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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">02131</article-id><article-id pub-id-type="doi">10.7554/eLife.02131</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>Plant biology</subject></subj-group></article-categories><title-group><article-title>Delivery of endocytosed proteins to the cell–division plane requires change of pathway from recycling to secretion</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-10019"><name><surname>Richter</surname><given-names>Sandra</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10020"><name><surname>Kientz</surname><given-names>Marika</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10021"><name><surname>Brumm</surname><given-names>Sabine</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10022"><name><surname>Nielsen</surname><given-names>Mads Eggert</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10023"><name><surname>Park</surname><given-names>Misoon</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10024"><name><surname>Gavidia</surname><given-names>Richard</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10025"><name><surname>Krause</surname><given-names>Cornelia</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10026"><name><surname>Voss</surname><given-names>Ute</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa2">‡</xref><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10027"><name><surname>Beckmann</surname><given-names>Hauke</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10028"><name><surname>Mayer</surname><given-names>Ulrike</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10029"><name><surname>Stierhof</surname><given-names>York-Dieter</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-9816"><name><surname>Jürgens</surname><given-names>Gerd</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Developmental Genetics, The Center for Plant Molecular Biology (ZMBP)</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff><aff id="aff2"><institution content-type="dept">Microscopy, The Center for Plant Molecular Biology (ZMBP)</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Hardtke</surname><given-names>Christian S</given-names></name><role>Reviewing editor</role><aff><institution>University of Lausanne</institution>, <country>Switzerland</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>gerd.juergens@zmbp.uni-tuebingen.de</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark</p></fn><fn fn-type="present-address" id="pa2"><label>‡</label><p>Plant Sciences Division, School of Biosciences, University of Nottingham, Nottingham, United Kingdom</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>08</day><month>04</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02131</elocation-id><history><date date-type="received"><day>19</day><month>12</month><year>2013</year></date><date date-type="accepted"><day>27</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Richter et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Richter 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="elife02131.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.02747"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02131.001</object-id><p>Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1–BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell–division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.001">http://dx.doi.org/10.7554/eLife.02131.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02131.002</object-id><title>eLife digest</title><p>Cells are surrounded by a plasma membrane, and when a cell divides to create two new cells, it must grow a new membrane to keep the two new cells apart. Animal cells and plant cells tackle this challenge in different ways: in animal cells the new membrane grows inwards from the surface of the cell, whereas the new membrane grows outwards from the centre of the cell in plant cells.</p><p>The materials needed to make the plasma membrane are delivered in packages called vesicles: most of these materials arrive from a structure within the cell called the trans-Golgi network, but some materials are recycled from the existing plasma membrane. In plants the formation of the new cell membrane is orchestrated by scaffold-like structure that forms in the plant cell called the ‘phragmoplast’. It is widely thought that this structure guides the vesicles bringing materials from the trans-Golgi network, but the details of this process are not fully understood.</p><p>Now, Richter et al. have discovered four proteins, called BIG1 to BIG4, that control the formation of the new cell membrane in the flowering plant <italic>Arabidopsis thaliana</italic>, a species that is routinely studied by plant biologists. These four proteins belong to a larger family of proteins that control the trafficking of vesicles within a cell. Richter et al show that a plant cell can lose up to three of these four proteins and still divide, as the plant can still grow and develop as normal. Thus, BIG1 to BIG4 appear to perform essentially the same role in the plant.</p><p>Richter et al. also show that, when a plant cell is not dividing, these proteins are involved in controlling the delivery of new materials to surface membrane, and not the recycling of material. However, when a cell is dividing, these proteins switch to regulate both processes, but direct all the material to a new destination—the newly forming membrane, instead of the established surface membrane. Richter et al. suggest that this switch is important to stop any recycling to the plasma membrane that might move material away from the new membrane. The next challenge will be to identify the molecular signals and mechanisms that enable the proteins BIG1 to BIG4 to re-route the recycling of membrane material during cell division.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.002">http://dx.doi.org/10.7554/eLife.02131.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>post-Golgi trafficking</kwd><kwd>gegulation of vesicle traffic</kwd><kwd>ARF-GEF</kwd><kwd>cell division</kwd><kwd>secretion</kwd><kwd>recycling</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Arabidopsis</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>German Research Foundation (DFG)</institution></institution-wrap></funding-source><award-id>SFB446/TP A9</award-id><principal-award-recipient><name><surname>Jürgens</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>German Research Foundation (DFG)</institution></institution-wrap></funding-source><award-id>JU 179/18-1</award-id><principal-award-recipient><name><surname>Jürgens</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Carlsberg Foundation</institution></institution-wrap></funding-source><award-id>2011_01_0789</award-id><principal-award-recipient><name><surname>Nielsen</surname><given-names>Mads Eggert</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 same membrane trafficking cargo can change pathways mediated by different members of ARF-GEF family of vesicle formation regulators.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>In post-Golgi membrane trafficking, cargo proteins are dynamically distributed between trans-Golgi network (TGN), various endosomes, lysosome/vacuole and plasma membrane (<xref ref-type="bibr" rid="bib34">Surpin and Raikhel, 2004</xref>). In contrast to animals, the TGN also functions as an early endosome in plants and is a major trafficking hub where secretory, endocytic, recycling and vacuolar pathways intersect (<xref ref-type="bibr" rid="bib40">Viotti et al., 2010</xref>; <xref ref-type="bibr" rid="bib25">Reyes et al., 2011</xref>). Therefore, it has been notoriously difficult to functionally delineate the recycling vs secretory pathways in plants. Sorting of cargo proteins occurs during the formation of transport vesicles, involving activation of small ARF GTPases by ARF guanine-nucleotide exchange factors (ARF-GEFs) and recruitment of specific coat proteins (<xref ref-type="bibr" rid="bib2">Casanova, 2007</xref>). Arabidopsis ARF-GEFs are related to human large ARF-GEFs, GBF1 or BIG1. Whereas the three GBF1-related members GNOM, GNL1 and GNL2 have been characterised in detail (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>; <xref ref-type="bibr" rid="bib26">Richter et al., 2007</xref>, <xref ref-type="bibr" rid="bib27">2012</xref>), of the 5 BIG1-related ARF-GEFs only BIG5 has been analysed so far and implicated in pathogen response (MIN7) and endocytic traffic (BEN1) (<xref ref-type="bibr" rid="bib20">Nomura et al., 2006</xref>, <xref ref-type="bibr" rid="bib21">2011</xref>; <xref ref-type="bibr" rid="bib35">Tanaka et al., 2009</xref>; <xref ref-type="bibr" rid="bib36">Tanaka et al., 2013</xref>). Here, we show that ARF-GEFs BIG1-4 play a crucial role in post-Golgi traffic, which enables us to dissect the regulation of secretory and recycling pathways in interphase and cytokinesis.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>ARF-GEFs BIG1 to BIG4 are redundantly required in development</title><p>Up to three of ARF-GEFs BIG1 to BIG4 (BIG1-4) were knocked out without recognisable phenotypic effect except for <italic>big1,2,3</italic>, which was retarded in growth because BIG4 is predominantly expressed in root and pollen (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). Other triple mutants were growth-retarded only if the activity of the respective fourth gene was reduced to 50%. No quadruple mutants were recovered because BIG1-4 were essential in male reproduction, sustaining pollen tube growth (<xref ref-type="fig" rid="fig1">Figure 1B</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>). BIG1-4 functional redundancy would be consistent with the occurrence of <italic>BIG1-4</italic>-like single-copy or closely related sister genes in lower plants (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>). Although large ARF-GEFs are often inhibited by the fungal toxin brefeldin A (BFA), the SEC7 domain of BIG3 (At1g01960; formerly named BIG2 in <xref ref-type="bibr" rid="bib18">Nielsen et al., 2006</xref>; see nomenclature used by <xref ref-type="bibr" rid="bib4">Cox et al., 2004</xref>) displayed BFA-insensitive GDP/GTP exchange activity in vitro (<xref ref-type="bibr" rid="bib18">Nielsen et al., 2006</xref>). BFA treatment of <italic>big3</italic> mutants impaired seed germination and seedling root growth, in contrast to wild-type (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref>). We engineered a BFA-resistant variant of the naturally BFA-sensitive ARF-GEF BIG4 by replacing amino acid residue methionine at position 695 with leucine, as previously described for the recycling ARF-GEF GNOM (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>). Engineered BFA-resistant BIG4-YFP rescued BFA-inhibited seed germination of <italic>big3</italic> (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). The rescue activity of BFA-resistant BIG4 was comparable to that of BIG3 when both were expressed from the ubiquitin 10 (<italic>UBQ10</italic>) promoter whereas BFA-sensitive BIG4 did not at all rescue BFA-inhibited primary root growth of <italic>big3</italic> mutant seedlings (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D,E</xref>). Thus, BFA treatment of <italic>big3</italic> single mutants effectively causes conditional inactivation of BIG1-4 ARF-GEF function, providing us with a unique tool for studying BIG1-4-dependent trafficking in an organismic context.