuron. Animals grown with a MT depolymerizing drug caused synthetic defects in neurite branching in the absence of <italic>ptrn-1</italic> function, indicating that PTRN-1 promotes MT stability. Further, <italic>ptrn-1</italic> null mutants exhibited aberrant neurite morphology and synaptic vesicle localization that is partially dependent on <italic>dlk-1</italic>. Our results suggest that PTRN-1 represents an important mechanism for promoting MT stability in neurons.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.001">http://dx.doi.org/10.7554/eLife.01498.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01498.002</object-id><title>eLife digest</title><p>Microtubules are tiny tubular structures made from many copies of proteins called tubulins. Microtubules have a number of important roles inside cells: they are part of the cytoskeleton that provides structural support for the cell; they help to pull chromosomes apart during cell division; and they guide the trafficking of proteins and molecules around inside the cell. Most microtubules are relatively unstable, undergoing continuous dis-assembly and re-assembly in response to the needs of the cell. The microtubules in the branches of nerve cells are an exception, remaining relatively stable over time. Now Richardson et al. and, independently, Marcette et al., have shown that a protein called PTRN-1 has an important role in stabilizing the microtubules in the nerve cells of nematode worms.</p><p>By tagging the PTRN-1 proteins with fluorescent molecules, Richardson et al. were able to show that these proteins were present along the length of the microtubules within the nerve cells. Further work showed that the PTRN-1 proteins stabilize the microtubule filaments within the branches of these nerve cells and also hold them in position.</p><p>Richardson et al. also found that worms that had been genetically modified to prevent them from producing PTRN-1 failed to traffic certain molecules to the synapses between nerve cells. Moreover, these mutants also had problems with the branching of their nerve cells; however, these defects were relatively mild, which suggests that other molecules and proteins act in parallel with PTRN-1 to stabilize microtubules in nerve cells. Further work should be able to identify these factors and elucidate how they work together to stabilize the microtubules in nerve cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.002">http://dx.doi.org/10.7554/eLife.01498.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>neurons</kwd><kwd>microtubules</kwd><kwd>development</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>C. elegans</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Shen</surname><given-names>Kang</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>NS047715</award-id><principal-award-recipient><name><surname>Goodman</surname><given-names>Miriam B</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>EB006745</award-id><principal-award-recipient><name><surname>Goodman</surname><given-names>Miriam B</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>1F32NS083135-01</award-id><principal-award-recipient><name><surname>Richardson</surname><given-names>Claire E</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 protein PTRN-1 anchors microtubules in neuronal branches to promote proper neuron morphology and function in <italic>Caenorhabditis elegans</italic>.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>In neurons, microtubules (MTs) provide structural support, provide tracks that molecular motors use to transport cargo from the cell body to the synapses, and promote the establishment and maintenance of neuronal polarity. The MT bundles in neuronal processes, especially axons, are exceptionally stable compared to those present in most cell types (<xref ref-type="bibr" rid="bib18">Conde and Cáceres, 2009</xref>). Many proteins bind along the side or at the plus end of neuronal MTs to promote MT stability (<xref ref-type="bibr" rid="bib18">Conde and Cáceres, 2009</xref>). Additionally, tubulin posttranslational modifications contribute to the structure and function of neuronal MTs (<xref ref-type="bibr" rid="bib32">Janke and Kneussel, 2010</xref>). A long-standing question is what mechanisms prevent depolymerization from the MT minus ends.</p><p>MTs are polarized, cylindrical structures assembled from α/β-tubulin heterodimers. Although tubulin dimers can be added and removed from the plus end of an MT, the minus end depolymerizes continuously if not stabilized (<xref ref-type="bibr" rid="bib41">Mimori-Kiyosue, 2011</xref>). In most cells, minus ends are anchored at the centrosome by the γ-tubulin ring complex (γ-TuRC). Ninein, another minus end-binding protein, both stabilizes MTs that have been released from the centrosome and anchors MTs at centrosomal and non-centrosomal sites (<xref ref-type="bibr" rid="bib42">Mogensen et al., 2000</xref>).</p><p>To produce the MT bundles in neurites, MTs are nucleated at the centrosome and transported into neurites by MT motor proteins (<xref ref-type="bibr" rid="bib65">Yu et al., 1993</xref>; <xref ref-type="bibr" rid="bib1">Ahmad et al., 1998</xref>; <xref ref-type="bibr" rid="bib61">Wang and Brown, 2002</xref>). Recently, <xref ref-type="bibr" rid="bib46">Ori-McKenney et al. (2012)</xref> showed that, in the dendritic arbor of <italic>D. melanogaster</italic> neurons, minus ends are also both nucleated and stabilized by γ-tubulin localized to Golgi outposts. Still, in at least some cell types, γ-tubulin could not be detected in neurites (<xref ref-type="bibr" rid="bib3">Baas and Joshi, 1992</xref>). Further, the centrosome is dispensable for promoting neuronal MT function in both <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib5">Basto et al., 2006</xref>) and cultured hippocampal neurons (<xref ref-type="bibr" rid="bib54">Stiess et al., 2010</xref>). These studies imply that additional factors stabilize the minus ends of MTs released from the centrosome and nucleate MTs in neurites.</p><p>The CAMSAP family of proteins has been identified as a group of conserved, MT minus end-binding proteins (<xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>). Patronin, the CAMSAP homolog in <italic>D. melanogaster</italic>, promotes MT stability by protecting minus ends released from the centrosome from depolymerization by kinesin-13 MT depolymerase (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>; <xref ref-type="bibr" rid="bib60">Wang et al., 2013</xref>). In <italic>H. sapiens</italic> epithelial cells, CAMSAP3 (Nezha) stabilizes MT minus ends at adherens junctions and throughout the cytosol (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). Along with the partially redundant CAMSAP2, CAMSAP3 promotes proper MT organization and organelle assembly (<xref ref-type="bibr" rid="bib55">Tanaka et al., 2012</xref>). Importantly, both Patronin and CAMSAP3 have been shown to bind the MT minus end directly in vitro (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Meng et al. purified and fluorescence-labeled the C-terminal half of CAMSAP3 and sequentially added rhodamin-labeled and rhodamin-unlabeled MTs (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). The CAMSAP3 fragment colocalized with the end of the MT with higher rhodamine fluorescence, which indicates that it was bound to the minus end (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). Goodwin and Vale found that purified GFP–Patronin, which was attached to a coverslip bound and anchored rhodamine-MTs by a single end (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Further, they used MT gliding assays in which either the plus-end motor kinesin or the minus-end motor dynein were added to the purified rhodamine-MT plus GFP–Patronin to show that the Patronin-bound end of the MT was the minus end (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Taken together, this literature suggests that the CAMSAP family of proteins plays important roles in stabilizing MTs in vivo.</p><p>We tested the hypothesis that CAMSAP proteins bind and stabilize MT minus ends in neuronal processes. We used <italic>C. elegans</italic> because neurite structure and function, along with subcellular protein localization, can be readily observed in vivo<italic>.</italic> Live imaging of the <italic>C. elegans</italic> CAMSAP homolog, PTRN-1, in cells co-labeled with fluorescence-tagged MTs indicates that PTRN-1 localizes to MT-binding puncta throughout neuronal processes. Using a combination of live imaging and electron microscopy, we implicate a role for PTRN-1 in promoting MT stability and polymerization in neurites. Finally, we show that the loss of <italic>ptrn-1</italic> function results in defective neurite branching and mislocalization of synaptic vesicles, indicating that it has an important role in neuron morphology and function. The loss of the DLK-1 pathway, which is known to function in synapse localization and neurite morphology (<xref ref-type="bibr" rid="bib43">Nakata et al., 2005</xref>; <xref ref-type="bibr" rid="bib56">Tedeschi and Bradke, 2013</xref>), partially suppresses these defects.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>PTRN-1 exhibits punctate localization throughout neuronal processes</title><p>The <italic>C. elegans</italic> genome encodes a single homolog of the CAMSAP family of MT minus end-binding proteins, PTRN-1. (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). PTRN-1a has a conserved domain structure with previously characterized CAMSAP proteins <italic>H. sapiens</italic> CAMSAP3 and <italic>D. melanogaster</italic> Patronin, consisting of a calponin homology domain near the N-terminus, a central region with predicted coiled-coil repeats, and a C-terminal CKK domain (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>) (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). As the other PTRN-1 isoform, PTRN-1b, lacks the CKK domain, which is the domain required for MT binding in other CAMSAP proteins, we focused on the PTRN-1a isoform (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Using a fosmid expressing mCherry from the <italic>ptrn-1</italic> promoter (<xref ref-type="bibr" rid="bib58">Tursun et al., 2009</xref>), we observed <italic>ptrn-1</italic> expression in many tissues throughout development, including neurons (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C–H</xref>).</p><p>We examined PTRN-1a subcellular localization in neurons using fluorescence-tagged PTRN-1a. Three fluorescence-tagged PTRN-1 constructs - PTRN-1a::YFP and PTRN-1a::tdTomato, which both used C-terminal tags, and GFP::PTRN-1, in which PTRN-1 was tagged at the N-terminus – localized to small, closely spaced puncta throughout neurites (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>, <xref ref-type="fig" rid="fig1">Figure 1</xref>). We focused on the PVD neuron, which elaborates a branching dendrite arbor from two primary dendrites that run laterally along the animal, as well as a single axon that extends ventrally to make presynaptic connections in the ventral nerve cord (VNC), thereby providing a useful system for visualizing multiple distinct processes (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Expressing <italic>ptrn-1a(cDNA)::tdTomato</italic> in a subset of cells including the PVD neuron, we observed irregularly spaced puncta of PTRN-1a::tdTomato throughout the PVD dendrites and axon (<xref ref-type="fig" rid="fig1">Figure 1B,C</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3A,B</xref>). A similar punctate localization was observed from PTRN-1 tagged with GFP at the N-terminus or with YFP at the C-terminus. We often observed continuous PTRN-1::tdTomato fluorescence in the primary dendrite adjacent to the cell body, and fewer, farther spaced puncta in the quaternary processes (<xref ref-type="fig" rid="fig1">Figure 1B</xref> and data not shown).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.003</object-id><label>Figure 1.</label><caption><title>PTRN-1 localizes to puncta throughout neurites and colocalizes with MTs.</title><p>(<bold>A</bold>) Schematic diagram of the central region of the PVD neuron. The cell body (blue oval) is in the posterior half of the animal. An elaborate dendritic arbor (blue lines) extends from the base of the head to the posterior of the animal, and the single axon (magenta) is extended into the ventral nerve chord (VNC). Black lines represent the outline of the animal. (<bold>B</bold>) PTRN-1a::tdTomato localization in the PVD neuron. The cell body is outside of the image, close to the left edge. (<bold>C</bold>) Confocal micrographs from 10 animals showing PTRN-1a::tdTomato localization in the PVD primary dendrite directly posterior to the cell body. (<bold>D</bold>–<bold>F</bold>). Colocalization of PTRN 1a::tdTomato (magenta) and EMTB::GFP (green) in the PVD neurites (<bold>D</bold>), at the sarcolemma of the body wall muscle cells (<bold>E</bold>), and in the cell interior of the body wall muscle cells (<bold>F</bold>). Data were acquired from <italic>wyEx5968</italic> and <italic>wyEx6022</italic> transgenes coexpressed in the <italic>ptrn-1(tm5597)</italic> mutant. Closed arrowhead indicates the primary dendrite, the open arrowheads indicate tertiary dendrite, and arrow points to axon of the PVD neuron (<bold>A</bold>, <bold>B</bold>, <bold>D</bold>). A, anterior; V, ventral. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.003">http://dx.doi.org/10.7554/eLife.01498.003</ext-link></p></caption><graphic xlink:href="elife01498f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>PTRN-1 is broadly expressed.</title><p>(<bold>A</bold>) The <italic>ptrn-1(F35B3.5)</italic> open reading frame, which encodes <italic>ptrn-1a</italic> and <italic>ptrn-1b</italic>. The <italic>tm5597</italic> allele contains a 604 nt deletion, resulting in Met136>Thr, Ala137>STOP. The <italic>wy560</italic> allele contains a 65.3-kb deletion spanning nucleotide 16,983,396-17,048,700 of LGX. (<bold>B</bold>) PTRN-1a has a conserved domain structure with CAMSAP proteins. CH, calponin homology; CC, coiled-coil; CKK, calmodulin-regulated spectrin-associated CKK domain. The PTRN-1a CKK domain is the most well-conserved portion of the protein, with 64% similarity to Patronin CKK. PTRN-1b lacks the CKK domain. (<bold>C–H</bold>) <italic>ptrn-1</italic> is expressed in many tissues throughout development. Fluorescence of mCherry expressed from a fosmid encoding <italic>ptrn-1::GFP::SL2::mCherry</italic> in the <italic>ptrn-1(tm5597)</italic> mutant (SL2: trans-splice leader 2, which causes mCherry to be transcribed as part of the <italic>ptrn-1</italic> transcript but translated independently). The head (<bold>C</bold>) and mid-body (<bold>E</bold>) of an adult, the posterior region of an L4 animal (<bold>D</bold> and <bold>F</bold>), and the whole body of an L1 animal (<bold>G</bold> and <bold>H</bold>) are shown. A, anterior; R, right; V, ventral; neu, neuron; int, intestine; pha, pharynx; DTC, distal tip cell; VNC, ventral nerve chord; PLM, the PLM touch receptor neuron; hyp, hypodermis; mus, muscle. Scale bar: 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.004">http://dx.doi.org/10.7554/eLife.01498.004</ext-link></p></caption><graphic xlink:href="elife01498fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>PTRN-1 exhibits punctate localization in neuronal processes and the body wall muscle cells.