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02131.003</object-id><label>Figure 1.</label><caption><title>BIG1 – BIG4 act redundantly at TGN and are involved in several physiological processes.</title><p>(<bold>A</bold>) <italic>big1,2,4 (big1 big2 big4)</italic>, <italic>big2,3,4 (big2 big3 big4)</italic>, <italic>big1,3,4 (big1 big3 big4)</italic> and <italic>big1,2,3/+,4 (big1 big2 big3/BIG3 big4)</italic> mutant plants without obvious phenotype but <italic>big1/+,2,3,4 (big1/BIG1 big2 big3 big4)</italic>, <italic>big1,2/+,3,4 (big1 big2/BIG2 big3 big4)</italic> and <italic>big1,2,3 (big1 big2 big3)</italic> were dwarfed (yellow arrowheads). Scale bar, 2 cm. (<bold>B</bold>) F1 of reciprocal crosses between wild-type (Col) and <italic>big1 big2 big3/BIG3 big4</italic> (<italic>1,2,3/+,4</italic>) mutants: 0% or 48% <italic>big3</italic> heterozygous seedlings derived from mutant male or female gamete, respectively. (<bold>C</bold>) BFA inhibited primary root growth of <italic>big3</italic> mutant seedlings with or without BFA-resistant GNOM (GN<sup>R</sup> <italic>big3</italic>). Numbers of analysed seedlings are indicated (<bold>B</bold> and <bold>C</bold>). (<bold>D</bold>-<bold>H</bold>) BFA treatment did not prevent seed germination in wild-type (Col; <bold>D</bold>) and BFA-resistant GN (GN<sup>R</sup>; <bold>G</bold>) but did so in <italic>big3</italic> mutants without (<bold>E</bold>) or with BFA-resistant GNOM (GN<sup>R</sup> <italic>big3</italic>; <bold>H</bold>). This defect was suppressed by BFA-resistant BIG4 (UBQ10::BIG4R-YFP <italic>big3</italic>; <bold>F</bold>). Scale bar, 5 mm. (<bold>I</bold>-<bold>L</bold>) Live imaging of BIG4-YFP (<bold>I</bold>) and TGN marker VHA-a1-RFP (<bold>J</bold>) revealed co- localization (<bold>K</bold>; <bold>L</bold>, intensity–line profile). (<bold>M</bold>–<bold>P</bold>) Immunolocalization of BIG4 (UBQ10::BIG4-YFP; M) and Golgi-marker γCOP (<bold>N</bold>) indicated no co-localization (<bold>O</bold>; <bold>P</bold>, intensity–line profile). (<bold>I</bold>–<bold>K</bold>, <bold>M</bold>–<bold>O</bold>) Scale bar, 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.003">http://dx.doi.org/10.7554/eLife.02131.003</ext-link></p></caption><graphic xlink:href="elife02131f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Expression and phylogeny of BIG ARF-GEFs.</title><p>(<bold>A</bold>) Expression profiles of BIG1–BIG5 and GNOM. Data from AtGenExpress (<xref ref-type="bibr" rid="bib32">Schmid et al., 2005</xref>) (<ext-link ext-link-type="uri" xlink:href="http://jsp.weigelworld.org">http://jsp.weigelworld.org</ext-link>). Note preferential expression of BIG4 in roots and floral organs. (<bold>B</bold>) Analysis of in vitro pollen tube growth of pollen from wild-type (Col) and <italic>big124 3/+</italic> plants. Note approximately 50% of germinated pollen from mutant plants produced short tubes (red column); asterisks: red, short; green, long pollen tubes). (<bold>C</bold>) Phylogenetic tree (ClustalW) of BIG ARF-GEFs from flowering plants (dicots Arabidopsis [At], poplar [Pt] and grapevine [Vv]; monocots rice [Os] and Brachypodium [Bd]), gymnosperm Picea abies (Pa), lower plants (lycopod Selaginella [Sm], moss Physcomitrella [Pp], and algae Chlamydomonas [Cr] and Volvox [Vc]), and outgroups (human [Hs], <italic>Saccharomyces cerevisiae</italic> [Sc]) (grey). Three distinct subclades (BIG1/4 [blue], BIG2/3 [red] and BIG5 [green]) are present in angiosperms. However, only BIG5 is distinct in all plant species whereas lower plants have a single subclade BIG1-4 corresponding to the two subclades BIG1/4 and BIG2/3 in angiosperms (orange). Accession numbers and source of data are listed in the table.(<bold>D</bold> and <bold>E</bold>) BFA-resistant BIG4 and BIG3 expressed from the Ubiquitin10-promoter (UBQ10::BIG4R/BIG3-YFP) can partially rescue the BFA-inhibited primary root growth of <italic>big3</italic> mutants.(<bold>E</bold>) Percentage of root growth of BFA-treated seedlings shown in (<bold>D</bold>) relative to untreated controls.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.004">http://dx.doi.org/10.7554/eLife.02131.004</ext-link></p></caption><graphic xlink:href="elife02131fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>BIG3 and BIG4 localize at the TGN.</title><p>(<bold>A</bold>–<bold>N</bold>) Live imaging of YFP-tagged BIG3 (<bold>A</bold> and <bold>I</bold>), expressed from its own promoter, and BIG4 (<bold>E</bold> and <bold>L</bold>), expressed from the <italic>UBQ10</italic> promoter, in seedling roots counterstained with endocytic tracer FM4-64 (<bold>B</bold>, <bold>F</bold>, <bold>J</bold>, <bold>M</bold>). Both BIG3 and BIG4 co-localized with FM4-64, which visualized the TGN after 10 min of incubation (<bold>C</bold>, <bold>G</bold>; <bold>D</bold>, <bold>H</bold>, intensity profiles of lines numbered in <bold>C</bold>, <bold>G</bold>). (<bold>I</bold>–<bold>N</bold>) After BFA treatment, both BIG3 and BIG4 co-localized with FM4-64 in BFA compartments (<bold>K</bold> and <bold>N</bold>). Scale bars, 5 μm. (<bold>O</bold>–<bold>R</bold>) Co-localization of BIG4-YFP fluorescence (<bold>O</bold>) with immunofluorescence labeling of the TGN marker ARF1 (<bold>P</bold>) in 350 nm thin cryosections (= high axial resolution) revealed by overlay (<bold>Q</bold>) and image frames shifted by 5 pixels (<bold>R</bold>). Blue, DAPI-stained nuclei. Scale bar (<bold>O</bold>), 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.005">http://dx.doi.org/10.7554/eLife.02131.005</ext-link></p></caption><graphic xlink:href="elife02131fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>Ultrastructural localization of BIG4-YFP and ultrastructural abnormalities in BFA-treated <italic>big3</italic> mutant seedling root cells.</title><p>(<bold>A</bold> and <bold>B</bold>) Anti-GFP immunogold labeling of YFP-tagged BIG4 in root epidermal cells of UBQ10::YFP-BIG4 transgenic (<bold>A</bold>) and Col-0 wild-type control (<bold>B</bold>) seedlings (thawed cryosection labeling). (<bold>C</bold>–<bold>F</bold>) Ultrastructural TEM analysis of BFA-treated <italic>big3</italic> (<bold>C</bold> and <bold>D</bold>) and Col-0 wild-type (<bold>E</bold> and <bold>F</bold>) root epidermal cells. Note Golgi stacks are abnormally shaped (<bold>C</bold> and <bold>G</bold>) or reduced (<bold>D</bold>, arrowheads) near endosomal BFA aggregates (<bold>C</bold> and <bold>D</bold>), in contrast to well-formed Golgi stacks in wild-type (<bold>E</bold> and <bold>F</bold>). (<bold>F</bold>) Higher magnification of boxed area in (<bold>E</bold>). g, Golgi stack; m, mitochondrion; mvb, multivesicular body; tgn, trans-Golgi network; v, vacuole. Scale bars, 500 nm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.006">http://dx.doi.org/10.7554/eLife.02131.006</ext-link></p></caption><graphic xlink:href="elife02131fs003"/></fig></fig-group></p></sec><sec id="s2-2"><title>BIG1 to BIG4 regulate membrane trafficking at the TGN</title><p>BIG4-YFP co-localized with TGN markers vacuolar H<sup>+</sup>-ATPase (VHA) subunit a1 and ARF1 GTPase (<xref ref-type="fig" rid="fig1">Figure 1I–L</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2O–R</xref>; <xref ref-type="bibr" rid="bib6">Dettmer et al., 2006</xref>; <xref ref-type="bibr" rid="bib33">Stierhof and El Kasmi, 2010</xref>) but not with Golgi marker COPI subunit γCOP (<xref ref-type="fig" rid="fig1">Figure 1M–P</xref>; <xref ref-type="bibr" rid="bib15">Movafeghi et al., 1999</xref>). TGN localization of BIG4-YFP was confirmed by immunogold labeling on EM sections (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3A,B</xref>). BIG3-YFP and BIG4-YFP co-localized with endocytic tracer FM4-64, labeling TGN after brief uptake (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A–H</xref>; <xref ref-type="bibr" rid="bib39">Ueda et al., 2001</xref>; <xref ref-type="bibr" rid="bib6">Dettmer et al., 2006</xref>). BIG3 and BIG4 also accumulated together with FM4-64 in BFA-induced post-Golgi membrane vesicle aggregates (‘BFA compartments’), consistent with ultrastructural abnormalities in these aggregates and Golgi stacks in BFA-treated <italic>big3</italic> mutant (<xref ref-type="fig" rid="fig1s2 fig1s3">Figure 1—figure supplement 2I–N, 3C–F</xref>). Together, these data suggest a role for BIG1-4 in post-Golgi membrane trafficking.