</title><p>(<bold>A</bold>) PTRN-1a::YFP expressed from the <italic>Pptrn-1</italic> promoter in the <italic>ptrn-1(tm5597)</italic> mutant. Pictured is a young adult animal. (<bold>B-D</bold>) Expanded view with a subset of confocal slices of region in the solid box (<bold>B</bold> and <bold>C</bold>) and dashed box (<bold>D</bold>) from A. (<bold>B</bold>) A single confocal slice at the sarcolemma of the body wall muscle cell. (<bold>C</bold>) A single confocal slice of the interior of the same body wall muscle cell as B. (<bold>D</bold>) Commissures from the ventral nerve chord (VNC) intersecting a sublateral neuronal process; a maximum projection of ∼6 μm is shown. (<bold>E</bold>) GFP::PTRN-1 expressed from the <italic>Punc-86</italic> promoter in the <italic>ptrn-1(tm5597)</italic> mutant exhibits similar localization to PTRN-1a::YFP in neurites in the ventral nerve cord. The cell body is the HSN neuron. As PTRN-1b is produced by removing an intron for which the splice sites are within two ptrn-1a exons, the gfp::ptrn-1a(cDNA) construct is expected to produce both GFP::PTRN-1a and GFP::PTRN1b. The comparable localization between these two constructs indicates that the <italic>ptrn-1a</italic> isoform is sufficient to achieve punctate localization in neurites. For comparison, Chalfie and Thomson used electron microscopy of MTs in the VNC to show that the average distance between MT minus ends is approximately 1.7 μm. (<bold>F</bold>) Expanded view of boxed region from <bold>E</bold>. Solid arrow head: PVD cell body, open arrow: VNC. A: anterior, R: right, V: ventral. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.005">http://dx.doi.org/10.7554/eLife.01498.005</ext-link></p></caption><graphic xlink:href="elife01498fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>PTRN-1 localizes to puncta throughout the neurites in the PVD and PHC neurons.</title><p>(<bold>A–C</bold>) Confocal images of 8–10 <italic>ptrn-1(tm5597)</italic> mutants expressing PTRN-1a::tdTomato in a subset of neurons, including PVD and PHC, were straightened and aligned. Regions shown are the tertiary dendrite (posterior the cell body) (<bold>A</bold>) and the axon (<bold>B</bold>) of the PVD neuron, as well as the dendrite of the PHC neuron (<bold>C</bold>). The PHC neuron with indicated approximate region of interest on the dendrite is diagramed in <xref ref-type="fig" rid="fig3">Figure 3A</xref>. A, anterior; D, dorsal. Scale bar: 5 μm. (<bold>D</bold> and <bold>E</bold>) Schematic diagrams of animals shown in <xref ref-type="other" rid="video1">Video 1</xref> (<bold>D</bold>) and <xref ref-type="other" rid="video2">Video 2</xref> (<bold>E</bold>). Gray lines outline of the animal, black lines show PVD dendrites: the filled arrowhead indicates the PVD primary dendrite, the open arrowheads point to tertiary dendrites. Pink lines are PVD axons. The PVD cell body is out of frame to the right in both images. PTRN-1::tdTomato is also expressed in the body wall muscle of these animals, marked with the dashed blue lines. A, anterior; V, ventral; scale bar: 10 μm. We could not resolve the ends of the tertiary dendrites, but there are usually gaps between the tertiary branches from each secondary branch (see <xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.006">http://dx.doi.org/10.7554/eLife.01498.006</ext-link></p></caption><graphic xlink:href="elife01498fs003"/></fig><fig id="fig1s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.007</object-id><label>Figure 1—figure supplement 4.</label><caption><title>EMTB::GFP binds MTs in the PVD neuron and the body wall muscles.</title><p>(<bold>A</bold> and <bold>B</bold>) EMTB::GFP in the PVD neuron of wild-type (<bold>A</bold>) and <italic>ptrn-1(tm5597) mutant</italic> (<bold>B</bold>) animals. Note that fluorescence intensity becomes progressively dimmer from the primary to the quaternary dendritic processes, as compared to the cytosolic GFP shown in <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>. (<bold>C–F</bold>) EMTB::GFP fluorescence in the body wall muscle cells of wild-type (<bold>C</bold> and <bold>E</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>D</bold> and <bold>F</bold>) animals. (<bold>C</bold> and <bold>D</bold>) Confocal slice at the sarcolemma of the body wall muscle; (<bold>E</bold> and <bold>F</bold>) the interior of the body wall muscle cell.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.007">http://dx.doi.org/10.7554/eLife.01498.007</ext-link></p></caption><graphic xlink:href="elife01498fs004"/></fig><fig id="fig1s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.008</object-id><label>Figure 1—figure supplement 5.</label><caption><title>Highly expressed PTRN-1a::tdTomato binds along MT filaments.</title><p>PTRN-1a::tdTomato near the membrane (<bold>A</bold>) and in the interior (<bold>B</bold>) of a body wall muscle cell in an animal carrying a highly expressing <italic>ptrn-1a::tdTomato</italic> transgene. Compare to MT localization in <xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4</xref>. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.008">http://dx.doi.org/10.7554/eLife.01498.008</ext-link></p></caption><graphic xlink:href="elife01498fs005"/></fig></fig-group></p><p>We also examined PTRN-1a::tdTomato localization in the PHC sensory neuron, which has the simple bipolar morphology more typical of <italic>C. elegans</italic> neurons (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). In the PHC dendrite, PTRN-1::tdTomato localized to puncta with spacing similar to that of the PVD neuron (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3C</xref>), suggestive that the PTRN-1a localization pattern in neurites is similar across different neuron classes.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.009</object-id><label>Figure 2.</label><caption><title>PTRN-1 stabilizes MT foci in neurons and muscles.</title><p>(<bold>A</bold>–<bold>D</bold>) PTRN-1a::tdTomato and EMTB::GFP at the sarcolemma (<bold>A</bold> and <bold>C</bold>) and cell interior (<bold>B</bold> and <bold>D</bold>) of body wall muscle cells after acute colchicine exposure (<bold>C</bold> and <bold>D</bold>) or M9 control (<bold>A</bold> and <bold>B</bold>). (<bold>E</bold> and <bold>F</bold>) PTRN-1a(ΔCKK)::tdTomato and EMTB::GFP at the sarcolemma (<bold>E</bold>) and cell interior (<bold>F</bold>) of body wall muscle cells after acute colchicine exposure. (<bold>G</bold>–<bold>H</bold>) Localization of PTRN-1a::tdTomato and EMTB::GFP in the PVD dendrite after acute colchicine exposure (<bold>H</bold>) or M9 control (<bold>G</bold>). (<bold>I</bold>) PTRN-1a(ΔCKK)::tdTomato and EMTB::GFP in the PVD primary dendrite after acute colchicine exposure. All data acquired from <italic>wyEx5968</italic> with either <italic>wyEx6022</italic> (<bold>A</bold>–<bold>D</bold> and <bold>G</bold> and <bold>H</bold>), <italic>wyEx6092</italic> (<bold>I</bold>), or <italic>wyEx6165</italic> (<bold>E</bold> and <bold>F</bold>) co-expressed in <italic>bus-17(e2800)</italic> mutant animals. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.009">http://dx.doi.org/10.7554/eLife.01498.009</ext-link></p></caption><graphic xlink:href="elife01498f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.010</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Acute colchicine exposure changes EMTB::GFP localization in body wall muscles.</title><p>(<bold>A</bold> and <bold>B</bold>) EMTB::GFP localization in the body wall muscle after acute colchicine treatment in the <italic>bus-17(e2800)</italic> genetic background. (<bold>A</bold>) Confocal slice at the sarcolemma; (<bold>B</bold>) the interior of the body wall muscle cell. Note that this animal, unlike those shown in <xref ref-type="fig" rid="fig2">Figure 2</xref>, does not have the <italic>wyEx6022 ptrn-1::tdTomato</italic> transgene, so the EMTB::GFP puncta from <italic>wyEx5968</italic> that remain after the acute colchicine treatment are not caused by PTRN-1::tdTomato overexpression.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.010">http://dx.doi.org/10.7554/eLife.01498.010</ext-link></p></caption><graphic xlink:href="elife01498fs006"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.011</object-id><label>Figure 2—figure supplement 2.</label><caption><title>PTRN-1::tdTomato colocalizes with EMTB::GFP puncta after MT depolymerization by colchicine.</title><p><italic>bus-17(e2800)</italic> mutant animals co-expressing EMTB::GFP with either PTRN-1::tdTomato or PTRN-1a(ΔCKK)::tdTomato were imaged after acute colchicine treatment (example images shown in <xref ref-type="fig" rid="fig2">Figure 2</xref>), and the Pearson's colocalization coefficient (PCC) between the two fluorescent proteins was calculated for body wall muscle cells (<bold>A</bold>) and PVD dendrites (<bold>B</bold>). In <bold>A</bold>, plotted is the average PCC calculated from maximum projection images of confocal stacks from four (PTRN-1a::tdTomato) or three (PTRN-1a(ΔCKK)::tdTomato) body wall muscle cells. In <bold>B</bold>, plotted is the average PCC calculated from linescans of PVD dendrites from four animals each for both PTRN-1a::tdTomato and PTRN-1a(ΔCKK)::tdTomato. (*p<0.05, one-tailed students <italic>t</italic> test). The number above each bar indicates the p value from a one-tailed, one-sample <italic>t</italic> test comparing the calculated PCC against 0 (no colocalization) (<xref ref-type="bibr" rid="bib38">Mcdonald and Dunn, 2013</xref>). In the PVD neurites, PTRN-1a(ΔCKK)::tdTomato exhibited a small but significant colocalization with EMTB::GFP, which was contrary to the hypothesis that the CKK domain is necessary for MT binding and stabilization. This colocalization might be due to the presence of other MT-protecting proteins or endogenous PTRN-1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.011">http://dx.doi.org/10.7554/eLife.01498.011</ext-link></p></caption><graphic xlink:href="elife01498fs007"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.012</object-id><label>Figure 2—figure supplement 3.</label><caption><title>PTRN-1a(ΔCKK) exhibits punctate localization in body wall muscle cells and neurons.</title><p>(<bold>A</bold> and <bold>B</bold>) PTRN-1a(Δ CKK)::tdTomato, in which the MT-binding CKK domain has been deleted, co-expressed with EMTB::GFP in <italic>ptrn-1(tm5597)</italic> mutant animals. Images show the body wall muscle cells at the sarcolemma (<bold>A</bold>) and in the cell interior (<bold>B</bold>). Expanded boxes highlight regions where PTRN-1(ΔCKK)::tdTomato and EMTB::GFP do not colocalize. (<bold>C</bold>) PTRN-1a(ΔCKK)::tdTomato expressed in the <italic>ptrn-1(tm5597)</italic> mutant exhibits punctate localization in the processes of the PVD neuron. A, anterior; V, ventral. Filled arrowheads point to PVD primary dendrite, open arrowheads point to PVD tertiary dendrites. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.012">http://dx.doi.org/10.7554/eLife.01498.012</ext-link></p></caption><graphic xlink:href="elife01498fs008"/></fig></fig-group><fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.013</object-id><label>Figure 3.</label><caption><title>Immobility of PTRN-1a::tdTomato puncta contrasts with EBP-2::GFP movements in the PVD dendrite.</title><p>(<bold>A</bold>) Live imaging was performed on a <italic>ptrn-1(tm5597)</italic> mutant animal co-expressing EBP-2::GFP (green, from the <italic>wyEx4828</italic> transgene), which labels growing plus-end of MTs, and PTRN-1a::tdTomato (magenta, from the <italic>wyEx6022</italic> transgene) along a section of the tertiary dendrite of the PVD neuron. (<bold>B</bold>–<bold>D</bold>). Kymographs of EBP-2::GFP (<bold>B</bold>), PTRN-1a::tdTomato (<bold>C</bold>), and overlay of EBP-2::GFP (green) with PTRN-1a::dtTomato (magenta) (<bold>D</bold>) from a 110 s video acquired from the PVD process shown in <bold>A</bold>. Time runs top to bottom. Arrows point to start of EBP-2::GFP movements. A, anterograde; R, retrograde. Scale bar: 5 μm, ∼22 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.013">http://dx.doi.org/10.7554/eLife.01498.013</ext-link></p></caption><graphic xlink:href="elife01498f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.014</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Immobility of PTRN-1a::tdTomato puncta contrasts with EBP-2::GFP movements in the PVD dendrite.</title><p>Replicate 2 (A–D) and replicate 3 (E–H) of experiment in <xref ref-type="fig" rid="fig2">Figure 2</xref>. (<bold>A</bold> and <bold>E</bold>) Live imaging was performed on <italic>ptrn-1(tm5597)</italic> mutant animals coexpressing EBP-2::GFP (green, from the <italic>wyEx4828</italic> transgene), and PTRN-1a::tdTomato (magenta, from the <italic>wyEx6022</italic> transgene) along a section of the tertiary dendrite of the PVD neuron. (<bold>B</bold>–<bold>D</bold> and <bold>F</bold>–<bold>H</bold>). Kymographs of EBP-2::GFP (B,F), PTRN- 1a::tdTomato (<bold>C</bold> and <bold>G</bold>), and overlay of EBP-2::GFP (green) with PTRN-1a::dtTomato (magenta) (<bold>D</bold> and <bold>H</bold>) from a 110 s video acquired from the PVD process shown in <bold>A</bold> and <bold>E</bold>. (<bold>I</bold>–<bold>K</bold>) Live imaging was performed as above on an animal lacking the <italic>ptrn-1a::tdTomato</italic> transgene. Time runs top to bottom. A, anterograde; R, retrograde. Scale bar: 5 μm, ∼22 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.014">http://dx.doi.org/10.7554/eLife.01498.014</ext-link></p></caption><graphic xlink:href="elife01498fs009"/></fig></fig-group></p><p>We used live imaging to examine the dynamics of the PTRN-1::tdTomato puncta in the PVD neurites (<xref ref-type="other" rid="video1">Video 1</xref>, <xref ref-type="other" rid="video2">Video 2</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3D,E</xref>). In 40-min videos, the majority of the PTRN-1::tdTomato puncta exhibit some slow movement. The puncta can be seen dividing, appearing and growing, dissolving, and merging. The movements of each punctum are not obviously correlated with those of the surrounding puncta.<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife01498v001.avi"><object-id pub-id-type="doi">10.7554/eLife.01498.015</object-id><label>Video 1.</label><caption><title>PTRN-1::tdTomato movements in the PVD neuron.</title><p>First example video of a L4 animal showing 40 min with 90 s/frame. See <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3D</xref> for diagram of neuron morphology.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.015">http://dx.doi.org/10.7554/eLife.01498.015</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife01498v002.avi"><object-id pub-id-type="doi">10.7554/eLife.01498.016</object-id><label>Video 2.</label><caption><title>PTRN-1::tdTomato movements in the PVD neuron.</title><p>Second example video of a L4 animal showing 40 min with 90 s/frame. See <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3E</xref> for diagram of neuron morphology.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.016">http://dx.doi.org/10.7554/eLife.01498.016</ext-link></p></caption></media></p></sec><sec id="s2-2"><title>PTRN-1 stabilizes MT foci in neuronal processes and body wall muscle</title><p>To examine whether PTRN-1 binds MTs in neurites, we co-expressed PTRN-1a::tdTomato with EMTB::GFP, the MT-binding domain of ensconsin fused to GFP (<xref ref-type="bibr" rid="bib37">Masson and Kreis, 1993</xref>; <xref ref-type="bibr" rid="bib10">Bulinski and Bossler, 1994</xref>; <xref ref-type="bibr" rid="bib22">Faire et al., 1999</xref>). EMTB::GFP, which binds dynamically along the side of MTs, has been previously used to visualize MTs in vivo (<xref ref-type="bibr" rid="bib11">Bulinski et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Lechler and Fuchs, 2007</xref>; <xref ref-type="bibr" rid="bib59">von Dassow et al., 2009</xref>; <xref ref-type="bibr" rid="bib62">Wühr et al., 2010</xref>). In <italic>C. elegans</italic> neurons, it generally exhibited continuous fluorescence throughout neuronal processes. In the PVD neuron, the dendritic arbor consists of processes that branch perpendicularly from each other, getting progressively thinner with each branching event (<xref ref-type="bibr" rid="bib2">Albeg et al., 2011</xref>). Accordingly, EMTB::GFP fluorescence in the PVD neuron was strong in the primary dendrites but weak and sometimes discontinuous in the tertiary and quaternary dendrites, likely because these narrow neurites contain few MTs (<xref ref-type="fig" rid="fig1">Figure 1D</xref>, <xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4A,B</xref>) (<xref ref-type="bibr" rid="bib2">Albeg et al., 2011</xref>).</p><p>We assessed the relationship between MTs and PTRN-1 by examining the fluorescence of these two fusion proteins in the body wall muscle cells, which have larger cell bodies than neurons, providing more space in which MTs are organized. EMTB::GFP-labeled MTs were strung throughout the cytosol of these cells (<xref ref-type="fig" rid="fig1">Figure 1F</xref>, <xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4E,F</xref>). They also formed parallel lines along the sarcolemma, from which emanated rows of evenly-spaced puncta visible in the next confocal slice or two closer to the membrane (slices were 0.4 μm apart) (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4C,D</xref>). These puncta appeared to be MT ends. Hence, the disorganized MT strands are anchored at the regularly spaced loci on or near the plasma membrane. The angle and spacing of the MT lines at the sarcolemma suggest that they run parallel and perhaps adjacent to the dense bodies (the <italic>C. elegans</italic> equivalent of Z-discs) and M-lines. This microtubule organization in the body wall muscle cells resembles the pattern observed by fluorescence-labeled ELP-1, the <italic>C. elegans</italic> EMAP (Echinoderm Microtubule-Associated Protein)-like protein (<xref ref-type="bibr" rid="bib31">Hueston et al., 2008</xref>). In mammalian muscle fibers, MTs filaments form both a grid-like organization aligned with the Z-discs, which is dependent on dystrophin (<xref ref-type="bibr" rid="bib49">Prins et al., 2009</xref>), and squiggles in the cytosol with less apparent organization (<xref ref-type="bibr" rid="bib50">Ralston et al., 1999</xref>).</p><p>Whether fused to YFP or tdTomato, PTRN-1 localized to evenly spaced puncta at the sarcolemma and to irregularly spaced puncta throughout the interior of the body wall muscle cells (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A–C</xref>, <xref ref-type="fig" rid="fig1">Figure 1E,F</xref>). The PTRN-1a::tdTomato puncta within the muscle cytosol always co-localized with the EMTB::GFP cytosolic threads (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), confirming that PTRN-1 localizes to MTs. Further, PTRN-1a puncta colocalized with EMTB::GFP puncta at the sarcolemma (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), a finding which suggests that, like its homologs in fruitflies and humans (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>; <xref ref-type="bibr" rid="bib55">Tanaka et al., 2012</xref>), PTRN-1a localizes to MT ends. It is unclear how PTRN-1::tdTomato is localized at either the sarcolemma or in the muscle cell interior. Interestingly, in mammalian muscle, MTs are nucleated from the immobile Golgi elements strung throughout the cytoplasm (<xref ref-type="bibr" rid="bib45">Oddoux et al., 2013</xref>).</p><p>Although CAMSAP proteins preferentially bind to the minus ends of MTs, when they are overexpressed, CAMSAPs have also been shown to bind along the side of MTs (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Similarly, highly expressed PTRN-1a::tdTomato localized along the side of MTs in the body wall muscle cells (<xref ref-type="fig" rid="fig1">Figure 1F</xref> (bottom left of main panel), <xref ref-type="fig" rid="fig1s5">Figure 1—figure supplement 5</xref>).</p><p>We next sought to determine whether PTRN-1 localizes to sites where MTs are stabilized. We treated animals with the MT depolymerizing drug colchicine for 1 hr and examined the effect on EMTB::GFP localization. Because the <italic>C. elegans</italic> cuticle is largely impermeable to colchicine, we performed this experiment in the <italic>bus-17(e2800)</italic> genetic background, which has increased permeability to drugs, including colchicine (<xref ref-type="bibr" rid="bib35">Leung et al., 2008</xref>; <xref ref-type="bibr" rid="bib26">Gravato-Nobre et al., 2005</xref>; <xref ref-type="bibr" rid="bib7">Bounoutas et al., 2009</xref>). Acute colchicine treatment dramatically altered the distribution of EMTB::GFP such that fibers were no longer visible. Small EMTB::GFP foci remained at the cell membrane and in the cell interior, along with a haze of fluorescence throughout the cytosol (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A,B</xref>). Since EMTB::GFP normally binds to the sidewalls of MTs (<xref ref-type="bibr" rid="bib22">Faire et al., 1999</xref>), this change in its distribution confirms that the colchicine treatment led to MT depolymerization, as expected. The localization of PTRN-1a::tdTomato appeared to be unaffected by the acute colchicine exposure, and the PTRN-1a::tdTomato puncta co-localized with the EMTB::GFP puncta both at the sarcolemma and in the cell interior (<xref ref-type="fig" rid="fig2">Figure 2A–D</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). These data show that, in the body wall muscle cells, the localization of PTRN-1a puncta is not dependent on MTs. They further imply that PTRN-1a localizes to sites where MTs are stabilized, even under conditions that cause complete depolymerization of all other MTs in the cell. Finally, because PTRN-1a::tdTomato and EMTB::GFP puncta exhibit a regular, repeating pattern at the sarcolemma, the fact that these rows are unaffected by the acute colchicine treatment indicates that these puncta represent sites of MT anchorage.</p><p>We used acute colchicine treatment to examine whether PTRN-1a likewise localizes to sites of MT stabilization in neurons. In the PVD neuron, acute colchicine exposure caused the continuous EMTB::GFP staining to dissolve into closely spaced puncta (<xref ref-type="fig" rid="fig2">Figure 2G,H</xref>). As in the body wall muscle cells, the PTRN-1a::tdTomato localization in the PVD neurites appeared unaffected by the MT depolymerization, and the remaining EMTB::GFP colocalized with the PTRN-1a::tdTomato puncta (<xref ref-type="fig" rid="fig2">Figure 2G,H</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). We interpret these data as suggestive that PTRN-1a localizes to sites where MTs are stabilized in the neurites.</p><p>As previous studies have shown that the CKK domain of CAMSAP proteins is involved in MT binding (<xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>), we analyzed PTRN-1a(ΔCKK)::tdTomato to determine whether PTRN-1a itself stabilizes MTs. PTRN-1a(ΔCKK)::tdTomato exhibited similar localization in body wall muscle cells as full-length PTRN-1a::tdTomato, though these PTRN-1a(ΔCKK)::tdTomato puncta sometimes did not colocalize with EMTB::GFP (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3A,B</xref>).</p><p>Performing acute colchicine treatment on animals co-expressing EMTB::GFP and PTRN-1a(ΔCKK)::tdTomato in the body wall muscle cells, we found that GFP-stained MT filaments were transformed into GFP puncta, but these puncta did not colocalize with PTRN-1a(ΔCKK)::tdTomato (<xref ref-type="fig" rid="fig2">Figure 2E,F</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). There appeared to be fewer puncta in the muscle cells after the acute colchicine treatment, particularly at the sarcolemma. This may indicate that PTRN-1a(ΔCKK)::tdTomato localization in the muscle cells is dependent on MTs. The EMTB::GFP foci present after acute colchicine treatment in this strain might be stabilized by endogenous PTRN-1 and/or other MT binding proteins.</p><p>Finally, although PTRN-1a(ΔCKK)::tdTomato localized to puncta in the PVD processes (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3C</xref>), after acute colchicine exposure, PTRN-1a(ΔCKK)::tdTomato puncta exhibited reduced colocalization with EMTB::GFP (<xref ref-type="fig" rid="fig2">Figure 2I</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Taken together, these data indicate that one of the MT ends is more stable than the rest of the MT in vivo, possibly due to end-binding proteins, and PTRN-1a::tdTomato itself stabilizes MT foci.</p></sec><sec id="s2-3"><title>PTRN-1 supports MT polymerization initiation in neuronal processes</title><p>Live imaging of EBP-2 (EB1), an MT-binding protein that specifically associates with growing plus ends, has been used to visualize MT polymerization in <italic>C. elegans</italic> neurites (<xref ref-type="bibr" rid="bib40">Mimori-Kiyosue et al., 2000</xref>; <xref ref-type="bibr" rid="bib36">Maniar et al., 2012</xref>). To examine the relationship between PTRN-1 and dynamic MT plus ends, we performed live imaging on PVD tertiary dendritic processes co-expressing EBP-2::GFP with PTRN-1a::tdTomato (<xref ref-type="fig" rid="fig3">Figure 3</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). As reported for other neurons, each EBP-2::GFP punctum appeared, migrated in a single direction, and disappeared (blue arrowheads, <xref ref-type="fig" rid="fig3">Figure 3B</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B,E</xref>). In some cases, multiple EBP-2::GFP movements emanated from the same position in the course of a video, suggestive of a local factor that promotes MT polymerization from these loci. There were also motionless EBP-2::GFP puncta that were present whether the neuron expressed the EBP-2::GFP transgene alone or with the PTRN-1a::tdTomato (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C,F,I–K</xref>).</p><p>PTRN-1a::tdTomato, in contrast, was localized almost exclusively to immobile puncta for the duration of these 110 s videos (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C,G</xref>). Many of the EBP-2::GFP movements appeared to emanate from PTRN-1a::tdTomato puncta, though the close spacing of the PTRN-1a::tdTomato puncta makes quantification of this observation impracticable (<xref ref-type="fig" rid="fig3">Figure 3D</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1D,H</xref>).</p><p>To investigate the requirement for PTRN-1 in neurite MT dynamics, we performed live imaging of EBP-2::GFP movements in wild-type vs <italic>ptrn-1</italic> mutant animals. We obtained two <italic>ptrn-1(null)</italic> alleles: <italic>tm5597,</italic> which carries an intragenic deletion that introduces an early nonsense mutation, and <italic>wy560,</italic> a 65 kb deletion that spans the entire <italic>ptrn-1</italic> locus (<xref ref-type="bibr" rid="bib53">Spilker et al., 2012</xref>). Strains carrying either of the <italic>ptrn-1(null)</italic> alleles exhibited grossly wild-type growth, development, and neuronal morphology (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>, and data not shown).</p><p>As the PHC dendrite has been used previously to monitor EBP-2::GFP movements (<xref ref-type="bibr" rid="bib64">Yan et al., 2013</xref>), we used this system to examine EBP-2::GFP movements in the <italic>ptrn-1</italic> mutants (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In wild-type animals, EBP-2::GFP comets move predominantly toward the cell body (<xref ref-type="bibr" rid="bib64">Yan et al., 2013</xref>), consistent with the known minus-end-out polarity of MTs in dendrites (<xref ref-type="bibr" rid="bib12">Burton, 1985</xref>). In both of the <italic>ptrn-1</italic> mutant strains, we observed fewer total EBP-2::GFP movements than in the wild-type strain (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), but the direction of EBP-2::GFP movements was like that of wild-type (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). Expression of <italic>ptrn-1a::tdTomato</italic> in the PHC neuron of <italic>ptrn-1(tm5597)</italic> mutant animals rescued the decreased number of EBP-2::GFP movements (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), indicating that the requirement for PTRN-1 in promoting EBP-2::GFP movements is cell-autonomous. These data implicate PTRN-1 in promoting MT polymerization in the dendrite but not directly organizing MT polarity. This loss of dynamic MTs in the <italic>ptrn-1</italic> mutants could be indicative of a reduction in the total number of MTs in the neurite, which would be suggestive of a role for PTRN-1 in MT nucleation or stabilization. These data do not quantify the stable MT population, however, so an alternate explanation for the reduction in EBP-2::GFP movements is that there is an increase in neurite MT stability in the <italic>ptrn-1</italic> mutants.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.017</object-id><label>Figure 4.</label><caption><title>PTRN-1 promotes MT polymerization in neurites.