</p></sec><sec id="s2-3"><title>Secretory and vacuolar trafficking depend on BIG1 to BIG4 function</title><p>To identify trafficking routes regulated by BIG1-4, pathway-specific soluble and membrane-associated cargo proteins were analysed in BFA-treated wild-type and <italic>big3</italic> mutant seedlings (for a list of markers used, see <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1S,T</xref>). Secretory GFP (secGFP) (<xref ref-type="bibr" rid="bib40">Viotti et al., 2010</xref>), which is normally secreted from the cell, and plasma membrane (PM)-targeted syntaxin SYP132 were trapped in BFA compartments and did not reach the plasma membrane of <italic>big3</italic> seedlings, in contrast to wild-type, suggesting a role for BIG1-4 in late secretory traffic, that is from the TGN to the plasma membrane (<xref ref-type="fig" rid="fig2">Figure 2A–D</xref>). There was a slight retention of SYP132 in the BFA compartments of wild-type seedling roots, which probably reflects slowed-down passage of newly-synthesized proteins through the TGN. This becomes apparent upon BFA treatment because of TGN aggregation into BFA compartments, as has been reported earlier for <italic>HS::secGFP</italic> (<xref ref-type="bibr" rid="bib40">Viotti et al., 2010</xref>). Vacuolar cargo proteins also pass through the TGN via multivesicular bodies (MVBs) to the vacuole (<xref ref-type="bibr" rid="bib25">Reyes et al., 2011</xref>). Soluble RFP fused to phaseolin vacuolar sorting sequence AFVY accumulated in BFA compartments in <italic>big3</italic> mutant, in contrast to wild-type (<xref ref-type="bibr" rid="bib30">Scheuring et al., 2011</xref>; <xref ref-type="fig" rid="fig2">Figure 2E–J</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A–F</xref>). Endocytosed PM proteins are delivered to the vacuole for degradation, for example boron transporter BOR1 in response to high external boron concentration (<xref ref-type="bibr" rid="bib37">Takano et al., 2005</xref>; <xref ref-type="fig" rid="fig2">Figure 2K–N</xref>). BFA treatment prevented boron-induced trafficking of BOR1 to the vacuole in <italic>big3</italic> mutant, but not in wild-type (<xref ref-type="fig" rid="fig2">Figure 2L,N</xref>). BOR1 was rapidly turned over in the vacuole of wild-type, leaving no trace of GFP (<xref ref-type="fig" rid="fig2">Figure 2L</xref>). As expected, ARF-GEF BIG4 and its putative cargo BOR1 co-localized in BFA compartments (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1G–I</xref>). Thus, BIG1-4 mediate both late secretory and vacuolar trafficking from the TGN.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02131.007</object-id><label>Figure 2.</label><caption><title>BIG1 – BIG4 regulate secretory and vacuolar trafficking by recruiting AP-1 adaptor complex.</title><p>(<bold>A</bold> and <bold>B</bold>) BFA inhibited secretion of heat shock (HS)-induced secGFP in <italic>big3</italic> mutants (<bold>B</bold>) but not in wild-type (Col; <bold>A</bold>). (<bold>C</bold> and <bold>D</bold>) BFA inhibited trafficking of estradiol (Est)-induced YFP-SYP132 to the plasma membrane in <italic>big3</italic> mutants (<bold>D</bold>) but not in wild-type (Col; <bold>C</bold>). (<bold>E</bold>–<bold>J</bold>) BFA inhibited trafficking of soluble cargo AFVY-RFP to the vacuole (v), labeled by FM1-43 (<bold>F</bold> and <bold>I</bold>), in <italic>big3</italic> mutants (<bold>H</bold>–<bold>J</bold>) but not in wild-type (Col, <bold>E</bold>–<bold>G</bold>). (<bold>K</bold>–<bold>N</bold>) Live imaging of BOR1-GFP localization. Without boron (−B), BOR1-GFP localized at the plasma membrane in wild-type (<bold>K</bold>) and <italic>big3</italic> mutants (<bold>M</bold>). After BFA and boron treatment (+B), BOR1-GFP was degraded in the vacuole of wild-type (<bold>L</bold>) but accumulated in BFA compartments of <italic>big3</italic> mutants (<bold>N</bold>). (<bold>O</bold>–<bold>T</bold>) Immunostaining of 3xHA-tagged muB2 subunit of AP-1 complex (AP1M2; <bold>O</bold>, <bold>R</bold>) and COPI subunit γCOP (<bold>P</bold> and <bold>S</bold>) in BFA-treated seedlings. AP1M2 accumulated in BFA compartments surrounded by γCOP in wild-type (Col; <bold>Q</bold>). In <italic>big3</italic> mutants, γCOP was still recruited to Golgi membranes whereas AP1M2 was cytosolic (<bold>R</bold>–<bold>T</bold>). Blue, DAPI-stained nuclei. Scale bars, 5 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.007">http://dx.doi.org/10.7554/eLife.02131.007</ext-link></p></caption><graphic xlink:href="elife02131f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>BIG1 – BIG4 regulate trafficking of secretory and vacuolar cargo by recruiting AP-1 complex.</title><p>(<bold>A</bold>–<bold>F</bold>) Live imaging of vacuolar cargo AFVY-RFP (<bold>A</bold> and <bold>D</bold>) and vacuolar membrane marker Wave9Y (Wave9-YFP/YFP-VAMP711; <bold>B</bold>, <bold>E</bold>; <xref ref-type="bibr" rid="bib8">Geldner et al., 2009</xref>) in root cells of <italic>big3</italic> mutant seedlings. (<bold>C</bold> and <bold>F</bold>) Overlays. Traffic of AFVY-RFP to the Wave9-labeled vacuole (v; <bold>A</bold>–<bold>C</bold>) was blocked by BFA treatment, with AFVY-RFP accumulating in small compartments distinct from vacuoles (<bold>D</bold>–<bold>F</bold>). (<bold>G</bold>–<bold>I</bold>) Live imaging of BOR1-GFP in boron (+B) and BFA-treated <italic>big3</italic> mutants expressing BIG3-promoter driven BIG4-RFP (BIG3::BIG4-RFP). BOR1-GFP was not transported to the vacuole but co-localized with BFA-sensitive BIG4 in BFA compartments. (<bold>J</bold>–<bold>O</bold>) Immunolocalization of HA-tagged AP1M2 and TGN marker SYP61 in BFA-treated seedling roots. AP1M2 co-localized with SYP61 in BFA compartments in wild-type (<bold>J</bold>–<bold>L</bold>) but was cytosolic in <italic>big3</italic>, in contrast to TGN-associated SYP61 (<bold>M</bold>–<bold>O</bold>). (<bold>P</bold>–<bold>R</bold>) Immunostaining of <italic>UBQ10</italic> promoter-driven BIG4-YFP and SYP61 in untreated <italic>big3</italic> mutant seedling roots. BIG4 (<bold>P</bold>) co-localized with SYP61 (<bold>Q</bold>) at TGN (<bold>R</bold>). (<bold>S</bold> and <bold>T</bold>) YFP-SYP132 (<bold>S</bold>) and PIN1-RFP (<bold>T</bold>) expressed from the estradiol-inducible promoter localize at the plasma membrane in an unpolar or polar fashion, respectively. Scale bars, 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.008">http://dx.doi.org/10.7554/eLife.02131.008</ext-link></p></caption><graphic xlink:href="elife02131fs004"/></fig></fig-group></p></sec><sec id="s2-4"><title>Recruitment of clathrin adaptor complex AP-1 to the TGN requires BIG1 to BIG4 function</title><p>ARF-GEFs activate ARF GTPases, resulting in recruitment of vesicular coat proteins to the respective endomembrane compartment, such as COPI complex to Golgi stacks or adaptor protein (AP) complexes to post-Golgi compartments (<xref ref-type="bibr" rid="bib28">Robinson, 2004</xref>). Like BIG1-4, AP-1 complex subunit muB2-adaptin (AP1M2) localizes to SYP61-labeled TGN and is required for late secretory and vacuolar trafficking (<xref ref-type="bibr" rid="bib22">Park et al., 2013</xref>; <xref ref-type="bibr" rid="bib38">Teh et al., 2013</xref>; <xref ref-type="bibr" rid="bib41">Wang et al., 2013</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1P–R</xref>). AP1M2 also co-localized with TGN marker SYP61 in BFA compartments (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1J–L</xref>). In BFA-treated <italic>big3</italic> mutant, however, AP1M2 was cytosolic whereas SYP61 was still TGN-associated (<xref ref-type="fig" rid="fig2">Figure 2O,R</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1J–O</xref>). In contrast to AP1M2, Golgi association of COPI subunit γCOP, which is mediated by BFA-resistant ARF-GEF GNL1 (<xref ref-type="bibr" rid="bib26">Richter et al., 2007</xref>), was not affected in BFA-treated <italic>big3</italic> mutant (<xref ref-type="fig" rid="fig2">Figure 2O–T</xref>). Thus, BIG1-4 specifically mediate AP-1 recruitment to the TGN.</p></sec><sec id="s2-5"><title>Secretion and recycling to the plasma membrane are independently regulated trafficking pathways</title><p>Another ARF-GEF in post-Golgi traffic, GNOM regulates polar recycling of auxin-efflux carrier PIN1 to the basal plasma membrane (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>). BFA treatment of wild-type and <italic>big3</italic> mutant seedlings inhibited recycling of PIN1, which accumulated in BFA compartments, and this defect was suppressed by engineered BFA-resistant GNOM (<xref ref-type="fig" rid="fig3">Figure 3A–D</xref>). Thus, BIG1-4 did not play any obvious role in PIN1 recycling. PIN1 is a stable protein such that most protein detectable at the plasma membrane is delivered via the recycling but not the secretory pathway (<xref ref-type="bibr" rid="bib9">Geldner et al., 2001</xref>). In order to analyse the behavior of newly-synthesized PIN1 protein, we generated transgenic plants expressing estradiol-inducible PIN1. In contrast to recycling PIN1, newly-synthesized PIN1 protein was trapped in BFA compartments of <italic>big3</italic> mutant, regardless of BFA-resistant GNOM (<xref ref-type="fig" rid="fig3">Figure 3E–H</xref>). In conclusion, secretory ARF-GEFs BIG1-4 and recycling ARF-GEF GNOM regulate different post-Golgi trafficking pathways to the plasma membrane that function independently of each other.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02131.009</object-id><label>Figure 3.</label><caption><title>Secretion and recycling to the plasma membrane are regulated by different ARF-GEFs.</title><p>(<bold>A</bold>–<bold>D</bold>) PIN1 localization in interphase cells of BFA-treated seedlings; apolar at the plasma membrane (PM) and in BFA compartments in wild-type (Col; <bold>A</bold>) and <italic>big3</italic> mutants (<bold>B</bold>); at the basal PM in BFA-resistant GN in wild-type (GN<sup>R</sup>, <bold>C</bold>) or <italic>big3</italic> mutant background (GN<sup>R</sup> <italic>big3</italic>, <bold>D</bold>). Blue, DAPI-stained nuclei. (<bold>E</bold>–<bold>H</bold>) After BFA treatment, estradiol (Est)-induced PIN1-RFP was trafficked to the PM in wild-type (<bold>E</bold>) and BFA-resistant GN seedlings (GN<sup>R</sup>, <bold>G</bold>) but not in <italic>big3</italic> mutants without (<bold>F</bold>) or with expression of BFA-resistant GN (GN<sup>R</sup> <italic>big3</italic>; <bold>H</bold>). Scale bars, 5 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.009">http://dx.doi.org/10.7554/eLife.02131.009</ext-link></p></caption><graphic xlink:href="elife02131f003"/></fig></p><p>Gravitropic growth response of the seedling root relies on GNOM-mediated PIN1 recycling (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>). We tested whether BIG1-4 are also required, using <italic>DR5::NLS-3xGFP</italic> expression to visualise auxin response (<xref ref-type="bibr" rid="bib42">Weijers et al., 2006</xref>). BFA-induced inhibition of auxin response in wild-type and <italic>big3</italic> mutant was overcome by BFA-resistant GNOM, suggesting that BIG1-4 mediated secretion plays no role in gravitropic growth response (<xref ref-type="fig" rid="fig4">Figure 4A–D</xref>). GNOM-dependent PIN1 recycling is also required for lateral root initiation (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>). Surprisingly, BFA-resistant GNOM failed to initiate lateral root primordia in BFA-treated <italic>big3</italic> mutant in spite of stimulation by NAA, in contrast to seedlings that expressed both BIG3 and BFA-resistant GNOM (<xref ref-type="fig" rid="fig4">Figure 4E–L</xref>). <italic>big3</italic> mutants displayed binucleate cells, suggesting an essential role for secretory traffic in cytokinesis required for lateral root initiation (<xref ref-type="fig" rid="fig4">Figure 4M–T</xref>). For comparison, the BFA-induced defects in seed germination and primary root growth of <italic>big3</italic> were not rescued by engineered BFA-resistant GNOM, thus depending on secretory traffic rather than recycling (<xref ref-type="fig" rid="fig1">Figure 1C,E,H</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02131.010</object-id><label>Figure 4.