</title><p>(<bold>A</bold>) Schematic diagram of the PHC neuron. The anterior process is the axon; the posterior process is the dendrite. Live imaging was used to monitor EBP-2::GFP movements in the boxed region of the PHC dendrite. A: anterior, V: ventral. (<bold>B</bold> and <bold>C</bold>) Quantification of EBP-2::GFP anterograde and retrograde movements in the PHC dendrite of wild-type (WT) vs <italic>ptrn-1(tm5597)</italic> and <italic>ptrn-1(wy560)</italic> mutant animals, and vs the <italic>ptrn-1(tm5597)</italic> mutant carrying the <italic>Pdes-2::ptrn-1::tdTomato</italic> transgene, which is expressed in a subset of neurons as well as the body wall muscle. (<bold>B</bold>) Total EBP-2::GFP movements in each strain normalized against the wild-type control. (<bold>C</bold>) Fraction of EBP-2::GFP movements in each strain that moved in the retrograde direction. Mean ± SEM. (n = 3 experiments, each with at least 10 animals/genotype, *p<0.05, **p<0.01, ANOVA with Bonferroni post test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.017">http://dx.doi.org/10.7554/eLife.01498.017</ext-link></p></caption><graphic xlink:href="elife01498f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.018</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Neuronal morphology is grossly unaffected by loss of <italic>ptrn-1</italic>.</title><p>(<bold>A</bold> and <bold>B</bold>) The PVD neuron visualized by cytosolic GFP in wild-type (<bold>A</bold>) versus <italic>ptrn-1(tm5597)</italic> mutant (<bold>B</bold>) animals. (<bold>C</bold> and <bold>D</bold>) The PHC neuron visualized by cytosolic GFP in wild-type (<bold>C</bold>) versus <italic>ptrn-1(tm5597)</italic> mutant (<bold>D</bold>) animals. In the schematic diagrams, the PHC neuron is represented in black. (<bold>E</bold> and <bold>F</bold>) The DD/VD-type motorneurons visualized by cytosolic mCherry in wild-type (<bold>E</bold>) versus <italic>ptrn-1(tm5597)</italic> mutant (<bold>F</bold>) animals. (<bold>G</bold> and <bold>H</bold>) The PLM neuron, along with other touch receptor neurons, visualized by cytosolic GFP in wild-type (<bold>G</bold>) versus <italic>ptrn-1(tm5597)</italic> mutant (<bold>H</bold>) animals. Arrow points to PLM cell body, open arrowhead points to PLM commissure. Fluorescence in the head is from the co-injection marker. A, anterior; V, ventral. Scale bar: 50 μm (<bold>A, B, G, H</bold>), 5 μm (<bold>C–F</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.018">http://dx.doi.org/10.7554/eLife.01498.018</ext-link></p></caption><graphic xlink:href="elife01498fs010"/></fig></fig-group></p></sec><sec id="s2-4"><title>PTRN-1 promotes MTs stability in neurites</title><p>To determine whether PTRN-1 promotes neurite MT stabilization, we examined the interaction between <italic>ptrn-1</italic> and the MT destabilizing drug colchicine. Although the impenetrability of the <italic>C. elegans</italic> cuticle at the L4 and older stages necessitated the use of the <italic>bus-17</italic> mutation for the acute colchicine treatment described above, wild-type animals reared from hatching in a low dose of colchicine exhibit defects in MT organization and neuronal function, indicating that the drug reaches the neurons in this longer timeframe (<xref ref-type="bibr" rid="bib16">Chalfie and Thomson, 1982</xref>). Furthermore, rearing animals in a low dose of colchicine has been shown to suppress neurite morphology defects caused by several dominant alleles of β-tubulin <italic>mec-7,</italic> supporting the hypothesis that these alleles caused increased MT stability (<xref ref-type="bibr" rid="bib51">Savage et al., 1994</xref>; <xref ref-type="bibr" rid="bib33">Kirszenblat et al., 2013</xref>)<italic>,</italic> Therefore, this method of administering colchicine can reveal pharmacogenetic interactions between colchicine and genes that affect neuronal MT stability.</p><p>Although the neurite morphology of many <italic>C. elegans</italic> neurons in the <italic>ptrn-1(tm5597)</italic> mutant all appeared grossly wild-type under normal growth conditions (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>), growth in a low dose of colchicine caused dramatic ectopic sprouting from the sides of neurites in the <italic>ptrn-1(tm5597)</italic> mutant but not wild-type animals (<xref ref-type="fig" rid="fig5">Figure 5</xref>). In the DD/VD-type motorneurons, the cell bodies are situated in the ventral nerve cord (VNC), and a single unbranched commissure per cell connects processes in the VNC with those in the dorsal nerve cord. In wild-type animals grown in the presence of colchicine, these processes generally appear to be largely morphologically normal (<xref ref-type="fig" rid="fig5">Figure 5A,C</xref>). In the <italic>ptrn-1(tm5597)</italic> mutant grown in colchicine, in contrast, we observed ectopic branching of the commissures, as well as additional neurites sprouting from processes in the VNC (<xref ref-type="fig" rid="fig5">Figure 5B,C</xref>). This ectopic branching could be rescued by tissue-specific <italic>ptrn-1</italic> expression either pan-neuronally or exclusively in the DD/VD neurons (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). This synthetic interaction between colchicine and <italic>ptrn-1</italic> is suggestive that <italic>ptrn-1</italic> promotes MT stabilization in the DD/VD-type neurons.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.019</object-id><label>Figure 5.</label><caption><title>PTRN-1 supports MT stability in neurites.</title><p>(<bold>A-C</bold>) Wild-type (<bold>A</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>B</bold>) animals were grown in 0.13 mM colchicine to the L4 stage, and cytosolic RFP was used to visualize the DD/VD neurons. Scale bar: 10 μm. (<bold>C</bold>) Fraction of animals with ectopic sprouting from the DD/VD neurons, scored based on severity as described in ‘Materials and methods’ (n = at least 80 animals/genotype, ***p<0.001, Chi-squared test with Šidák correction). (<bold>D</bold>–<bold>F</bold>) Wild-type (<bold>D</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>E</bold>) animals were grown in 0.035 mM colchicine to the L4 stage, and the PLM neuron was visualized with myr::GFP. Scale bar: 5 μm. (<bold>F</bold>) Fraction of animals exhibiting ectopic sprouting from the PLM neuron, scored based on severity (n = at least 60 animals/genotype, ***p<0.001, Chi-squared test with Šidák correction). In schematic diagrams, the light gray lines represent the outline of the animal, DD and PLM neurons are black, and other neurons (in <bold>D</bold> and <bold>E</bold> only, one short, unbranched process near the PLM cell body of each image) are dark gray. For tissue specific rescue, DD/VD (<italic>Punc-47L</italic>), Pan-neu: pan-neuronal (<italic>Prab-3</italic>), Hyp: hypodermal (<italic>Pdpy-7</italic>), Int: intestinal (<italic>Pvha-6</italic>), neu: a subset of neurons including PLM (<italic>Punc-86</italic>). (<bold>G</bold>) Touch sensitivity of wild-type vs <italic>ptrn-1(tm5597)</italic> mutant animals. Mean ± SEM. (n = 3 experiments, each with 8–12 animals/genotype, ns not significant (p=0.46), <italic>t</italic> test). (<bold>H</bold>) Touch sensitivity of wild-type vs <italic>ptrn-1(tm5597)</italic> mutant animals grown in the indicated concentrations of colchicine. Mean ± SEM. (n = 2 experiments, each with 10 animals/genotype, ***p<0.001, *p<0.05, <italic>t</italic> test for each drug concentration). (<bold>I</bold>) Average fluorescence of GFP expressed from the <italic>Pmec-7</italic> (β-tubulin) promoter in the PLM cell body of wild-type vs <italic>ptrn-1</italic> mutant animals. Mean ± SEM. (n = 2 experiments, each with at least 13 animals/genotype, **p<0.01, <italic>t</italic> test). (<bold>J</bold> and <bold>K</bold>) Transmission electron microscopy of the PLM neuron in wild-type (<bold>J</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>K</bold>) young adult animals, sectioned near the rectum. Note MTs with abnormally smaller diameters (Asterisks), and a MT sheet shaped like an ‘S’ (Arrow head). Scale bar: 100 nm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.019">http://dx.doi.org/10.7554/eLife.01498.019</ext-link></p></caption><graphic xlink:href="elife01498f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.020</object-id><label>Figure 5—figure supplement 1.</label><caption><title>PTRN-1 protects the ALM touch receptor neuron against ectopic neurite sprouting during growth in colchicine.</title><p>Wild-type (<bold>A</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>B</bold>) animals were grown in 0.035 mM colchicine to the L4 stage, and the ALM neuron was visualized with myr::GFP. Scale bar: 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.020">http://dx.doi.org/10.7554/eLife.01498.020</ext-link></p></caption><graphic xlink:href="elife01498fs011"/></fig></fig-group></p><p>Of note, we also tested the hypothesis that the colchicine-induced branching in the <italic>ptrn-1</italic> mutant could be due to increased in vivo colchicine levels compared to wild-type. The most likely cause of such an effect would be <italic>ptrn-1-</italic>dependent defects in the hypodermis or intestine, tissues involved in drug uptake in <italic>C. elegans</italic> (<xref ref-type="bibr" rid="bib35">Leung et al., 2008</xref>). We therefore created transgenic <italic>ptrn-1</italic> mutants expressing PTRN-1a in the hypodermis and intestine. This transgene had little or no effect on colchicine-induced ectopic branching in the DD/VD neurons (<xref ref-type="fig" rid="fig5">Figure 5C</xref>), indicating that the drug–gene interaction leading to ectopic neuronal branching is unlikely to be due to increased drug permeability.</p><p>Whereas most <italic>C. elegans</italic> neurons contain four to six 11-protofilament (pf) MTs, the six touch receptor neurons (TRNs) have a strikingly different MT organization. These mechanoreceptor neurons have 15-pf MTs produced from tubulin genes expressed predominantly in the TRNs (<xref ref-type="bibr" rid="bib15">Chalfie and Thomson, 1979</xref>; <xref ref-type="bibr" rid="bib28">Hamelin et al., 1992</xref>; <xref ref-type="bibr" rid="bib23">Fukushige et al., 1999</xref>). The TRNs have 25–50 MTs per neurite cross-section in young adult animals. Both the morphology and function of these cells are particularly sensitive to perturbations in MT stability (<xref ref-type="bibr" rid="bib16">Chalfie and Thomson, 1982</xref>). We focused on the two PLM neurons, which each elaborate both an anterior-directed process and a posterior-directed process from the cell bodies located at the base of the tail (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1G,H</xref>). A single commissure extends from each of the PLM anterior processes to the VNC, where PLM makes presynaptic connections with other neurons, making these anterior-directed processes axon-like. The posterior-directed processes make neither presynaptic nor postsynaptic connections.</p><p>Under normal growth conditions, the morphology of the PLM neurons in the <italic>ptrn-1(tm5597)</italic> mutant was largely wild-type (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>), with several more subtle defects described below. Growth in a low dose of colchicine, however, resulted in extensive ectopic sprouting from the PLM axon in the <italic>ptrn-1(tm5597)</italic> mutant but not in the wild-type strain (<xref ref-type="fig" rid="fig5">Figure 5D–F</xref>). Similar ectopic sprouting was observed in the ALM neuron, another TRN in the anterior half of the animal (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Tissue-specific <italic>ptrn-1</italic> expression either in all neurons or in subset of neurons that includes PLM but not in both the hypodermis and the intestine rescued this ectopic sprouting of the touch receptor neurons (<xref ref-type="fig" rid="fig5">Figure 5F</xref>).</p><p>The PLM axon is extended during embryogenesis. As the <italic>C. elegans</italic> eggshell is impermeable to colchicine (<xref ref-type="bibr" rid="bib7">Bounoutas et al., 2009</xref>), the ectopic sprouting occurs after neurogenesis has been completed. Therefore, the ectopic sprouting reflects a defect in neurite maintenance rather than neurite outgrowth during development. Taken together, these data indicate that the loss of <italic>ptrn-1</italic> enhances sensitivity to colchicine cell-autonomously in neurons containing either 11-pf or 15-pf MTs. This enhanced sensitivity to colchicine likely reflects reduced MT stability in the <italic>ptrn-1</italic> mutant, suggesting that PTRN-1 promotes MT stabilization.</p><p>TRNs mediate the behavioral response to light touch (<xref ref-type="bibr" rid="bib13">Chalfie, 2009</xref>). We assessed the functionality of the TRNs in the absence of <italic>ptrn-1</italic> function by quantifying light touch sensitivity in <italic>ptrn-1(tm5597)</italic> mutant vs wild-type animals. We found no significant difference in light touch response between the <italic>ptrn-1(tm5597)</italic> mutant and wild-type animals grown in the absence of colchicine, though our assay may have lacked sufficient sensitivity to parse subtle differences (<xref ref-type="fig" rid="fig5">Figure 5G</xref>). Growing the animals in several concentrations of colchicine, however, we found that the <italic>ptrn-1(tm5597)</italic> mutant lost light touch sensitivity at a lower concentration of colchicine than the wild-type strain (<xref ref-type="fig" rid="fig5">Figure 5H</xref>).</p><p>MT destabilization has long been known to negatively regulate tubulin production (<xref ref-type="bibr" rid="bib17">Cleveland, 1988</xref>). In the <italic>C. elegans</italic> TRNs, MT destabilization induced by genetic or pharmacological manipulations results in not only decreased levels β-tubulin <italic>mec-7</italic> mRNA but also a general decrease in protein production, including GFP driven by the <italic>mec-7</italic> promoter (<xref ref-type="bibr" rid="bib51">Savage et al., 1994</xref>; <xref ref-type="bibr" rid="bib6">Bounoutas et al., 2011</xref>). Similarly, the <italic>ptrn-1(tm5597)</italic> mutant exhibited decreased GFP fluorescence from a <italic>Pmec-7::gfp</italic> transgene relative to the wild-type strain (<xref ref-type="fig" rid="fig5">Figure 5I</xref>), further implicating PTRN-1 in promoting MT stability.</p><p>MT density and protofilament composition in the neurites of the TRNs have been well characterized by electron microscopy (<xref ref-type="bibr" rid="bib15">Chalfie and Thomson, 1979</xref>, <xref ref-type="bibr" rid="bib16">1982</xref>; <xref ref-type="bibr" rid="bib20a">Cueva et al., 2007</xref>; <xref ref-type="bibr" rid="bib20">Cueva et al., 2012</xref>). To better understand the function of PTRN-1, we used electron microscopy to compare the PLM MTs in the <italic>ptrn-1(tm5597)</italic> mutant to wild-type. In cross sections of the PLM neuron in wild-type animals reared at 25°C, there are 25–50 15-pf MTs and occasionally one or two 11-pf MTs (<xref ref-type="fig" rid="fig5">Figure 5J</xref>) (<xref ref-type="bibr" rid="bib15">Chalfie and Thomson, 1979</xref>; <xref ref-type="bibr" rid="bib20">Cueva et al., 2012</xref>; our unpublished data). We examined cross sections of the PLML/R axons from two <italic>ptrn-1(tm5597)</italic> mutant young adult animals sectioned at the rectum and found they had 13, 15, 2, and 14 MTs, respectively. The majority of these MTs had the characteristically large diameter of 15-pf MTs, but several MTs had smaller diameters indicative of 11-pf MTs (<xref ref-type="fig" rid="fig5">Figure 5K</xref>). Furthermore, in one of the PLML cross sections, we observed an irregular MT structure that persisted through three serial sections (150 nm) that was ‘S’ shaped instead of circular (<xref ref-type="fig" rid="fig5">Figure 5K</xref>). We speculate that such a structure might have formed from two circular MTs opening and then joining. The reduction in MT number found in all four cells, as well as the ‘S’ shaped MT structure in one, implicates a role for PTRN-1 in maintaining the integrity of MTs in neuronal processes.</p></sec><sec id="s2-5"><title>PTRN-1 is required for proper neurite morphology and synaptic material localization in the PLM neuron</title><p>Given the requirement for <italic>ptrn-1</italic> in MT stability in the PLM neuron, we examined the effect of <italic>ptrn-1</italic> deficiency on PLM morphology in greater detail. Roughly 20% of <italic>ptrn-1(tm5597)</italic> mutant L4 animals exhibited defective extension of the PLM commissure (<xref ref-type="fig" rid="fig6">Figure 6A–C</xref>). In wild-type animals, this commissure is extended during the L1 larval stage from the axon to the VNC posterior of and close to the vulva, and it is present in every L4 animal (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). In <italic>ptrn-1(tm5597)</italic> mutants with this defect, we generally observed one or several ventrally directed buds along the region of PLM axon, where the commissure is normally positioned (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). The defective PLM commissure extension observed in the <italic>ptrn-1(tm5597)</italic> mutant was fully rescued by <italic>ptrn-1a</italic> cDNA expressed in the PLM neuron, indicating that the role for PTRN-1 in commissure formation is cell-autonomous (<xref ref-type="fig" rid="fig6">Figure 6C</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.021</object-id><label>Figure 6.</label><caption><title>PTRN-1 promotes synapse localization and neurite morphology in the PLM neuron.</title><p>(<bold>A</bold> and <bold>B</bold>) myrGFP was used to visualize the PLM commissure in wild-type (<bold>A</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>B</bold>) animals. Arrows point to commissure or commissure bud. (<bold>C</bold>) Fraction of animals with a PLM commissure connecting the axon to the ventral nerve cord. Mean ± SEM. (n = 3 experiments, each with at least 30 animals/genotype, **p<0.01, ***p<0.001, one-way ANOVA with Bonferroni post test). (<bold>D</bold> and <bold>E</bold>) mCherry::RAB-3 at the synaptic patch of the PLM neurons of wild-type (<bold>D</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>E</bold>) animals. (<bold>F</bold> and <bold>G</bold>) mCherry::RAB-3 in the posterior process of the PLM neurons in wild-type (<bold>F</bold>) and <italic>ptrn-1(tm5597)</italic> mutant (<bold>G</bold>) animals. (<bold>H</bold>) Fraction of wild-type and <italic>ptrn-1</italic> mutant animals with visible accumulation of mCherry:RAB-3 at the synaptic patch and the posterior process of the PLM neuron. The <italic>Punc-86</italic> promoter is expressed in a subset of neurons including the TRNs; the <italic>Pmec-3</italic> promoter is expressed in the TRNs. Animals with two visible mCherry:RAB-3 patches in the PLM synaptic region were counted as having synaptic accumulation, and animals with one or no visible mCherry::RAB-3 patches were considered to have loss of synaptic accumulation. Mean ± SEM. (n = 2 experiments, each with 30 animals/genotype, *p<0.05, ***p<0.001, two-way ANOVA with Bonferroni post test). (<bold>I</bold>) Fraction of wild-type and <italic>ptrn-1(tm5597)</italic> mutant animals with visible accumulation of SNB-1::GFP at the synaptic patch and the posterior process of the PLM neuron. Synaptic patch accumulation was scored as in H. Mean ± SEM. (n = 2 experiments, each with at least 20 animals/trial. ***p<0.001, two-way ANOVA with Bonferroni post test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.021">http://dx.doi.org/10.7554/eLife.01498.021</ext-link></p></caption><graphic xlink:href="elife01498f006"/></fig></p><p>We next examined the localization of synaptic material in the PLM neuron. Each PLM neuron has a presynaptic specialization in the VNC at the end of the commissure, where synaptic vesicles (SVs) and associated proteins such as the small GTPase RAB-3 and SNB-1/synaptobrevin are localized (<xref ref-type="bibr" rid="bib14">Chalfie et al., 1985</xref>; <xref ref-type="bibr" rid="bib52">Schaefer et al., 2000</xref>). In wild-type animals, mCherry::RAB-3 localized to two patches along the VNC that correspond to the synaptic patches of the two PLM neurons (<xref ref-type="fig" rid="fig6">Figure 6D,H</xref>). In the <italic>ptrn-1</italic> mutants, there was an incompletely penetrant loss of mCherry::RAB-3 at the synaptic patch region, and in many <italic>ptrn-1</italic> mutant animals, at least one of the PLM neurons had no visible accumulation of mCherry::RAB-3 at the synaptic patch (<xref ref-type="fig" rid="fig6">Figure 6E,H</xref>). The <italic>ptrn-1(wy560)</italic> strain had a higher penetrance of this defect than the <italic>ptrn-1(tm5597)</italic> strain (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). The <italic>ptrn-1(wy560)</italic> allele is a deletion that removes not only the entire <italic>ptrn-1</italic> locus but also seven surrounding ORFs, including the Muscleblind homolog <italic>mbl-1</italic>. As <italic>mbl-1</italic> has been shown to promote the accumulation of synaptic material at the presynaptic region of other <italic>C. elegans</italic> neurons (<xref ref-type="bibr" rid="bib53">Spilker et al., 2012</xref>), we speculate that this difference in penetrance might be due to <italic>mbl-1</italic> deficiency in the <italic>wy560</italic> allele.</p><p>In addition to the loss of mCherry::RAB-3 from the synaptic patches, we observed a fully penetrant ectopic accumulation of mCherry::RAB-3 in the PLM posterior process in both <italic>ptrn-1</italic> mutant strains (<xref ref-type="fig" rid="fig6">Figure 6F–H</xref>). The localization of SNB-1::GFP in the PLM neuron of <italic>ptrn-1(tm5597)</italic> was similar to that of RAB-3::mCherry (<xref ref-type="fig" rid="fig6">Figure 6I</xref>). These data implicate a requirement for <italic>ptrn-1</italic> in proper SV localization.</p><p>To determine whether the requirement for <italic>ptrn-1</italic> in SV localization is cell-autonomous, we used two different promoters to drive <italic>ptrn-1a::yfp</italic> cDNA expression in the PLM neuron of the <italic>ptrn-1</italic> mutants. Both constructs rescued the defects in mCherry::RAB-3 localization (<xref ref-type="fig" rid="fig6">Figure 6H</xref>), indicating that PTRN-1 functions cell autonomously in the PLM neuron to promote proper SV localization.</p><p>What mechanism underlies the aberrant commissure formation and SV mislocalization in the <italic>ptrn-1</italic> mutant? DLK-1 is a conserved mitogen-activated protein kinase kinase kinase (MAPKKK) that functions in a variety of situations in neurons, including neurite outgrowth, synapse development, and axon regeneration (<xref ref-type="bibr" rid="bib56">Tedeschi and Bradke, 2013</xref>). In <italic>C. elegans</italic>, the DLK-1 pathway is required to mediate the response to MT destabilization in the PLM neuron, and it also promotes proper synapse localization (<xref ref-type="bibr" rid="bib43">Nakata et al., 2005</xref>; <xref ref-type="bibr" rid="bib6">Bounoutas et al., 2011</xref>). Indeed, hyperactivation of the DLK-1 pathway causes a defect in the PLM commissure similar to that observed in the <italic>ptrn-1(tm5597)</italic> mutant, albeit with a higher penetrance (<xref ref-type="bibr" rid="bib27">Grill et al., 2007</xref>). We examined PLM commissure formation and SV localization in a <italic>dlk-1(ju476); ptrn-1(tm5597)</italic> double mutant. In this double mutant, we found that <italic>dlk-1</italic> completely suppressed both the commissure extension defect and the loss of mCherry::RAB-3 from the synaptic patch (<xref ref-type="fig" rid="fig7">Figure 7A</xref>), indicating that <italic>dlk-1</italic> is required to mediate these aspects of the <italic>ptrn-1</italic> mutant phenotype. The PMK-3 p38 MAPK functions downstream of DLK-1 (<xref ref-type="bibr" rid="bib43">Nakata et al., 2005</xref>). We observed similar suppression of these <italic>ptrn-1</italic> phenotypes in a <italic>pmk-3(ok169); ptrn-1(tm5597)</italic> double mutant (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>), corroborating the role for the <italic>dlk-1</italic> pathway in mediating the <italic>ptrn-1</italic> PLM commissure formation and SV localization defects.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01498.022</object-id><label>Figure 7.</label><caption><title>Aberrant phenotype of the PLM neuron in the <italic>ptrn-1</italic> mutant is mediated partially by the DLK-1 pathway.</title><p>(<bold>A</bold>) The <italic>wyIs97(Punc-86::myrGFP, Punc-86::mCherry::rab-3)</italic> transgene was used to simultaneously visualize commissure formation and SV localization. Only animals with an intact PLM commissure were counted for mCherry:RAB-3 localization at the synaptic patch. Values represent mean ± SEM. (n = 3 experiments, each with at least 30 animals/genotype, **p<0.01, ***p<0.001, two-way ANOVA with Bonferroni post test). (<bold>B–F</bold>) Animals were grown in 0.13 mM colchicine (<bold>C</bold> and <bold>D</bold>) or 0.5 mM colchicine (<bold>E</bold> and <bold>F</bold>) to the L4 stage, and the PLM neuron was visualized with myr::GFP. In schematic diagrams, the PLM neurons are black, and other neurons in the image are dark gray. (<bold>B</bold>) Fraction of animals exhibiting ectopic sprouting from the PLM neuron, scored based on severity (n = at least 120 animals/genotype, ***p<0.001, Chi-squared test for each drug concentration). (<bold>G</bold> and <bold>H</bold>) Quantification of EBP-2::GFP anterograde and retrograde movements in the PHC dendrite. (<bold>G</bold>) Total EBP-2::GFP movements in each strain normalized against the wild-type control. (<bold>H</bold>) Fraction of EBP-2::GFP movements in each strain that moved in the retrograde direction. Mean ± SEM. (n = 3 experiments, each with at least 9 animals/genotype, *p<0.05, ns not significant, one-way ANOVA with Bonferroni post test. The data for the <italic>ptrn-1</italic> single mutant is the same as that shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.022">http://dx.doi.org/10.7554/eLife.01498.022</ext-link></p></caption><graphic xlink:href="elife01498f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01498.023</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Loss of <italic>pmk-3</italic> partially suppresses the aberrant phenotype of the PLM neuron in the <italic>ptrn-1</italic> mutant.</title><p><italic>wyIs97(Punc-86::myrGFP, Punc-86::mCherry::rab-3)</italic> transgene was used to simultaneously visualize commissure formation and SV localization. Only animals with an intact PLM commissure were counted for mCherry:RAB-3 localization at the synaptic patch. Values represent mean ± SEM. (n = 2 experiments, each with at least 30 animals/genotype, **p<0.01, ***p<0.001, two-way ANOVA with Bonferroni post test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01498.023">http://dx.doi.org/10.7554/eLife.01498.023</ext-link></p></caption><graphic xlink:href="elife01498fs012"/></fig></fig-group></p><p>We next asked whether the synthetic interaction between <italic>ptrn-1</italic> and colchicine that resulted in neurite sprouting from the PLM axon is also mediated through the DLK-1 pathway. Indeed, <italic>dlk-1(ju476)</italic> completely suppressed the neurite sprouting observed in <italic>ptrn-1(tm5597)</italic> during growth on colchicine (<xref ref-type="fig" rid="fig7">Figure 7B–D</xref>). Interestingly, the wild-type strain reared in a higher dose of colchicine exhibited neurite sprouting similar to that seen in the <italic>ptrn-1(tm5597)</italic> mutant at the lower colchicine concentration, and this sprouting was likewise suppressed by <italic>dlk-1(ju476)</italic> (<xref ref-type="fig" rid="fig7">Figure 7B,E,F</xref>). Similar effects of <italic>dlk-1</italic> were observed on colchicine-induced neurite sprouting in the ALM neurons (data not shown).</p><p>DLK-1 was not required to mediate all of the abnormal phenotypes caused by <italic>ptrn-1</italic> loss of function, however: the ectopic accumulation of mCherry::RAB-3 in the PLM posterior process was similar in the <italic>dlk-1(ju476); ptrn-1(tm5597)</italic> double mutant to that of the <italic>ptrn-1(tm5597)</italic> mutant strain (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Therefore, the accumulation of SVs in the PLM posterior process is separable from the loss of SVs at the synaptic patch, and it is mediated by a mechanism other than the DLK-1 pathway.</p><p>Finally, we asked whether the reduction in EBP-2::GFP movements in the <italic>ptrn-1</italic> mutant is also dependent on <italic>dlk-1.</italic> Interestingly, we observed an increase of roughly two-fold in EBP-2::GFP movements in the PHC dendrite in the <italic>dlk-1(ju476)</italic> single mutant relative to wild-type (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). Loss of <italic>dlk-1</italic> had no effect on the orientation of EBP-2 movements (<xref ref-type="fig" rid="fig7">Figure 7H</xref>). There was a trend for the <italic>dlk-1(ju476); ptrn-1(tm5597)</italic> double mutant to have increased EBP-2::GFP movements relative to the <italic>ptrn-1</italic> single mutant, though this difference was not statistically significant (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). However, the <italic>dlk-1(ju476); ptrn-1(tm5597)</italic> double mutant exhibited fewer movements than the <italic>dlk-1</italic> single mutant (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). This intermediate phenotype in the <italic>dlk-1(ju476); ptrn-1(tm5597)</italic> double mutant indicates that the DLK-1 pathway is not the only mechanism required for the <italic>ptrn-1</italic> mutant phenotypes, and it is suggestive that DLK-1 functions partially in parallel to PTRN-1 to influence EBP-2::GFP movements in the PHC neuron.