</label><caption><title>BIG1-4 in response to auxin application.</title><p>(<bold>A</bold>–<bold>D</bold>) Visualization of auxin distribution by DR5::NLS-3xGFP (green) in BFA-treated seedlings after gravistimulation. Arrows, gravity vector. Cell walls were stained by propidium iodide (PI; magenta). Wild-type (<bold>A</bold>) and <italic>big3</italic> mutant seedling roots (<bold>B</bold>) did not respond to gravity (open asterisks), in contrast to BFA-resistant GN either in wild-type (GN<sup>R</sup>, <bold>C</bold>) or <italic>big3</italic> mutant background (GN<sup>R</sup> <italic>big3</italic>, <bold>D</bold>). Asterisks, auxin response in epidermal cell layer on lower side (<bold>C</bold> and <bold>D</bold>). (<bold>E–H</bold>) NAA and BFA treatment led to proliferation of pericycle cells (arrows) in wild-type (<bold>E</bold>) but not <italic>big3</italic> mutants without (<bold>F</bold>) or with BFA-resistant GN (<bold>H</bold>). Normal lateral root primordia only formed in BFA-resistant GN (GN<sup>R</sup>, <bold>G</bold>). Scale bars, 25 µm. (<bold>I</bold>–<bold>L</bold>) Bright-field microcopy of developing lateral root primordia in NAA-treated seedlings; genotypes: wild-type (Col; <bold>I</bold>), <italic>big3</italic> (<bold>J</bold>), BFA-resistant GN (GN<sup>R</sup>; <bold>K</bold>) and BFA-resistant GN in <italic>big3</italic> mutant background (GN<sup>R</sup> <italic>big3</italic>; <bold>L</bold>). (<bold>M</bold>–<bold>T</bold>) Live imaging of DR5::NLS-3xGFP of seedling roots after NAA and BFA treatment. DR5::NLS-3xGFP signals (left panels <bold>M</bold>, <bold>O</bold>, <bold>Q</bold>, <bold>S</bold>) overlaid with Nomarski images (right panels <bold>N</bold>, <bold>P</bold>, <bold>R</bold>, <bold>T</bold>). Pericycle cells proliferated in wild-type (<bold>M</bold> and <bold>N</bold>) but became binucleate (asterisks) in <italic>big3</italic> (<bold>O</bold> and <bold>P</bold>) and GN<sup>R</sup> <italic>big3</italic> (<bold>S</bold> and <bold>T</bold>) mutants. Normal lateral root primordia were only formed in BFA-resistant GN (GN<sup>R</sup>; <bold>Q</bold>, <bold>R</bold>) mutant. Scale bars, 25 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.010">http://dx.doi.org/10.7554/eLife.02131.010</ext-link></p></caption><graphic xlink:href="elife02131f004"/></fig></p></sec><sec id="s2-6"><title>Trafficking of both endocytosed and newly-synthesized proteins to the plane of cell division is regulated by secretory ARF-GEFs BIG1 to BIG4</title><p>In plant cytokinesis, which is assisted by a dynamic microtubule array named phragmoplast, both newly-synthesized and endocytosed proteins traffic to the plane of cell division on post-Golgi membrane vesicles that fuse with one another to form the partitioning cell plate (<xref ref-type="bibr" rid="bib29">Samuels et al., 1995</xref>). This raises the problem of coordinating different trafficking routes in the brief period of mitotic division (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). Cell-plate formation requires cytokinesis-specific syntaxin KNOLLE, newly synthesized during late G2/M phase (<xref ref-type="bibr" rid="bib13">Lauber et al., 1997</xref>; <xref ref-type="bibr" rid="bib24">Reichardt et al., 2007</xref>). In contrast to wild-type, KNOLLE targeting to the division plane was inhibited in BFA-treated <italic>big3</italic> mutants, with KNOLLE accumulating in BFA compartments together with BIG4-YFP (<xref ref-type="fig" rid="fig5">Figure 5A–F</xref>, <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A–D</xref>). Cell-plate formation was disrupted, resulting in binucleate cells, which sometimes displayed cell-wall stubs (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2A–C</xref>). We used the non-cycling plasma-membrane syntaxin SYP132 expressed from the strong mitosis-specific <italic>KN</italic> promoter as another secretory marker for trafficking to the cell–division plane (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). SYP132 also accumulated, together with KN, in BFA compartments of BFA-treated <italic>big3</italic> mutants, in contrast to BFA-treated wild-type (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1E–J</xref>). We also analysed endocytosed plasma-membrane proteins PEN1 and PIN1 for BFA-sensitive trafficking to the cell plate in <italic>big3</italic> mutants. PEN1 syntaxin involved in non-host immunity accumulates at the pathogen entry site by GNOM-dependent relocation following endocytosis from other regions of the plasma membrane (<xref ref-type="bibr" rid="bib3">Collins et al., 2003</xref>; <xref ref-type="bibr" rid="bib19">Nielsen et al., 2012</xref>). PEN1 continually cycles between plasma membrane and endosomes in interphase and accumulates at the cell plate in cytokinesis (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). To make sure that we were only looking at endocytosed PEN1, PEN1 was expressed from a histone H4 expression cassette that limits protein synthesis to S phase (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). In wild-type, BFA treatment inhibited PEN1 recycling to the plasma membrane but not its trafficking to the cell plate (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>; <xref ref-type="fig" rid="fig5">Figure 5G–I</xref>). In contrast, in BFA-treated <italic>big3</italic> mutants, endocytosed PEN1 was not trafficked to the cell division plane but accumulated, together with KNOLLE, in BFA compartments (<xref ref-type="fig" rid="fig5">Figure 5J–L</xref>, asterisks). Endocytosed PIN1 trafficked, like KNOLLE, to the cell plate in BFA-treated wild-type but both PIN1 and KNOLLE were trapped in BFA compartments of <italic>big3</italic> mutants (<xref ref-type="fig" rid="fig5">Figure 5M–R</xref>). Expression of engineered BFA-resistant GNOM did not overcome the trafficking block to the division plane but rather diverted PIN1 to the basal plasma membrane (<xref ref-type="fig" rid="fig5">Figure 5S–X</xref>; compare <xref ref-type="fig" rid="fig5">Figure 5X</xref> with <xref ref-type="fig" rid="fig5">Figure 5R</xref>). Careful analysis of mitotic cells revealed polar accumulation of PIN1 at the plasma membrane of BFA-resistant GNOM seedling roots throughout mitosis while additional PIN1 accumulates at the forming and expanding cell plate, suggesting that trafficking to the plane of division and polar recycling to the plasma membrane occur simultaneously (<xref ref-type="fig" rid="fig5s3">Figure 5—figure supplement 3</xref>). Thus, both endocytosed and newly-synthesized plasma-membrane proteins require secretory ARF-GEF function BIG1-4 for trafficking to the plane of cell division.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02131.011</object-id><label>Figure 5.</label><caption><title>Trafficking to the plane of cell division is mediated by BIG1 – BIG4.</title><p>(<bold>A</bold>–<bold>F</bold>) Immunolocalization of KNOLLE (KN; <bold>A</bold>, <bold>D</bold>) and tubulin (<bold>B</bold> and <bold>E</bold>) in cytokinetic root cells of BFA-treated seedlings (50 µM for 3 hr). (<bold>A</bold>–<bold>C</bold>) KN was located at the cell plate (<bold>A</bold>) flanked by tubulin-positive phragmoplast (<bold>B</bold>) in wild-type. (<bold>D</bold>–<bold>F</bold>) In <italic>big3</italic> mutants, KN accumulated in BFA compartments separated from tubulin-positive phragmoplast, resulting in a binucleate cell. (<bold>G</bold>–<bold>L</bold>) Co-localization of GFP-tagged KN and endocytosed RFP-PEN1 (H4::RFP-PEN1) in BFA-treated seedlings. KN and PEN1 co-localized at the cell plate and in BFA compartments of wild-type (<bold>G</bold>–<bold>I</bold>) but only in BFA compartments in <italic>big3</italic> mutants (<bold>J</bold>–<bold>L</bold>). (<bold>M</bold>–<bold>X</bold>) Immunostaining of GFP-KN and PIN1 in cytokinetic root cells of BFA-treated seedlings. (<bold>M</bold>–<bold>R</bold>) PIN1 localized apolarly at the plasma membrane (PM) and co-localized with KN in BFA compartments and at the cell plate in wild-type (<bold>M</bold>–<bold>O</bold>) but only in BFA-compartments in <italic>big3</italic> mutants (<bold>P</bold>–<bold>R</bold>). (<bold>S</bold>–<bold>U</bold>) In GN<sup>R</sup>, PIN1 localized polarly at the plasma membrane (<bold>T</bold>) and co-localized with KN (<bold>S</bold>) at the cell plate (<bold>U</bold>). (<bold>V</bold>–<bold>X</bold>) Although PIN1 localized polarly at the PM (<bold>W</bold>) in GN<sup>R</sup> <italic>big3</italic>, neither PIN1 (<bold>W</bold>) nor KN (<bold>V</bold>) was located at the cell plate. Blue, DAPI-stained nuclei. Asterisks label nuclei of binucleate cells (<bold>F</bold>, <bold>L</bold>, <bold>R</bold>, <bold>X</bold>). Scale bars, 5 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.011">http://dx.doi.org/10.7554/eLife.02131.011</ext-link></p></caption><graphic xlink:href="elife02131f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.012</object-id><label>Figure 5—figure supplement 1.</label><caption><title>BIG4 and cargo proteins trapped in BFA compartments of dividing cells in BFA-treated <italic>big3</italic> mutant seedlings.</title><p>(<bold>A</bold>–<bold>C</bold>) Immunostaining of <italic>UBQ10</italic> promoter-driven BIG4-YFP and KN in BFA-treated <italic>big3</italic> mutant seedlings. BFA-sensitive YFP-tagged BIG4 (<bold>A</bold>) co-localized with its cargo KN (<bold>B</bold>) in BFA aggregates. (<bold>C</bold>) Overlay. (<bold>D</bold>) Intensity profile of line numbered in (<bold>C</bold>) indicated overlapping signals. (<bold>E</bold>–<bold>J</bold>) Immunostaining of Myc-tagged SYP132 expressed from the <italic>KN</italic> promoter (KN::Myc-SYP132; <bold>E</bold>, <bold>H</bold>) and KN (<bold>F</bold> and <bold>I</bold>) in BFA-treated cytokinetic cells of wild-type and <italic>big3</italic> mutant seedlings. (<bold>E</bold>–<bold>G</bold>) SYP132 localized at the plasma membrane and KN-labeled cell plate in wild-type whereas both SYP132 and KN were trapped in BFA compartments in <italic>big3</italic> mutant (<bold>H</bold>–<bold>J</bold>). Asterisks, binucleate cells (<bold>C</bold> and <bold>J</bold>). Scale bars, 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.012">http://dx.doi.org/10.7554/eLife.02131.012</ext-link></p></caption><graphic xlink:href="elife02131fs005"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.013</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Ultrastructural appearance of cryofixed, freeze-substituted and resin-embedded <italic>big3</italic> seedling root tips treated with BFA.