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The mechanisms preventing depolymerization from the MT minus ends within neuronal processes are a long-standing mystery. Previous studies have shown that CAMSAP family proteins directly bind MT minus ends (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Our data suggest that PTRN-1 binds to MT minus ends and protects them from depolymerization in neuronal processes. Multiple lines of evidence support this conclusion. First, PTRN-1 localized to puncta throughout the neuronal processes that directly bind and stabilize MTs. Of note, electron microscopy reconstruction of neurites in the VNC showed that MT ends are staggered, with an average distance between minus ends of roughly 1.7 μm (<xref ref-type="bibr" rid="bib15">Chalfie and Thomson, 1979</xref>). Second, our electron microscopy data revealed that the <italic>ptrn-1(tm5597)</italic> mutant strain had fewer total MTs and some MTs with abnormal structure in the PLM neuron. Third, the <italic>ptrn-1</italic> mutant exhibited a pharmacogenetic enhancement with colchicine in respect to neurite morphology. Fourth, the <italic>ptrn-1</italic> mutant exhibited a reduction in the number of neurite MT polymerization events as determined by counting EBP-2::GFP movements. Finally, the SV mislocalization and aberrant neurite branching observed in the <italic>ptrn-1</italic> mutant were suppressed by <italic>dlk-1,</italic> which is known to influence the effects of MT destabilization in <italic>C. elegans.</italic></p><p>The <italic>D. melanogaster</italic> CAMSAP protein Patronin localizes to MT minus ends throughout the cytoplasm in interphase S2 cells (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Acute MT depolymerization in this system resulted in puncta of mCherry-tubulin that co-localized with the GFP–Patronin foci, similar to our findings in <italic>C. elegans</italic> neurons and muscle cells in vivo<italic>.</italic> Importantly, by allowing MT repolymerization, Goodwin and Vale established that the GFP–Patronin foci represented MT nucleation centers. It is unclear from our studies whether PTRN-1 localizes to MT nucleation sites in neurites, though the colocalization between PTRN-1 and the beginning of EBP-2 movements is suggestive that it might be.</p><p><italic>H. sapiens</italic> CAMSAP3 (Nezha) was originally identified as a component of epithelial cell adherens junctions, where it is anchored by cadherins and p120-catenin (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). We showed that PTRN-1 puncta in neuronal processes were unaffected by drug-induced MT depolymerization, and PTRN-1 localization was independent of the CKK domain thought to bind MTs (<xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). These data suggest that, like CAMSAP3 at adherens junctions, PTRN-1 is localized in an MT-independent manner.</p><p>If PTRN-1 were the sole mechanism protecting minus ends in neurites, we would expect the phenotype of mutants carrying <italic>ptrn-1</italic> null alleles to include severe defects in neuronal morphogenesis. We observed, however, relatively mild defects under standard growth conditions. Because of these data, we speculate that PTRN-1 functions in parallel with other mechanisms that promote MT stability in <italic>C. elegans</italic> neurites. These are likely to include tubulin posttranslational modifications and MT-associated proteins (<xref ref-type="bibr" rid="bib48">Poulain and Sobel, 2010</xref>). Of particular interest, tubulin detyrosination protects MTs from depolymerization by kinesin-13 family motors in fibroblasts and neurites (<xref ref-type="bibr" rid="bib47">Peris et al., 2009</xref>; <xref ref-type="bibr" rid="bib24">Ghosh-Roy et al., 2012</xref>). Because <italic>D. melanogaster</italic> Patronin protects MTs from kinesin-13-mediated depolymerization (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>; <xref ref-type="bibr" rid="bib60">Wang et al., 2013</xref>), these modifications are an attractive candidate for how MTs are stabilized in the absence of <italic>ptrn-1.</italic> Defective regulation of α-tubulin acetylation, another prevalent posttranslational tubulin modification in neurites, causes abnormal neurite morphology and function in both mice and <italic>C. elegans</italic> (<xref ref-type="bibr" rid="bib19">Creppe et al., 2009</xref>; <xref ref-type="bibr" rid="bib57">Topalidou et al., 2012</xref>), though its effect on MT stability is uncertain and may be circumstance-dependent (<xref ref-type="bibr" rid="bib32">Janke and Kneussel, 2010</xref>)<italic>.</italic> In <italic>C. elegans,</italic> electron microscopy studies have shown that the loss of α-tubulin acetyltransferases causes a decrease in MT abundance, increase in MTs with irregular protofilament number, and appearance of MTs in which the protofilament lattice had opened into semicircular or sheet-like structures (<xref ref-type="bibr" rid="bib20">Cueva et al., 2012</xref>; <xref ref-type="bibr" rid="bib57">Topalidou et al., 2012</xref>). Further, the loss of the α-tubulin acetyltransferase MEC-17 in the PLM neuron causes an increase in dynamic MTs and, in older adult animals, loss of synaptic material at the synaptic region accompanied by accumulation of synaptic material in the posterior process (<xref ref-type="bibr" rid="bib44">Neumann and Hilliard, 2014</xref>).</p><p>Consistent with the notion that <italic>ptrn-1</italic> functions in parallel with other mechanisms to promote MT stability, the <italic>ptrn-1</italic> mutant strain grown in a low dose of colchicine exhibited aberrant neurite outgrowth. A higher dose of colchicine caused similar ectopic branching in the PLM neuron of the wild-type strain, indicating that MT destabilization is sufficient to elicit this phenotype. What is the mechanism by which MT destabilization leads to the neurite outgrowth from axons? When collateral branches form along an axon, the budding branch and surrounding axon have fewer, shorter MTs than regions of the axon with no collateral branching (<xref ref-type="bibr" rid="bib66">Yu et al., 1994</xref>). Further, pharmacological or genetic manipulations that decrease MT stability have been shown to cause neurite outgrowth along the length of mature neurites (<xref ref-type="bibr" rid="bib8">Bray et al., 1978</xref>; <xref ref-type="bibr" rid="bib67">Yu et al., 2008</xref>). Perhaps the combination of <italic>ptrn-1</italic> mutation with colchicine results in fewer, shorter MTs in these neurites, and this MT status promotes ectopic sprouting. Alternatively, the response to MT destabilization resulting from loss of PTRN-1 function may be more akin to the regeneration response to an axonal lesion, since this also results in ectopic branching and growth cone formation in motor and sensory neurons (<xref ref-type="bibr" rid="bib29">Hammarlund et al., 2009</xref>; <xref ref-type="bibr" rid="bib63">Yan et al., 2009</xref>).</p><p>Taken together, our data indicate that PTRN-1 represents one of the elusive factors that stabilize the MT minus ends in neurites, promoting both the stable and dynamic MTs during development and maintenance of the nervous system. Through regulation of MTs, PTRN-1 supports proper SV localization and the balance between neurite stability and remodeling.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Nematode strains and maintenance</title><p><italic>C. elegans</italic> strains were cultured on <italic>E. coli</italic> OP50 as described (<xref ref-type="bibr" rid="bib9">Brenner, 1974</xref>). Data were collected from L4 stage animals except where otherwise noted, and all experiments were performed at 25°C because this elevated temperature enhanced the neuronal defects in the <italic>ptrn-1</italic> mutants (data not shown), except for those shown in <xref ref-type="fig" rid="fig7">Figure 7B–F</xref>, which were performed at 20°C. The <italic>ptrn-1(tm5597)</italic> allele was obtained from the National Bioresource Project in Japan and backcrossed three times. The <italic>ptrn-1(wy560)</italic> was isolated from RB771 provided by the CGC (<xref ref-type="bibr" rid="bib53">Spilker et al., 2012</xref>), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The following additional strains were used in this study: N2 Bristol, TV13426 <italic>ptrn-1(tm5597)</italic>, TV15320 <italic>ptrn-1(tm5597); wyEx6181</italic> [<italic>ptrn-1::gfp::SL2::mCherry</italic> in fosmid WRM0615Ab03; <italic>Podr-1::gfp</italic>], TV14056 <italic>ptrn-1(wy560); wyEx5730</italic> [<italic>Pptrn-1::ptrn-1a::yfp; Podr-1::gfp</italic>], TV15195 <italic>ptrn-1(tm5597); wyEx6022</italic> [<italic>Pdes-2::ptrn-1a::tdTomato; Podr-1::gfp</italic>], TV15770 <italic>ptrn-1(tm5597); wyEx6022; wyEx5968</italic> [<italic>Pdes-2::EMTB::gfp; Podr-1::rfp</italic>], TV15399 <italic>ptrn-1(tm5997); wyEx6216</italic> [<italic>Pmyo-3::ptrn-1a::tdTomato; rol-6(d)</italic>]<italic>,</italic> TV15773 <italic>ptrn-1(tm5597); wyEx6165</italic> [<italic>Pmyo-3::ptrn-1a(ΔCKK)::tdTomato; rol-6(d)</italic>]<italic>; wyEx5968,</italic> TV15790 <italic>ptrn-1(tm5597); wyEx6092</italic> [<italic>Pdes-2::ptrn-1a(ΔCKK)::tdTomato; Podr-1::gfp; Pdes-2::bfp</italic>]<italic>; wyEx5968,</italic> TV14687 <italic>wyEx5968,</italic> TV15383 <italic>ptrn-1(tm5597); wyEx5968, ptrn-1(tm5597);</italic> [<italic>Punc-86::gfp::ptrn-1a; Podr-1::rfp</italic>], TV15774 <italic>bus-17(e2800); wyEx5968, bus-17(e2800); wyEx6022; wyEx5968,</italic> TV15772 <italic>bus-17(e2800); wyEx6092; wyEx5968,</italic> TV15776 <italic>bus-17(e2800); wyEx6165; wyEx5968, ptrn-1(tm5597); wyEx4828</italic> [<italic>Pdes-2::ebp-2::gfp; Podr-1::gfp</italic>]<italic>; wyEx6022</italic>, TV11781 <italic>wyEx4828,</italic> TV14069 <italic>ptrn-1(tm5597); wyEx4828,</italic> TV13424 <italic>ptrn-1(wy560); wyEx4828,</italic> TV16422 <italic>ptrn-1(tm5597); wyIs602; wyEx4828,</italic> TV12310 <italic>wyIs371</italic> [<italic>ser-2prom3::myrGFP, Prab-3::mCherry, Podr-1::rfp</italic>], TV15768 <italic>wyIs371; ptrn-1(tm5597),</italic> TV1204 <italic>wyIs75</italic> [<italic>Pexp-1::gfp, Punc-47L::rfp</italic>], TV15151 <italic>wyIs75; ptrn-1(tm5597),</italic> TV15314 <italic>wyEx6177</italic> [<italic>pPD117.01 Pmec-7::gfp, Podr-1::gfp</italic>], TV15317 <italic>ptrn-1(tm5597); wyEx6177,</italic> TV1838 <italic>wyIs97</italic> [<italic>Punc-86::myr-gfp, Punc-86::mCherry::rab-3</italic>], TV13422 <italic>wyIs97; ptrn-1(tm5597)</italic>, TV12134 <italic>wyIs348</italic> [<italic>Pmec-17::mCherry::rab-3, Pmec-17::CD4::spGFP1-10</italic>], TV13423 <italic>wyIs348; ptrn-1(tm5597),</italic> TV15322 <italic>wyIs348; ptrn-1(tm5597); wyEx6023</italic> [<italic>Punc-86::gfp::ptrn-1</italic>], TV13430 <italic>wyIs348; ptrn-1(wy560),</italic> TV14063 <italic>wyIs348; ptrn-1(wy560); wyEx5782</italic> [<italic>Pmec-3::ptrn-1::yfp</italic>], NM0664 <italic>jsIs37</italic> [<italic>Pmec-7::snb-1::gfp</italic>], TV14346 <italic>jsIs37; ptrn-1(tm5597),</italic> TV15777 <italic>dlk-1(ju476); wyIs97,</italic> TV15778 <italic>dlk-1(ju476); wyIs97; ptrn-1(tm5597),</italic> TV16093 <italic>pmk-3(ok169) wyIs97,</italic> TV16240 <italic>pmk-3(ok169) wyIs97; ptrn-1(tm5597),</italic> TV16396 <italic>wyIs75; wyEx6577</italic> [<italic>Podr-1::gfp</italic>], TV16394 <italic>wyIs75; ptrn-1(tm5597); wyEx6575</italic> [<italic>Podr-1::gfp</italic>], TV16398 <italic>wyIs75; ptrn-1(tm5597); wyEx6579</italic> [<italic>Prab-3::ptrn-1a, Podr-1::gfp</italic> line 1], TV16399 <italic>wyIs75; ptrn-1(tm5597); wyEx6580</italic> [<italic>Prab-3::ptrn-1a, Podr-1::gfp</italic> line 2], TV16395 <italic>wyIs75; ptrn-1(tm5597); wyEx6576</italic> [<italic>Punc-47L::ptrn-1, Podr-1::gfp</italic> line 1], TV16397 <italic>wyIs75; ptrn-1(tm5597); wyEx6578</italic> [<italic>Punc-47L::ptrn-1, Podr-1::gfp</italic> line 2], <italic>wyIs75; ptrn-1(tm5597);</italic> [<italic>Pdpy-7::ptrn-1, Pvha-6::ptrn-1, Podr-1::gfp</italic> lines 1-4], <italic>wyIs97; wyEx6582</italic> [<italic>Podr-1::rfp</italic>], TV16401 <italic>wyIs97; ptrn-1(tm5597); wyEx6582</italic> [<italic>Podr-1::gfp</italic>], TV16400 <italic>wyIs97; ptrn-1(tm5597); wyEx6589</italic> [<italic>Prab-3::ptrn-1, Podr-1::rfp</italic>], TV16402 <italic>wyIs97; ptrn-1(tm5597); wyEx6583</italic> [<italic>Punc-86::ptrn-1, Podr-1::rfp</italic>], <italic>wyIs97; ptrn-1(tm5597);</italic> [<italic>Pdpy-7::ptrn-1, Pvha-6::ptrn-1, Podr-1::rfp</italic> lines 1-4].</p></sec><sec id="s4-2"><title>Molecular biology and transgenic lines</title><p>Expression vectors were made in the pSM vector, a derivative of pPD49.26 (A Fire, unpublished data) with added cloning sites (S McCarroll and CI Bargmann, unpublished data) using standard techniques. Plasmids were coinjected with markers <italic>Podr-1::gfp</italic>, <italic>Podr-1::rfp,</italic> or <italic>rol-6(d).</italic></p></sec><sec id="s4-3"><title>Confocal imaging and fluorescence microscopy</title><p>Images were acquired with a Zeiss LSM510 confocal microscope using a Plan-Apochromat 63x/1.4 objective. Data were analyzed using ImageJ software. Visual inspection and quantification of the penetrance of fluorescence localization were performed using a Zeiss Axioplan 2 microscope with a 63x/1.4NA objective and Chroma HQ filter sets for GFP, YFP, and RFP. Animals were immobilized in 2.5 mM levamisol +0.225 mM BDM (2,3-butanedione monoxime) or 2 mM levamisole for confocal imaging or fluorescence microscopy, respectively (Sigma, St Louis, MO). Colocalization was assessed using the Colocalization Finder in ImageJ.</p></sec><sec id="s4-4"><title>Dynamic imaging</title><p>Dynamic imaging was performed on an inverted Zeiss Azio Observer Z1 microscope using a Plan-Apochromat 63x/1.4 objective. L4 stage animals were anesthetized in 0.1% tricane +0.01% tetramisole for 15–30 min, then mounted on a 5% agarose pad on a slide for imaging (<xref ref-type="bibr" rid="bib54a">Sulston and Horvitz, 1977</xref>). All videos were acquired with a Quantum 512C camera. Videos of animals co-labeled with EBP-2::GFP and PQN-34a::tdTomato were 110-s videos with roughly 2 frames per second. Videos used to quantify EBP-2::GFP movements were 25-s videos with 8 frames per second. Kymographs were generated with ImageJ. <xref ref-type="other" rid="video1">Video 1</xref> and <xref ref-type="other" rid="video2">Video 2</xref> were acquired over 40 min with 90 s/frame. Z-stacks were acquired at each time point and maximum projections are shown.</p></sec><sec id="s4-5"><title>Colchicine treatment</title><p>For prolonged colchicine treatment, animals were grown from eggs on NGM plates containing colchicine as described (<xref ref-type="bibr" rid="bib16">Chalfie and Thomson, 1982</xref>). For acute colchicine treatment, L4-stage animals were soaked in a drop of 10 mM colchicine in M9 or M9 alone for 1 hr. Animals were alive and thrashing at the end of the treatment.