</title><p>(<bold>A</bold>–<bold>C</bold>) Binucleate cells in <italic>big3</italic> seedling roots treated with BFA. (<bold>A</bold>) Overview of ultrastructural TEM analysis. (<bold>B</bold>) Higher magnification of boxed area in (<bold>A</bold>). Note absence of cell-wall remnants or membrane vesicles between the daughter nuclei (<bold>B</bold>). (<bold>C</bold>) Another cell showing cell wall remnants (stubs, blue asterisks). Nuclei (n) have been false-colored; second nucleus in (<bold>C</bold>) is in a different focal plane. cw, cell wall; er, endoplasmic reticulum; m, mitochondrion; n, nucleus; v, vacuole. Scale bars, 2.5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.013">http://dx.doi.org/10.7554/eLife.02131.013</ext-link></p></caption><graphic xlink:href="elife02131fs006"/></fig><fig id="fig5s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.014</object-id><label>Figure 5—figure supplement 3.</label><caption><title>PIN1 recycling in mitotic cells.</title><p>BFA-treated seedlings expressing PIN1-GFP (green) were counterstained with tubulin (magenta) and DAPI (blue chromatin). (<bold>A</bold>–<bold>I</bold>) In wild-type, PIN1-GFP localizes apolarly at the plasma membrane and at the cell plate in different stages during cytokinesis (BFA 50 μM 3h). (<bold>J</bold>–<bold>U</bold>) In BFA-resistant GNOM lines (GN<sup>R</sup>), PIN1-GFP localizes polarly at the plasma membrane and at the cell plate at the same time (BFA 50 μM 3h). (<bold>V</bold>–<bold>X</bold>) Even after prolonged BFA-treatment (6h) of BFA-resistant GNOM seedlings, PIN1 polarity and cell-plate localization are maintained. Mitotic stages: arrowhead, metaphase (panels <bold>F</bold> and <bold>O</bold>); asterisk, anaphase (panels <bold>C</bold>, <bold>L</bold>, <bold>X</bold>); circle, telophase (panels <bold>C</bold>, <bold>I</bold>, <bold>R</bold>, <bold>U</bold>, <bold>X</bold>); cross, late cytokinesis (panels <bold>F</bold>, <bold>I</bold>, <bold>O</bold>, <bold>R</bold>). Scale bar, 5 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.014">http://dx.doi.org/10.7554/eLife.02131.014</ext-link></p></caption><graphic xlink:href="elife02131fs007"/></fig><fig id="fig5s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02131.015</object-id><label>Figure 5—figure supplement 4.</label><caption><title>Highly schematic model of secretory and recycling trafficking pathways in interphase and cytokinesis.</title><p>In interphase, proteins are synthesized at the ER and are transported via the Golgi to the TGN. The TGN serves as a sorting station. From there, cargo can be transported to the plasma membrane (PM; secretion, green) and this pathway requires the ARF-GEFs BIG1-BIG4. When plasma membrane localized proteins are endocytosed (endocytic pathway, red), they can return to the plasma membrane via the GNOM-dependent recycling pathway (orange). During cytokinesis, newly synthesized proteins are transported from the ER to the Golgi/TGN and from there to the cell plate (CP) in BIG1-BIG4 dependent fashion. Not only newly synthesized cargo but also endocytosed proteins follow this secretory route to the cell plate. PIN1 appears to be exceptional among endocytosed proteins, being recycled to the basal plasma membrane in a GNOM-dependent manner during cytokinesis (?).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.015">http://dx.doi.org/10.7554/eLife.02131.015</ext-link></p></caption><graphic xlink:href="elife02131fs008"/></fig></fig-group></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>It is a particularity of Arabidopsis and some other flowering-plant species that the secretory pathway of membrane traffic is comparatively insensitive to BFA treatment whereas endosomal recycling of endocytosed plasma-membrane proteins is rather sensitive (<xref ref-type="bibr" rid="bib9">Geldner et al., 2001</xref>, <xref ref-type="bibr" rid="bib7">2003</xref>; <xref ref-type="bibr" rid="bib37a">Teh and Moore, 2007</xref>; <xref ref-type="bibr" rid="bib26">Richter et al., 2007</xref>). The BFA insensitivity of the secretory pathway depends on the BFA resistance of ARF-GEF GNL1, which mediates COPI-vesicle formation in retrograde Golgi-ER traffic (<xref ref-type="bibr" rid="bib37a">Teh and Moore, 2007</xref>; <xref ref-type="bibr" rid="bib26">Richter et al., 2007</xref>), and also requires another BFA-resistant ARF-GEF acting in post-Golgi traffic to the plasma membrane. Here we show that ARF-GEFs BIG1-4 act at the TGN to mediate secretion of newly synthesized proteins to the plasma membrane in interphase but not recycling of endocytosed plasma-membrane proteins, and that BIG3 is BFA-resistant, unlike GNOM involved in recycling to the plasma membrane. Thus, there are two distinct trafficking pathways from the TGN to the plasma membrane in interphase. This is best illustrated by the trafficking of auxin-efflux carrier PIN1 - whereas newly synthesized PIN1 requires BIG1-4 on the late secretory pathway for non-polar delivery to the plasma membrane, polar PIN1 recycling to the basal plasma membrane solely depends on ARF-GEF GNOM (see model in <xref ref-type="fig" rid="fig5s4">Figure 5—figure supplement 4</xref>).</p><p>Like newly synthesized proteins, endocytosed proteins are targeted to the division plane during cytokinesis (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). Proteins that cycle between endosomes and the plasma membrane in interphase accumulate, preferentially or even exclusively, at the cell plate (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>). In general, recycling to the plasma membrane appears to be switched off during cytokinesis. Here we show that secretory ARF-GEFs BIG1-4 are essential for protein trafficking to the plane of cell division, regardless of proteins being newly synthesized or endocytosed from the plasma membrane (see model in <xref ref-type="fig" rid="fig5s4">Figure 5—figure supplement 4</xref>).</p><p>Although trafficking to the plane of cell division appears to override recycling of endocytosed proteins to the plasma membrane, we noticed one clear exception—auxin-efflux carrier PIN1, which accumulates polarly at the plasma membrane in interphase and during cell division when both BFA-resistant BIG3 and engineered BFA-resistant GNOM were expressed. Rather than substituting for BIG1-4 in traffic to the plane of cell division, recycling ARF-GEF GNOM appeared to counteract that process by promoting PIN1 recycling to the basal plasma membrane. Of course, the critical question is whether both processes occur at the same time or whether GNOM-dependent PIN1 recycling only sets in after trafficking to the cell plate has come to an end. Although there are no time-course studies, which would be difficult to perform because the process is very fast, detailed analysis of dividing cells at different mitotic stages revealed that polar recycling mediated by BFA-resistant GNOM occurs throughout mitosis and cytokinesis. Furthermore, only in the absence of both BFA-resistant BIG3 and BFA-resistant GNOM is PIN1 trapped in BFA compartments. If then BFA-resistant GNOM is expressed PIN1 is not delivered to the plane of division but rather polarly recycled to the plasma membrane, again suggesting that the latter pathway is a direct route bypassing the cell plate. PIN1 might be exceptional because continuous recycling of PIN1 is required for maintaining the polar transport of auxin across tissues (<xref ref-type="bibr" rid="bib7">Geldner et al., 2003</xref>). If PIN1 recycling were shut down during cytokinesis this would disrupt the polar auxin transport required in specific developmental situations such as forming lateral root primordia when essentially all cells proliferate (<xref ref-type="bibr" rid="bib10">Geldner et al., 2004</xref>). Another problem in auxin flow arises from cell division when the partitioning membrane has physically separated the two daughter cells: one daughter suddenly has PIN1 located at opposite ends. Obviously, PIN1 has to be removed from the wrong end in order to sustain polar auxin transport. This seems to be a fast process and has been studied for the related auxin-efflux carrier PIN2 in detail (<xref ref-type="bibr" rid="bib14">Men et al., 2008</xref>).</p><p>Animal and plant cytokinesis differ in the way the partitioning membrane is laid down. In animals, secretory and recycling pathways contribute to the ingrowth of the plasma membrane mediated by a contractile actomyosin ring and to the subsequent abscission of the daughter cells (<xref ref-type="bibr" rid="bib31">Schiel and Prekeris, 2013</xref>). In plants, a massive flow of membrane vesicles from TGN/early endosome to the plane of cell division sustains, by fusion, the rapid formation and outward expansion of the partitioning cell plate (<xref ref-type="bibr" rid="bib29">Samuels et al., 1995</xref>). This process is orchestrated by a specialised cytoskeletal array termed phragmoplast that delivers those membrane vesicles to the division plane. Phragmoplast-assisted trafficking might be required for completing the partitioning membrane on time, in the absence of a cytokinesis-interphase checkpoint, and would thus effectively rule out recycling of endocytosed proteins to the plasma membrane. However, our results make clear that this is not the case because recycling to the plasma membrane is not switched off during cytokinesis. Rather, endocytosed proteins enter the late-secretory pathway to reach the division plane at the expense of being recycled to the plasma membrane, which requires the late-secretory ARF-GEFs BIG1-4. In conclusion, our results raise the possibility that in general, different ARF-GEFs have different specificity of action during vesicle formation such that the same cargo protein can be delivered to different destinations.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Plant material and growth conditions</title><p>Plants were grown on soil or agar plates in growth chambers under continuous light conditions at 23°C. <italic>big</italic> mutant lines: <italic>big1</italic> (GK-452B06) and <italic>big2</italic> (GK-074F08) T-DNA lines were from GABI-KAT (<ext-link ext-link-type="uri" xlink:href="http://www.gabi-kat.de">http://www.gabi-kat.de</ext-link>), <italic>big3</italic> (SALK_044617) and <italic>big4</italic> (SALK_069870) T-DNA lines from the SALK collection (<ext-link ext-link-type="uri" xlink:href="http://signal.salk.edu/cgi-bin/tdnaexpress">http://signal.salk.edu/cgi-bin/tdnaexpress</ext-link>). <italic>big3</italic> mutant lines were selected on MS plates using kanamycin.