</p></sec><sec id="s4-6"><title>Mechanosensory assays</title><p>Mechanosensory assays were performed as described (<xref ref-type="bibr" rid="bib30">Hobert et al., 1999</xref>). Briefly, L4 animals were tapped 10 times each with an eyelash, alternating between the anterior and posterior half of the body. The reaction was scored as either a ‘response’, if the animal reversed direction of movement, or ‘no response’, if it did not. The fraction of touches resulting in a response were averaged for each animal to give the ‘fraction touch sensitive’.</p></sec><sec id="s4-7"><title>Quantification of neurite ectopic branching</title><p>Animals from 3 to 6 plates/genotype were scored blind to genotype according to the following categories. For DD neurons, the categories were None: no ectopic branches or sprouting from any of the DD commissures or from the nerve cords, Mild: at least one branch or sprout from a commissure, Moderate: at least four ectopic branches, or three branches total from two different commissures, Severe: at least eight branching events, often with large growth cones projecting multiple filopodia. For the PLM axon, the categories were None: no ectopic branches, Mild: at least one branch or at least 4 large bulges along the axon, Severe: At least three branches, often many more accompanied by expanses of distended axon in the region containing the ectopic branches. For the tissue specific rescue experiments, four independent lines were initially assessed per each expression construct. We observed no rescue from any of the intestine-plus-hypodermis lines, and so all four lines were used for the experiments shown in <xref ref-type="fig" rid="fig5">Figure 5C,F</xref>. For the lines expressing <italic>ptrn-1</italic> in the neurons, we observed some lines provided stronger rescue than others, likely due to differences in expression level or mosaicism of the transgene. We therefore included the 1–3 lines with the most rescue per construct in the experiments shown in <xref ref-type="fig" rid="fig5">Figure 5C,F</xref>.</p></sec><sec id="s4-8"><title>Electron microscopy</title><p>Young adult wild-type N2 and <italic>ptrn-1(tm5597)</italic> animals were prepared as described (<xref ref-type="bibr" rid="bib20">Cueva et al., 2012</xref>). Briefly, animals were frozen in an EMPACT2 high-pressure freezer system, and a Leica AFS freeze substitution apparatus (Vienna, Austria) was used to preserve in 2% glutaraldehyde plus 1% osmium tetroxide and embed in Epon/Araldite. A Leica Ultracut S microtome equipped with a diamond knife was used to cut 50-nm serial sections, which were collected on Formvar-coated copper slot grids. The grids were poststained to enhance contrast in 3.5% uranyl acetate (30 s) and Reynold’s lead citrate preparation (3 min). The grids were imaged on a transmission electron microscope (JEOL TEM 1230, Tokyo, Japan), and images were acquired with an 11 megapixel bottom-mounted cooled CCD camera (Orius SC1000, Gatan, Pleasanton, CA).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the <italic>Caenorhabditis</italic> Genetics Center and the National Bioresource Project-Japan for strains. We thank EV Stewart for technical advice. This work was supported by the Howard Hughes Medical Institute and NIH.</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>CER, 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>KAS, Conception and design, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>JGC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>JP, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>MBG, Analysis and interpretation 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pub-id-type="doi">10.1091/mbc.E07-09-0878</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.01498.024</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Hobert</surname><given-names>Oliver</given-names></name><role>Reviewing editor</role><aff><institution>Columbia 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://elife.elifesciences.org/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 “PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in <italic>C. elegans</italic> neurons” for consideration at <italic>eLife</italic>.</p><p>Two reviewers were clearly more positive than a third reviewer but the positive reviewers maintained their essentially positive stance even after being made aware of the more negative comments. Therefore, we would like to invite you to submit a revised version of your manuscript in which we hope you can address a number of issues raised by the reviewers, as summarized below.</p><p>1) The conclusion that PRTN-1 protein is found at minus ends of microtubule rests to a large degree on work on patronin in other species; this needs to be clearly acknowledged throughout the manuscript.</p><p>2) The extensive fluorescence within the muscle cell needs to be explained. This fluorescence could presumably from ends of microtubules, but no information is given about the organization of microtubules within muscles in <italic>C. elegans</italic>.</p><p>3) It is stated that PTRN-1 binds to immobile puncta throughout the manuscript, but the authors look at most for less than two minutes. The authors need to look over longer times to assert that the puncta are immobile.</p><p>4) At several places in the manuscript the authors state that PTRN-1 stabilizes microtubules. Their main argument is that colchicine acts at a lower concentration. This result only says that the loss of this protein makes the microtubules more sensitive to the drug. Elsewhere the authors state that they see no difference in structure or number in microtubules except in the PLM. One would expect that the number of microtubules would go down. This needs to be explained.</p><p>5) The authors fail to provide statistics for their assertions. The note inadvertently left in the methods shows that at least one of the authors was aware of this problem.</p><p>6) A large part of the manuscript uses two novel fluorescent markers-labeled patronin and a labeled microtubule-binding domain. It would significantly strengthen the paper to validate these markers. Further, experiments using these markers (colocalization) are descriptive-some quantitative data should be provided.</p><p>7) The authors should more clearly define in the abstract and elsewhere which prtn-1 phenotypes are derived from the upregulation of DLK-1 signaling, i.e., which phenotypes are rescued by loss of DLK-1. DLK-1 is not mentioned in abstract or introduction, which gives the impression that the neurite morphology and synaptic vesicle location defects result directly from changes in MT structure caused by loss of PTRN-1. The authors state that PTRN-1 ‘anchors’ microtubules in neurons and muscle based on the observations that PTRN-1 puncta colocalize with MT puncta following colchicine treatment and that the PTRN-1 CKK deletion mutant often does not colocalize with MTs. As stated, this conclusion is perhaps too strong given that ptrn-1 mutants have a relatively minor, but significant, MT defect. I believe that the conclusion is reasonable, but that it should be stated more conservatively.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01498.025</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The conclusion that PRTN-1 protein is found at minus ends of microtubule rests to a large degree on work on patronin in other species; this needs to be clearly acknowledged throughout the manuscript</italic>.</p><p>The following alterations were made to the text to address this issue:</p><p>- To the Introduction, we added the following more thorough description of the previous work that established that CAMSAP proteins directly bind the MT minus end: “Meng et al. purified and fluorescence-labeled the C-terminal half of CAMSAP3 and sequentially added rhodamin-labeled and unlabeled MTs (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). The CAMSAP3 fragment colocalized with the end of the MT with higher rhodamine fluorescence, which indicates that it was bound to the minus end (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>). Goodwin et al. found that purified GFP-Patronin which was attached to a coverslip bound and anchored rhodamine-MTs by a single end (<xref ref-type="bibr" rid="bib25">Goodwin and Vale, 2010</xref>). Further, they used MT gliding assays in which either the plus-end motor kinesin or the minus-end motor dynein were added to the purified rhodamine-MT plus GFP-Patronin to show that the Patronin-bound end of the MT was the minus end (Goodwin et al., 2010).”</p><p>- The sentence “Further, the regularly spaced PTRN-1a::tdTomato puncta at the sarcolemma co-localized with the EMTB::GFP puncta at the sarcolemma (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), suggestive that PTRN-1a localizes to MT ends” was changed to “Further, PTRN-1a puncta colocalized with EMTB::GFP puncta at the sarcolemma (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), a finding which suggests that, like its homologs in fruitflies and humans (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; Goodwin et al. 2010; <xref ref-type="bibr" rid="bib55">Tanaka et al., 2012</xref>), PTRN-1a localizes to MT ends”.</p><p>- The following was added to the Results:</p><p>“As previous studies have shown that the CKK domain of CAMSAP proteins is involved in MT binding (<xref ref-type="bibr" rid="bib4">Baines et al., 2009</xref>; Goodwin et al., 2010), we analyzed PTRN- 1a(ΔCKK)::tdTomato to determine whether PTRN-1a itself stabilizes MTs.”</p><p>- The following sentence was added to the Discussion:</p><p>“Previous studies have shown that CAMSAP family proteins directly bind MT minus ends (<xref ref-type="bibr" rid="bib39">Meng et al., 2008</xref>; Goodwin et al., 2010).”</p><p>- References were added as shown below in the Discussion:</p><p>“We showed that PTRN-1 puncta in neuronal processes were unaffected by drug-induced MT depolymerization, and PTRN-1 localization was independent of the CKK domain thought to bind MTs (<xref ref-type="bibr" rid="bib4">Baines et al. 2009</xref>; Goodwin et al. 2010).”</p><p><italic>2) The extensive fluorescence within the muscle cell needs to be explained. This fluorescence could presumably from ends of microtubules, but no information is given about the organization of microtubules within muscles in</italic> C. elegans<italic>.</italic></p><p>There is little reported about the organization of MTs in <italic>C. elegans</italic> muscle, but the EMTB::GFP pattern we observe appears quite similar to the MT pattern shown by <xref ref-type="bibr" rid="bib31">Hueston et al. (2008)</xref> using fluorescence-labeled ELP-1, the <italic>C. elegans</italic> EMAP-like homolog. We have added a reference to this paper. We have also added a brief description regarding MTs in mammalian muscle to the Results section.</p><p>The following sentences were added to the Results section, after the description of EMTB::GFP in the muscle cells, to better describe the known MT organization in muscle cells:</p><p>“This microtubule organization in the body wall muscle cells corroborates the pattern observed by fluorescence-labeled ELP-1, the <italic>C. elegans</italic> EMAP (Echinoderm Microtubule-Associated Protein)-like protein (<xref ref-type="bibr" rid="bib31">Hueston et al., 2008</xref>). In mammalian muscle fibers, MTs filaments form both a grid-like organization aligned with the Z-discs that is dependent on dystrophin (<xref ref-type="bibr" rid="bib49">Prins et al., 2009</xref>) and squiggles in the cytosol with less apparent organization (<xref ref-type="bibr" rid="bib50">Ralston et al., 1999</xref>).”</p><p>And later:</p><p>“It is unclear how PTRN-1::tdTomato is localized at either the sarcolemma or in the muscle cell interior. Interestingly, in mammalian muscle, MTs are nucleated from the immobile Golgi elements strung throughout the cytoplasm (<xref ref-type="bibr" rid="bib45">Oddoux et al., 2013</xref>).”</p><p>We use the muscle cells in this study because the large, three-dimensional cytoplasm makes the interaction between fluorescence-labeled PTRN-1 and MTs much more apparent than it is in the thin neurites. Because the import of this study is in describing the function of PTRN-1 in neurons, we prefer to keep the Discussion focused on this without additional speculation about the function of MTs and/or PTRN-1 in muscles.</p><p><italic>3) It is stated that PTRN-1 binds to immobile puncta throughout the manuscript, but the authors look at most for less than two minutes. The authors need to look over longer times to assert that the puncta are immobile</italic>.</p><p>We followed this suggestion and performed live imaging on the PTRN-1::tdTomato puncta in the PVD neuron for 40 minutes. Interestingly, in these longer movies, although some of the PTRN-1::tdTomato puncta do not change their positions, the majority can be seen moving slowly. These slow movements of each PTRN-1::tdTomato punctum appear to be independent of the movements of the neighboring puncta, leading to some puncta merging with each other. Others can be seen dividing, and others disappear. We have incorporated two of these movies into the manuscript along with accompanying description in the text.</p><p>Although the PTRN-1::tdTomato puncta are stable compared to EBP-2::GFP, it seems from these movies that they are not<italic>,</italic> in fact, immobile over this longer timeframe. Before taking these movies, we had imagined that PTRN-1 might be part of a protein complex that linked MT minus ends to some anchor that attached the neurite to the surrounding tissue. Because of these movies, we suspect that the PTRN-1 puncta might not anchored in such a manner; rather, they might be free-floating within the neurite but slow-moving due to crosslinking between bundled MTs and perhaps interactions with other structures within the neurite.</p><p>We have therefore altered sentences that had said that PTRN-1 “anchors” MTs to state instead that it “stabilizes” them. From the Introduction, we have also removed the word “immobile” from the following sentence: “Live imaging of the <italic>C. elegans</italic> CAMSAP homolog, PTRN-1, in cell co-labeled with fluorescence-tagged MTs indicates that PTRN-1 localizes to MT associated puncta throughout neuronal processes.” We changed “anchored” to “localized” in describing the implications of the similar localization between PTRN-1::tdTomato and PTRN-1(ΔCKK)::tdTomato in the Discussion.</p><p><italic>4) At several places in the manuscript the authors state that PTRN-1 stabilizes microtubules. Their main argument is that colchicine acts at a lower concentration. This result only says that the loss of this protein makes the microtubules more sensitive to the drug</italic>.</p><p>To strengthen our claim that the increased sensitivity of the <italic>ptrn-1</italic> mutant to neurite sprouting during growth on colchicine indicates that PTRN-1 stabilizes MTs in neurites, we have added tissue-specific rescue to these experiments (<xref ref-type="fig" rid="fig5">Figure 5C, 5F</xref>). These data show that PTRN-1 functions in the neurons to protect MTs from the effects of growth on colchicine.</p><p>Next, to avoid over-stating the implication of the grow on colchicine experiments, we have made the following alterations to the text that, we hope, clarify the interpretation of these experiments:</p><p>The sentence “Again, these synthetic interactions between <italic>ptrn-1</italic> loss-of-function and colchicine indicates that <italic>ptrn-1</italic> promotes MT stabilization in both the 11-pf and the 15-pf neurons” was replaced with “Taken together, these data indicate that loss of <italic>ptrn-1</italic> enhances sensitivity to colchicine cell-autonomously in both the 11-pf and the 15-pf neurons. This enhanced sensitivity to colchicine likely reflects reduced MT stability in the <italic>ptrn-1</italic> mutant, suggestive that PTRN-1 promotes MT stabilization.”</p><p>In addition, our deduction that PTRN-1 stabilizes MTs is inferred from the consideration of various other data in addition to the neurite sprouting on colchicine experiments. First, previous studies indicate that <italic>Drosophila</italic> Patronin and the human CAMSAP proteins promote MT stabilization (Meng et al., 2008; Goodwin et al., 2010; Tanaka et al., 2012; Wang et al., 2013). In adjusting the manuscript to answer Major Point 1, we hope we have also more clearly attributed the known function of CAMSAP proteins to these previous works. Second, PTRN-1::tdTomato puncta colocalize with EMTB::GFP foci after acute colchicine treatment whereas PTRN-1(ΔCKK)::tdTomato puncta generally do not. As we state in the text, we believe these data, when considered alongside the excellent characterizations of <italic>Drosophila</italic> Patronin and human CAMSAP proteins, indicate that PTRN-1 itself binds and stabilizes MTs.</p><p><italic>Elsewhere the authors state that they see no difference in structure or number in microtubules except in the PLM. One would expect that the number of microtubules would go down. This needs to be explained</italic>.</p><p>We performed detailed characterization of MTs with EM in the PLM neuron but not in other neurons for two reasons. First, PLM has a very well characterized MT number and organization. Previous studies from the Goodman lab and the Chalfie lab have shown that the PLM neuron in wild-type animals has 25-50 MTs per cross-section in young adult animals sectioned between the gonad and the anus (Chalfie & Thomson 1979; Chalfie & Thomson 1982; Cueva et al. 2012, our unpublished data). Second, the unique position and shape of the PLM neurites allow us to unambiguously identify them without reconstructing the entire neuron.</p><p>In contrast to PLM, neurons in the ventral nerve cord generally have around 4 MTs per cross section (Chalfie & Thomson, 1979), and we observed as few as one MT in some VNC processes in a wild-type animal. In addition, because there are many neurites in the nerve cord, it is impossible to identify them without a complete reconstruction, which is a huge effort that was only achieved a couple of times in the history. Therefore, due to the large variability of MT numbers between neurites and the inability to identify individual neurons, we did not attempt to compare the number of MTs in the VNC between wild-type and the <italic>ptrn-1</italic> mutant. We did state that MT shape and protofilament number appeared grossly wild-type, but with so few MTs examined, perhaps we should not have attempted to make any statement regarding the VNC MTs from our EM data. Therefore, we have removed <xref ref-type="fig" rid="fig5s1">Figure 5–figure supplement 1</xref> and the following sentence:</p><p>“Electron microscopy of a section of the VNC in the <italic>ptrn-1(tm5597)</italic> mutant did not reveal any gross abnormalities in MT shape or protofilament number (<xref ref-type="fig" rid="fig5s1">Figure 5–figure supplement 1</xref>), but more extensive analysis will be required to determine if there are differences in MT number or length.”</p><p>Additionally, in the Abstract, the sentence “Electron microscopy revealed that <italic>ptrn-1</italic> null mutants have a reduced number of MTs and abnormal MT organization” was amended to “Electron microscopy revealed that <italic>ptrn-1</italic> null mutants have a reduced number of MTs and abnormal MT organization in the PLM neuron.”</p><p>Of note, the animals prepared for our EM were grown at 25°C to be consistent with all of the other experiments in the manuscript, whereas Chalfie & Thomson, (1979), used animals grown at 20°C. The reduction in MT number at higher temperature is consistent with our previous unpublished observations.</p><p><italic>5) The authors fail to provide statistics for their assertions. The note inadvertently left in the methods shows that at least one of the authors was aware of this problem</italic>.</p><p>The Statistics section that summarized the statistical analyses in the Methods was deleted, and the test used in each case was stated in the figure legends. Also, statistical analyses were added to <xref ref-type="fig" rid="fig2">Figure 2</xref> (through the addition of <xref ref-type="fig" rid="fig2s2">Figure 2–figure supplement 2</xref>), <xref ref-type="fig" rid="fig5">Figure 5C</xref>, <xref ref-type="fig" rid="fig5">Figure 5F</xref>, and <xref ref-type="fig" rid="fig7">Figure 7B</xref>.</p><p><italic>6) A large part of the manuscript uses two novel fluorescent markers-labeled patronin and a labeled microtubule-binding domain. It would significantly strengthen the paper to validate these markers. Further, experiments using these markers (colocalization) are descriptive-some quantitative data should be provided</italic>.</p><p>We had taken several steps to confirm that the label PTRN-1 is not aberrantly localized. First, we show three different fluorescence labeled PTRN-1 constructs: PTRN-1a::YFP, GFP::PTRN-1a, and PTRN-1a::tdTomato. PTRN-1 tagged at either the N-terminus or the C-terminus and using different fluorophores with different amino acid linkers all exhibited similar localization in multiple neurons of different classes (<xref ref-type="fig" rid="fig1">Figure 1</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1–figure supplement 2</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1–figure supplement 3</xref>). This consistency in the localization of labeled PTRN-1 is suggestive that this localization is biologically relevant. Second, we used transgenic lines expressing <italic>ptrn-1a::yfp</italic> to show cell-autonomous rescue for the mislocalized synaptic vesicles and incomplete commissure in the PLM neuron (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). These data showed that at least some of the labeled PTRN-1 constructs were functional.</p><p>To provide additional validation of the labeled PTRN-1, we used the <italic>ptrn-1a::tdTomato</italic> transgene, which is shown in <xref ref-type="fig" rid="fig1">Figure 1</xref>, <xref ref-type="other" rid="video1">Video 1</xref>, <xref ref-type="other" rid="video2">Video 2</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1–figure supplement 3</xref>, <xref ref-type="fig" rid="fig2">Figure 2</xref>, <xref ref-type="fig" rid="fig3">Figure 3</xref>, and <xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1</xref>, for cell-autonomous rescue of the reduction of EBP-2 movements (<xref ref-type="fig" rid="fig4">Figure 4</xref>). We hope that by showing the transgene we used the most for localization data is able to rescue the <italic>ptrn-1</italic> phenotype, we alleviate the concern that the localization is spurious.</p><p>The ensconsin microtubule binding domain (EMTB) has been well characterized (<xref ref-type="bibr" rid="bib22">Faire et al., 1999</xref>; <xref ref-type="bibr" rid="bib11">Bulinski et al., 2001</xref>), and it has been used in numerous studies to label microtubules in vivo (<xref ref-type="bibr" rid="bib34">Lechler and Fuchs, 2007</xref>; <xref ref-type="bibr" rid="bib59">von Dassow et al., 2009</xref>; <xref ref-type="bibr" rid="bib62">Wühr et al., 2010</xref>). Our data show that EMTB::GFP localization in the muscle cell is strikingly similar to the localization of the <italic>C. elegans</italic> EMAP protein ELP-1, as described above (Hueston et al., 2009). To further confirm that EMTB::GFP labels MTs in our system, we constructed a strain coexpressing EMTB::GFP and ELP-1::mCherry in the body wall muscle (<xref ref-type="fig" rid="fig8">Author response image 1</xref>, below)<bold>.</bold> Both fluorescent proteins localize along filaments. We found EMTB::GFP to more consistently label long stretches of the filamentous structures, but the ELP-1::mCherry generally colocalized with the EMTB::GFP, labeling short stretches of the EMTB::GFP filaments. These filaments are MTs. We feel that the request to validate EMTB::GFP was likely elicited by inadequate explanation and citationin the text. We have therefore expanded the introduction of the EMTB::GFP marker in the text as follows:</p><p>“To examine whether PTRN-1 binds MTs in neurites, we co-expressed PTRN- 1a::tdTomato with the MT-binding domain of ensconsin fused to GFP (EMTB::GFP)(<xref ref-type="bibr" rid="bib37">Masson & Kreis 1993</xref>; <xref ref-type="bibr" rid="bib10">Bulinski & Bossler 1994</xref>; <xref ref-type="bibr" rid="bib22">Faire et al., 1999</xref>). EMTB::GFP, which binds dynamically along the side of MTs, has been previously used to visualize MTs in vivo (<xref ref-type="bibr" rid="bib11">Bulinski et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Lechler & Fuchs, 2007</xref>; <xref ref-type="bibr" rid="bib59">von Dassow et al., 2009</xref>; <xref ref-type="bibr" rid="bib62">Wühr et al., 2010</xref>). In <italic>C. elegans</italic> neurons, it generally exhibited continuous fluorescence throughout neuronal processes.”</p><p>We also added the reference included below in the Results:</p><p>“Since EMTB::GFP normally binds to the sidewalls of MTs (<xref ref-type="bibr" rid="bib22">Faire et al., 1999</xref>), this change in its distribution confirms that colchicine treatment led to MT depolymerization, as expected.”<fig id="fig8" position="float"><label>Author response image 1.</label><caption><p>The body wall muscle of an animal oc-expressing EMTB::GFP and ELP-1::mCherry. The two MT-binding proteins colocalize to the MT filaments throughout the muscle cells. Scale bar = 5 μm.</p></caption><graphic xlink:href="elife01498f008"/></fig></p><p><italic>7) The authors should more clearly define in the abstract and elsewhere which prtn-1 phenotypes are derived from the upregulation of DLK-1 signaling, i.e., which phenotypes are rescued by loss of DLK-1. DLK-1 is not mentioned in abstract or introduction, which gives the impression that the neurite morphology and synaptic vesicle location defects result directly from changes in MT structure caused by loss of PTRN-1</italic>.</p><p>To better understand the interaction between <italic>dlk-1</italic> and <italic>ptrn-1,</italic> we examined the affect of <italic>dlk-1</italic> on EBP-2::GFP movements in the PHC dendrite (<xref ref-type="fig" rid="fig7">Figure 7G,H</xref>). The following description of these data was added to the Results section:</p><p>“Finally, we asked whether the reduction in EBP-2::GFP movements in the <italic>ptrn-1</italic> mutant is also dependent on <italic>dlk-1.</italic> Interestingly, we observed an increase of roughly two-fold in EBP-2::GFP movements in the PHC dendrite in the <italic>dlk-1</italic> single mutant relative to wild- type but had no affect on the orientation (<xref ref-type="fig" rid="fig7">Figure 7G,H</xref><bold>)</bold>. There was a trend for the <italic>dlk-1; ptrn-1</italic> double mutant to have increased EBP-2::GFP movements relative to the <italic>ptrn-1</italic> single mutant, though this difference was not statistically significant. However, the <italic>dlk-1; ptrn-1</italic> double mutant exhibited fewer movements than the <italic>dlk-1</italic> single mutant (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). This intermediate phenotype in the <italic>dlk-1; ptrn-1</italic> double mutant indicates that the DLK-1 pathway is not the only mechanism required for the <italic>ptrn-1</italic> mutant phenotypes, and it is suggestive that <italic>dlk-1</italic> functions partially in parallel to <italic>ptrn-1</italic> to influence EBP- 2::GFP movements in the PHC neuron.”</p><p>To further strengthen the DLK-1 section, we have added <xref ref-type="fig" rid="fig7s1">Figure 7–figure supplement 1</xref>, which shows that <italic>pmk-3,</italic> like <italic>dlk-1</italic>, suppresses two of the defects observed in the <italic>ptrn-1</italic> mutant – the incomplete PLM commissure formation and the loss of synaptic vesicles from the synaptic patch.</p><p>We have edited the Abstract as follows:</p><p>“Further, <italic>ptrn-1</italic> null mutants exhibited aberrant neurite morphology and synaptic vesicle localization that is partially dependent on <italic>dlk-1</italic>.” Slight changes were made to the wording of the abstract to keep the length to 150 words.</p><p><italic>The authors state that PTRN-1 ‘anchors’ microtubules in neurons and muscle based on the observations that PTRN-1 puncta colocalize with MT puncta following colchicine treatment and that the PTRN-1 CKK deletion mutant often does not colocalize with MTs. As stated, this conclusion is perhaps too strong given that ptrn-1 mutants have a relatively minor, but significant, MT defect. I believe that the conclusion is reasonable, but that it should be stated more conservatively</italic>.</p><p>We appreciate the point that we do not present evidence that PTRN-1 itself anchors MTs, and we should not have implied that does. We think the data show that PTRN-1 stabilizes MT foci in both neurites and muscles, since full length PTRN-1 puncta colocalize with EMTB::GFP puncta after acute colchicine exposure whereas PTRN-1(ΔCKK) puncta generally do not. At the sarcolemma, the rows of PTRN-1a::tdTomato and EMTB::GFP puncta can be seen both before and after the acute colchicine exposure, indicating that PTRN-1 localizes to sites of MT anchorage near the muscle membrane. Considering this, along with the fact that the size, spacing, and abundance of PTRN-1 puncta plus EMTB::GFP in the PVD dendrite appears unaffected by acute colchicine exposure, we might have inferred that MTs are likewise anchored at (though not necessarily by) PTRN-1 puncta in the neurites. However, because of the useful suggestion that we take longer movies of PTRN-1::puncta, we now know that many PTRN-1::tdTomato puncta drift in the PVD dendrite, moving at a rate of roughly 1-2 μm per minute. In light of this Major Point and the 40 minute movie of PTRN-1::tdTomato, wherever we described the MTs as being “anchored” in the neurites, we replaced “anchored” with “stabilized.”</p></body></sub-article></article> |