</p><p>The following transgenic marker lines were used: H4::RFP-PEN1 (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>) (expressed from <italic>HISTONE4</italic> (<italic>H4</italic>) promoter during S phase), KN::Myc-SYP132 (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>) (expressed during lateG2/M phase), HS::secGFP (<xref ref-type="bibr" rid="bib40">Viotti et al., 2010</xref>) (expressed from heat shock promoter), GFP-KN (<xref ref-type="bibr" rid="bib24">Reichardt et al., 2007</xref>), BOR1-GFP (<xref ref-type="bibr" rid="bib37">Takano et al., 2005</xref>), DR5::NLS-3xGFP (<xref ref-type="bibr" rid="bib42">Weijers et al., 2006</xref>), VHA-a1-RFP (<xref ref-type="bibr" rid="bib40">Viotti et al., 2010</xref>), AP1M2-3xHA (<xref ref-type="bibr" rid="bib22">Park et al., 2013</xref>).</p></sec><sec id="s4-2"><title>T-DNA genotyping of <italic>big</italic> mutant lines</title><p>Primers used to test for <italic>big1</italic> heterozygosity:</p><p>5′GCAAGATCAGGGAAGACG 3′ and 5′ACCAGAGGAAGGTGCTTCTTC 3′</p><p>Primers used to test for <italic>big1</italic> homozygosity:</p><p>5′TCGTCCCATCTTCTTCATTTG 3′ and 5′ACCAGAGGAAGGTGCTTCTTC 3</p><p>Primers used to test for <italic>big2</italic> heterozygosity:</p><p>5′GCAAGATCAGGGAAGACG 3′ and 5′TTGAGGGGTTCATATGACAGC 3′</p><p>Primers used to test for <italic>big2</italic> homozygosity:</p><p>5′TTTCCCACTTTTTCCACTGTG 3′ and 5′TTGAGGGGTTCATATGACAGC 3′</p><p>Primers used to test for <italic>big3</italic> heterozygosity:</p><p>5′AAACTCTCCACTGGCTAAGCC 3′ and 5′ATTTTGCCGATTTCGGAAC 3′</p><p>Primers used to test for <italic>big3</italic> homozygosity:</p><p>5′AAACTCTCCACTGGCTAAGCC 3′ and 5′GCAAGTTTTCTTGCGCAATAC 3′</p><p>Primers used to test for <italic>big4</italic> heterozygosity:</p><p>5′ATTTTGCCGATTTCGGAAC 3′ and 5′CTATCTTGCGCTGGAGACAAC 3′</p><p>Primers used to test for <italic>big4</italic> homozygosity:</p><p>5′TCCTCTTCAAACTCGTCAACG 3′ and 5′CTATCTTGCGCTGGAGACAAC 3′</p></sec><sec id="s4-3"><title>Generating transgenic plants</title><p>Genomic BIG4 was amplified and introduced into pDONR221 (Invitrogen, Darmstadt, Germany) and afterwards into <italic>UBQ10::YFP</italic> destination vector (<xref ref-type="bibr" rid="bib11">Grefen et al., 2010</xref>). For generation of BFA-resistant <italic>UBQ10::BIG4</italic><sup><italic>R</italic></sup><italic>-YFP</italic>, methionine at position 695 was exchanged with leucine by site-directed mutagenesis. <italic>BIG3</italic> promoter was amplified and introduced into pUC57L4 via <italic>Kpn</italic>I and <italic>Sma</italic>I restriction sites. Multistep gateway cloning was performed using pUC57L4-<italic>BIG3</italic>-promoter, pEntry221-<italic>BIG4</italic> and R4pGWB553 (<xref ref-type="bibr" rid="bib17">Nakagawa et al., 2008</xref>) yielding <italic>BIG3::BIG4-RFP</italic>. Cloning the CDS from <italic>BIG3</italic> into pGREENII via A<italic>paI</italic> and <italic>Sma</italic>I restriction sites generated pGII-<italic>BIG3</italic>. The 1 kb <italic>BIG3</italic> promoter was amplified and introduced into pGII-<italic>BIG3</italic> via <italic>Apa</italic>I. 1 kb of 3′UTR was amplified and introduced into pGII-<italic>BIG3::BIG3</italic> via <italic>Sma</italic>I and <italic>Spe</italic>I. C-terminal YFP was inserted via <italic>Sma</italic>I and <italic>Spe</italic>I. <italic>AFVY-RFP</italic> was amplified from <italic>35S::AFVY-RFP</italic> (<xref ref-type="bibr" rid="bib30">Scheuring et al., 2011</xref>) and introduced into pDONR221 (Invitrogen) generating a pEntry clone. Afterwards, LR reaction was performed introducing <italic>AFVY-RFP</italic> into the estradiol-inducible destination vector pMDC7 (<xref ref-type="bibr" rid="bib5">Curtis and Grossniklaus, 2003</xref>). <italic>PIN1</italic> cDNA was cloned into pGem-T (Promega, Mannheim, Germany). <italic>RFP</italic> was inserted in <italic>PIN1</italic> via the <italic>Xho</italic>I site. <italic>PIN1-RFP</italic> was amplified and introduced first into pDONR221 and then into pMDC7. <italic>YFP-SYP132</italic> was amplified and introduced into pDONR221 and then into pMDC7.</p><p>All constructs were transformed into <italic>big3</italic> mutants and BFA-resistant GN (GN<sup>R</sup>) in <italic>big3</italic> mutant background. T1 plants of <italic>UBQ10::BIG4-YFP</italic>, <italic>UBQ10::BIG4</italic><sup><italic>R</italic></sup><italic>-YFP</italic> and <italic>BIG3-YFP</italic> were selected by spraying with Basta. T1 seeds of estradiol-inducible lines and <italic>BIG3::BIG4-RFP</italic> were selected with hygromycin. Experiments were performed using T2 or T3 seedlings. At least three independent lines were analysed.</p></sec><sec id="s4-4"><title>Immunofluorescence localization and live imaging in seedling roots</title><p>5 days old seedlings were incubated in 1 ml liquid growth medium (0.5x MS medium, 1% sucrose, pH 5.8) containing 50 µM BFA (Invitrogen, Molecular Probes) for 1 hr or 3 hr at room temperature in 24-well cell-culture plates. Seedlings treated with 50 µM BFA for (a) 1 hr or (b) 3 hr, respectively, were used for the following immunolocalisation studies: (a) AP1M2 vs γCOP, AP1M2 vs SYP61, PIN1; (b) KNOLLE vs Tubulin, KNOLLE vs PIN1, H4::RFP-PEN1 vs GFP-KN and KN::Myc-SYP132 vs KN. Incubation was stopped by fixation with 4% paraformaldehyde in MTSB. Immunofluorescence staining was performed as described (<xref ref-type="bibr" rid="bib13">Lauber et al., 1997</xref>) or with an InsituPro machine (Intavis, Cologne, Germany) (<xref ref-type="bibr" rid="bib16">Müller et al., 1998</xref>).</p><p>Antibodies used: mouse anti-MYC (Santa Cruz Biotechnology, Heidelberg, Germany) 1:600, mouse anti-HA 1:1000 (BAbCO, Richmond, CA, USA), rat anti-tubulin 1:600 (Abcam, Cambridge, UK), rabbit anti-PIN1 1:1000 (<xref ref-type="bibr" rid="bib9">Geldner et al., 2001</xref>), rabbit anti-γCOP 1:1000 (Agrisera, Vännäs, Sweden), rabbit anti-KNOLLE 1:2000 (<xref ref-type="bibr" rid="bib24">Reichardt et al., 2007</xref>) and rabbit anti-SYP61 1:700 (<xref ref-type="bibr" rid="bib22">Park et al., 2013</xref>). Alexa-488 or Cy3-conjugated secondary antibodies (Dianova, Hamburg, Germany) were diluted 1:600.</p><p>Live-cell imaging was performed with 2 µM FM4-64 or FM1-43 (Invitrogen, Molecular Probes) or propidium iodide (10 µg/ml).</p><p>Estradiol induction was performed using 10 or 20 µM estradiol. BFA incubation (25 µM) was done together with estradiol for 6 hr.</p><p>Heat-shock inducible secGFP (HS::secGFP) lines were first incubated for 30 min at 37°C in MS at pH8.1. BFA treatment (50 µM) in MS at pH8.1 followed for 4 hr at plant room conditions.</p><p>Analysis of BOR1 degradation was performed according to <xref ref-type="bibr" rid="bib37">Takano et al. (2005)</xref> . In addition, we treated the seedlings with BFA, 5 µM, for 1 hr together with boron.</p></sec><sec id="s4-5"><title>Electron microscopy</title><p>For ultrastructural analysis, root tips were high-pressure frozen (Bal-Tec HPM010; Balzers) in hexadecene (Merck Sharp and Dohme, Haar, Germany), freeze-substituted in acetone containing 2.5% osmium tetroxide, washed at 0°C with acetone, and embedded in Epon. For immunogold labeling of ultrathin thawed cryosections, root tips were fixed with 8% formaldehyde (2 hr), embedded in gelatin, and infiltrated with 2.1 M sucrose in PBS as previously described (<xref ref-type="bibr" rid="bib6">Dettmer et al., 2006</xref>). Thawed ultrathin sections were labeled with rabbit anti-GFP antibodies (1:300; Abcam) and silver-enhanced (HQ Silver, 8 min; Nanoprobes, Yaphank, NY, USA) goat anti-rabbit IgG coupled to Nanogold (no. 2004; Nanoprobes). Antibodies and markers were diluted in blocking buffer (PBS supplemented with 0.5% BSA and 1% milk powder).</p></sec><sec id="s4-6"><title>Acquisition and processing of fluorescence images</title><p>Fluorescence images were acquired at 512 × 512 or 512 × 256 pixels with the confocal laser scanning microscope TCS-SP2 or TCS-SP8 from Leica, using the 63x water-immersion objective and Leica software. All images were processed with Adobe Photoshop CS3 only for adjustment of contrast and brightness. Intensity line profile was performed with Leica software.</p></sec><sec id="s4-7"><title>Pollen germination</title><p>Pollen medium was prepared as described (<xref ref-type="bibr" rid="bib1">Boavida and McCormick, 2007</xref>). Pollen germinated over night or for 5 hr before microscopic analysis.</p></sec><sec id="s4-8"><title>Physiological tests</title><p>To investigate primary root growth, 5–6 days old seedlings were transferred to plates with 10 µM BFA and analysed after 5–7 additional days using ImageJ. DR5::NLS-GFP expressing seedlings analysed for lateral root formation were treated with 5 µM NAA or 5 µM NAA plus 10 µM BFA over night. Roots were cleared according to <xref ref-type="bibr" rid="bib10">Geldner et al. (2004)</xref>. Gravitropic response was investigated by transferring 5 days old seedlings, expressing DR5::NLS-GFP, to BFA plates (5 µM). Seedlings were grown vertically for 1 hr on BFA plates before rotated by 135° for 4 hr.</p><p>For analysis of seed germination, seeds were sown out on MS medium containing 5 µM BFA. Images were taken after 5 days of growth.</p></sec><sec id="s4-9"><title>Phylogenetic tree</title><p>Full-length protein sequence of BIG3 was used to search for related sequences from different plant species with sequenced genomes that are available at the phytozome homepage (<ext-link ext-link-type="uri" xlink:href="http://www.phytozome.net/">http://www.phytozome.net/</ext-link>). ARF-GEFs from different species were aligned by ClustalW (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/clustalw">www.ebi.ac.uk/clustalw</ext-link>) and the phylogenetic tree was drawn with Dendroscope (<xref ref-type="bibr" rid="bib12">Huson et al., 2007</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Lukas Sonnenberg and Marlene Ballbach for technical assistance, Toru Fujiwara, Niko Geldner, Christopher Grefen, Ueli Grossniklaus, Sumie Ishiguru, Peter Pimpl, Masao H. Sato and Karin Schumacher for sharing published materials, Joop Vermeer (Univ. Lausanne) for cloning vector pUC57L4, and NASC for T-DNA insertion lines. We also thank Martin Bayer, Niko Geldner, Christopher Grefen, Michael Hothorn and Steffen Lau for critical reading of the manuscript.</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>SR, 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>MK, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>MEN, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>MP, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>RG, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>CK, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>UV, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>HB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>UM, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con10"><p>Y-DS, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con11"><p>SB, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con12"><p>GJ, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.02131.016</object-id><label>Supplementary file 1.</label><caption><title>Localization of vesicle trafficking markers.</title><p>This table summarizes the localization of different vesicle trafficking markers without BFA (1<sup>th</sup> column) and with BFA in wild-type (Col; 2<sup>th</sup> column), <italic>big3</italic> (3<sup>th</sup> column), BFA-resistant GNOM (GN<sup>R</sup>; 4<sup>th</sup> column) and BFA-resistant GNOM in <italic>big3</italic> mutant background (GN<sup>R</sup> <italic>big3</italic>; 5th column). Abbreviations: PM, plasma membrane; CP, cell plate; BFA-comp., BFA-compartment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02131.016">http://dx.doi.org/10.7554/eLife.02131.016</ext-link></p></caption><media mime-subtype="pdf" mimetype="application" xlink:href="elife02131s001.pdf"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boavida</surname><given-names>LC</given-names></name><name><surname>McCormick</surname><given-names>S</given-names></name></person-group><year>2007</year><article-title>Temperature as a determinant factor for increased and reproducible in vitro pollen germination in <italic>Arabidopsis thaliana</italic></article-title><source>Plant Journal</source><volume>52</volume><fpage>570</fpage><lpage>582</lpage><pub-id 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Hardtke</surname><given-names>Christian S</given-names></name><role>Reviewing editor</role><aff><institution>University of Lausanne</institution>, <country>Switzerland</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://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 “Recycling-to-secretory ARF-GEF switch mediating protein traffic to the cell division plane” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor, a Reviewing editor, and 3 reviewers.</p><p>The Reviewing editor and the reviewers discussed their comments before reaching this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>All three reviewers agree that your paper addresses important unresolved issues and that both experiments and data presentation are of exceptional quality. There is however some discussion regarding your conclusion that “endocytosed proteins enter the late secretory pathway to reach the division plane at the expense of being recycled to the plasma membrane, which is regulated by the switch to the late secretory ARF-GEFS BIG1-4” that we would like you to address. The reviewers consider this is a significant claim because the identity of the cell-plate pathway and its relationship to pathways that operate in interphase has been a longstanding unresolved controversy. However, they fail to see how these specific conclusions can be drawn from the present data. We would like to ask you to consider the following reviewer comments in detail:</p><p>1) Although the data show that traffic of all cargoes to the cell plate requires BIG1-4, I see no evidence that BIG1-4 REGULATE a switch of cargoes from one pathway to another.</p><p>2) Second, an important piece of evidence is that in BFA-treated <italic>big3</italic> GN<sup>R</sup> plants PIN1 proteins recycle to their normal polar position at the basal PM during cytokinesis (<xref ref-type="fig" rid="fig5">Figure 5V-X</xref>). This implies that recycling continues during cytokinesis so traffic to the cell plate requires sorting of PIN1 away from that pathway into the BIG1-4 pathway. I think the authors are using the presence of the cytokinesis-specific syntaxin KNOLLE to identify cells undergoing cytokinesis. Normally this is reasonable because KNOLLE is so rapidly degraded in the vacuole after cytokinesis but I am concerned here that in BFA-treated <italic>big3</italic> plants KNOLLE may be artificially stabilised as trafficking to the vacuole is inhibited under these conditions (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Indeed the extensive labelling of whole files of cells is unusual for a marker of cytokinesis suggesting that there may be temporal stability. Treatment seems to have been for 3 h so the majority of cells that contain KNOLLE may have gone through (failed) cytokinesis some time ago. Isn't it possible that recycling to the plasma membrane does not in fact occur during cytokinesis, that endocytosed PIN1 accumulated in BFA-compartments during cytokinesis, but was recycled back to the PM in the subsequent G1. If so the important change could be the inhibition of the GN-pathway during cytokinesis causing endocytosed proteins to enter the default secretory pathway at the TGN, rather than any regulatory role for BIGs.</p><p>3) Even if recycling to the PM does occur during cytokinesis, I do not think the data shows compellingly that there is indeed such a switch (during cell plate assembly) from the GN-dependent recycling pathway to the BIG1-4-dependent secretory pathway.</p><p>a) Although the data show that GNOM is sufficient to recycle a majority of endocytosed protein, can it be excluded that a fraction of endocytosed protein that enters the TGN in interphase is returned by the BIG1-4 secretory pathway? If so, it could be this fraction that arrives in the cell plate during cytokinesis. This might explain why endocytosed PIN1 in BFA-treated wild-type appears to accumulate in BFA-compartments (cell plate associated and detached) as well as the cell plate.</p><p>b) Although some endocytosed protein arrives in the cell plate, is it clear that all or most endocytosed protein goes there as proposed?</p><p>4) To resolve question 2 convincingly I think the authors will need at least to show time-lapse data on cells of relevant genotypes undergoing cytokinesis with and without BFA treatments. There probably needs to be an independent marker of cell cycle stage such as tubulin-GFP, which would also provide evidence that phragmoplasts are normal in the BFA-treated cells. I also think that it would be important to ask whether newly-synthesised PIN1 is trapped in interphase BFA compartments in <italic>big3</italic> and <italic>big3</italic> GN<sup>R</sup>. To resolve the second question, perhaps photoswitchable fluorescent proteins which are available may also be suitable though I realise that they are not easy to image. Protein could be photoconverted in the BFA compartment of BFA-treated Col and followed over time to look at its rate of return to the PM versus the cell plate when cells enter cytokinesis – I don't recall this specific experiment being been done before.</p><p>5) From our perspective, the time-lapse (or equivalent) data would be necessary to establish the crucial point about recycling during cytokinesis. We imagine you should be in a position to do this without much delay and to establish the stability of KN in <italic>big3</italic>+BFA - one reviewer suggests that the conditionality of the BFA-induced phenotype is a perfect system for such experiments. The photoswitch experiment or similar is much more demanding but without it there is ambiguity about whether anything really changes with respect to PIN1 trafficking during cytokinesis, a major claim of the paper. If this point remains unclarified, we think the Discussion and title will need to change to reflect this.</p><p>6) The reviewers also question the coining of BIG1-4 as “regulatory switches”. It seems that the paper does not demonstrate a direct molecular mechanism in which BIG1-4 display a switch-like function, rather you seem to provide genetic evidence that BIG1-4 are necessary for the observed changes in trafficking. At this point, alternative hypotheses, e.g., that GN or something else on the GN pathway is somehow inactivated during cytokinesis, also appear plausible. We understand that this is partly a semantic issue, but we still would like to ask you to re-word appropriately.</p><p>We hope you will be able to address the above issue, but let us clarify that the remaining points that cell plate assembly requires BIG1-4 and so is derived from a biosynthetic pathway at the TGN, and that there are two distinct classes of TGN with distinct ARF-GEFs that keep biosynthetic and recycling cargo separate, are valuable contributions. For these alone, your paper merits publication, but it would have to be revised accordingly in the Discussion and title.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02131.018</article-id><title-group><article-title>Author Response</article-title></title-group></front-stub><body><p><italic>1) Although the data show that traffic of all cargoes to the cell plate requires BIG1-4, I see no evidence that BIG1-4 REGULATE a switch of cargoes from one pathway to another</italic>.</p><p>This is partly a semantic issue but we understand the concern and have toned down the statement, avoiding “regulation” and “switch”. In our view, nonetheless, BIG1-4 are absolutely required for trafficking to the cell plate. They cannot be substituted by e.g., GNOM, which rather mediates recycling to the plasma membrane (see also reply to the next comment). Thus, there is a specific trafficking pathway to the cell plate mediated by secretory ARF-GEFs and taken by both newly synthesised and endocytosed cargo proteins.</p><p><italic>2) Second, an important piece of evidence is that in BFA-treated</italic> big3 <italic>GN</italic><sup><italic>R</italic></sup> <italic>plants PIN1 proteins recycle to their normal polar position at the basal PM during cytokinesis (</italic><xref ref-type="fig" rid="fig5"><italic>Figure 5, V-X</italic></xref><italic>). This implies that recycling continues during cytokinesis so traffic to the cell plate requires sorting of PIN1 away from that pathway into the BIG1-4 pathway. I think the authors are using the presence of the cytokinesis-specific syntaxin KNOLLE to identify cells undergoing cytokinesis. Normally this is reasonable because KNOLLE is so rapidly degraded in the vacuole after cytokinesis but I am concerned here that in BFA-treated</italic> big3 <italic>plants KNOLLE may be artificially stabilised as trafficking to the vacuole is inhibited under these conditions (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref><italic>). Indeed the extensive labelling of whole files of cells is unusual for a marker of cytokinesis suggesting that there may be temporal stability. Treatment seems to have been for 3 h so the majority of cells that contain KNOLLE may have gone through (failed) cytokinesis some time ago</italic>.</p><p>We agree that KNOLLE might not be a reliable marker in this situation. The experiment has been repeated, using anti-tubulin immunostaining of mitotic microtubule arrays and DAPI staining of nuclear chromatin as reliable markers for mitotic staging of dividing cells. The new results reveal polar localisation of PIN1 at the plasma membrane in dividing cells of GN-BFAr at successive stages of mitosis (metaphase to telophase) and cytokinesis (cell plate being formed, laterally expanded or nearly complete), this being accompanied by PIN1 accumulation at the cell plate during cytokinesis (see new <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>). The control images (wild-type, i.e., GNOM inactivated by BFA) also show PIN1 accumulation at the cell plate but no polar accumulation at the basal plasma membrane. These data suggest competitive traffic to both cell plate and basal plasma membrane during cytokinesis (see last paragraph of the Results, and Discussion).</p><p><italic>Isn't it possible that recycling to the plasma membrane does not in fact occur during cytokinesis, that endocytosed PIN1 accumulated in BFA-compartments during cytokinesis, but was recycled back to the PM in the subsequent G1. If so the important change could be the inhibition of the GN-pathway during cytokinesis causing endocytosed proteins to enter the default secretory pathway at the TGN, rather than any regulatory role for BIGs</italic>.</p><p>See reply to preceding comment.</p><p><italic>3) Even if recycling to the PM does occur during cytokinesis, I do not think the data shows compellingly that there is indeed such a switch (during cell plate assembly) from the GN-dependent recycling pathway to the BIG1-4-dependent secretory pathway</italic>.</p><p>We agree that the underlying mechanism is unknown. We have replaced “switch” by “change” from recycling to secretory trafficking pathway.</p><p><italic>a) Although the data show that GNOM is sufficient to recycle a majority of endocytosed protein, can it be excluded that a fraction of endocytosed protein that enters the TGN in interphase is returned by the BIG1-4 secretory</italic> <italic>pathway</italic>?</p><p>This is a rather theoretical possibility. It has been documented many times since the first analysis published in <xref ref-type="bibr" rid="bib7">Geldner et al. (2003)</xref> that GNOM is sufficient for PIN1 recycling and here we show that BIG3 does not make any difference.</p><p><italic>If so, it could be this fraction that arrives in the cell plate during cytokinesis. This might explain why endocytosed PIN1 in BFA-treated wild-type appears to accumulate in BFA-compartments (cell plate associated and detached) as well as the cell plate</italic>.</p><p>When BIG3 is deleted and GNOM rendered BFA-resistant PIN1 does not reach the cell plate but is properly recycled to the basal plasma membrane (see also reply to preceding comment).</p><p><italic>b) Although some endocytosed protein arrives in the cell plate, is it clear that all or most endocytosed protein goes there</italic> <italic>as proposed</italic>?</p><p>A previous quantitative study of endocytosed PEN1 distribution in cytokinesis cells indicated that most PEN1 accumulates at the cell plate with little, if any, signal remaining at the plasma membrane (<xref ref-type="bibr" rid="bib23">Reichardt et al., 2011</xref>).</p><p><italic>4) To resolve question 2 convincingly I think the authors will need at least to show time-lapse data on cells of relevant genotypes undergoing cytokinesis with and without BFA treatments</italic>.</p><p>This is technically challenging and beyond the scope of the present paper. However, we think that our detailed analysis of dividing cells at different stages of mitosis and cytokinesis essentially provides the relevant information. We have now used anti-tubulin immunostaining of mitotic microtubule arrays and DAPI staining of nuclear chromatin as reliable markers for mitotic staging of dividing cells. The new results reveal polar localisation of PIN1 at the plasma membrane in dividing cells of GN-BFAr at successive stages of mitosis (metaphase to telophase) and cytokinesis (cell plate being formed, laterally expanded or nearly complete), this being accompanied by PIN1 accumulation at the cell plate during cytokinesis (see new <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>). The control images (wild-type, i.e., GNOM inactivated by BFA) also show PIN1 accumulation at the cell plate but no polar accumulation at the basal plasma membrane. These data suggest competitive traffic to both cell plate and basal plasma membrane during cytokinesis (see last paragraph of Results, and Discussion).</p><p><italic>There probably needs to be an independent marker of cell cycle stage such as tubulin-GFP, which would also provide evidence that phragmoplasts are normal in the BFA-treated cells</italic>.</p><p>We have used both the DAPI-visualised nuclear cycle and anti-tubulin staining of microtubule arrays (see new <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>).</p><p><italic>I also think that it would be important to ask whether newly-synthesised PIN1 is trapped in interphase BFA compartments in</italic> big3 <italic>and</italic> big3 <italic>GN</italic><sup><italic>R</italic></sup>.</p><p>That was done (<xref ref-type="fig" rid="fig3">Figure 3E-H</xref>): PIN1 expressed from the estradiol-inducible promoter was trapped in <italic>big3</italic> and <italic>big3</italic> GN<sup>R</sup>.</p><p><italic>To resolve the second question, perhaps photoswitchable fluorescent proteins which are available may also be suitable though I realise that they are not easy to image. Protein could be photoconverted in the BFA compartment of BFA-treated Col and followed over time to look at its rate of return to the PM versus the cell plate when cells enter cytokinesis</italic> – <italic>I don't recall this specific experiment being been done before</italic>.</p><p>As mentioned in our reply above, this is technically challenging and beyond the scope of the present paper. However, we have used both the DAPI-visualised nuclear cycle and anti-tubulin staining of microtubule arrays (see new <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>).</p><p><italic>5) From our perspective, the time-lapse (or equivalent) data would be necessary to establish the crucial point about recycling during cytokinesis. We imagine you should be in a position to do this without much delay and to establish the stability of KN in</italic> big3 <italic>+BFA</italic>.</p><p>Just to summarise our replies here, we have repeated the experiment, using DAPI-visualised nuclear cycle and anti-tubulin staining of microtubule arrays for staging of dividing cells (see new <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>). Unlike KN, these markers are not likely affected by changes in trafficking conditions. We detected PIN1 polar accumulation at the basal plasma membrane at all mitotic stages from metaphase to anaphase to telophase as well as during initiation, expansion and completion of the cell plate, revealing progression of cytokinesis. During cytokinesis, polar accumulation at the PM was accompanied by accumulation at the cell plate when both BFA-resistant BIG3 was present and GNOM was rendered BFA-resistant.</p><p><italic>One reviewer suggests that the conditionality of the BFA-induced phenotype is a perfect system for such experiments</italic>.</p><p>We agree, in principle. However, the dynamics of the process is enormous, and our previous experience with GFP-tagged KN suggests that the short duration of cytokinesis makes live-imaging challenging. Thus, the effort required may well be beyond the scope the present paper.</p><p><italic>The photoswitch experiment or similar is much more demanding but without it there is ambiguity about whether anything really changes with respect to PIN1 trafficking during cytokinesis, a major claim of the paper. If this point remains unclarified, we think the Discussion and Title will need to change to reflect this</italic>.</p><p>See our new data on Col vs. GN-BFAr and the expanded Discussion. We have modified the Results and Discussion sections accordingly.</p><p><italic>6) The reviewers also question the coining of BIG1-4 as “regulatory switches”. It seems that the paper does not demonstrate a direct molecular mechanism in which BIG1-4 display a switch-like function, rather you seem to provide genetic evidence that BIG1-4 are necessary for the observed changes in trafficking. At this point, alternative hypotheses, e.g., that GN or something else on the GN pathway is somehow inactivated during cytokinesis, also appear plausible. We understand that this is partly a semantic issue, but we still would like to ask you to re-word appropriately</italic>.</p><p>We agree that we cannot present a molecular mechanism regulating the pathway change of endocytosed cargo proteins in cytokinesis. However, our results strongly suggest that GNOM is still functional during mitotic and cytokinetic stages. This implies to us that the destination of cargo proteins, division plane or plasma membrane, depends on whether they are recruited to membrane vesicles whose formation is mediated by BIG1-4 or GNOM, respectively.</p><p><italic>We hope you will be able to address the above issue, but let us clarify that the remaining points that cell plate assembly requires BIG1-4 and so is derived from a biosynthetic pathway at the TGN, and that there are two distinct classes of TGN with distinct ARF-GEFs that keep biosynthetic and recycling cargo separate are valuable contributions. For these alone, your paper merits publication, but it would have to be revised accordingly in the Discussion and Title</italic>.</p><p>In order to avoid confusion for the reader, we have changed the title and the Discussion as suggested and make no claim regarding switches or molecular regulation of the process.</p></body></sub-article></article> |