The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

Institute of Experimental Botany Academy of Sciences of the Czech Republic Prague 6 Czech Republic

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Allersma MW, Wang L, Axelrod D, Holz RW. Visualization of regulated exocytosis with a granule-membrane probe using total internal reflection microscopy. Mol Biol Cell. 2004;15:4658–4668. PubMed PMC

Barg S, Knowles M, Chen X, Midorikawa M, Almers W. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells. Proc Natl Acad Sci USA. 2010;107:20804–20809. PubMed PMC

Batoko H, Zheng HQ, Hawes C, Moore I. A rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell. 2000;12:2201–2218. PubMed PMC

Battey N, James N, Greenland A, Brownlee C. Exocytosis and endocytosis. Plant Cell. 1999;11:643–660. PubMed PMC

Bendezú FO, Martin SG. Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast. Mol Biol Cell. 2010;22:44–53. PubMed PMC

Bendezú FO, Vincenzetti V, Martin SG. Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles. PLoS One. 2012;7:e40248. PubMed PMC

Boyd C, Hughes T, Pypaert M, Novick P. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J Cell Biol. 2004;167:889–901. PubMed PMC

Bröcker C, Engelbrecht-Vandré S, Ungermann C. Multisubunit tethering complexes and their role in membrane fusion. Curr Biol. 2010;20:R943–R952. PubMed

Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev Cell. 2007;12:671–682. PubMed

Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16:735–743. PubMed

Cole RA, Synek L, Žárský V, Fowler JE. SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol. 2005;138:2005–2018. PubMed PMC

Crowell EF, Bischoff V, Desprez T, Rolland A, Stierhof Y-D, Schumacher K, Gonneau M, Höfte H, Vernhettes S. Pausing of Golgi bodies on microtubules regulates secretion of cellulose synthase complexes in Arabidopsis. Plant Cell. 2009;21:1141–1154. PubMed PMC

Dixit R, Cyr R, Gilroy S. Using intrinsically fluorescent proteins for plant cell imaging. Plant J. 2006;45:599–615. PubMed

Enami K, Ichikawa M, Uemura T, Kutsuna N, Hasezawa S, Nakagawa T, Nakano A, Sato MH. Differential expression control and polarized distribution of plasma membrane-resident SYP1 SNAREs in Arabidopsis thaliana. Plant Cell Physiol. 2009;50:280–289. PubMed

Fendrych M, et al. The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell. 2010;22:3053–3065. PubMed PMC

Finger FP, Hughes TE, Novick P. Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell. 1998;92:559–571. PubMed

Fujimoto M, Arimura S, Ueda T, Takanashi H, Hayashi Y, Nakano A, Tsutsumi N. Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis. Proc Natl Acad Sci USA. 2010;107:6094–6099. PubMed PMC

Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G. The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell. 2003;112:219–230. PubMed

Genre A, Ivanov S, Fendrych M, Faccio A, Žárský V, Bisseling T, Bonfante P. Multiple exocytotic markers accumulate at the sites of perifungal membrane biogenesis in Arbuscular mycorrhizas. Plant Cell Physiol. 2012;53:244–255. PubMed

Gromley A, Yeaman C, Rosa J, Redick S, Chen C-T, Mirabelle S, Guha M, Sillibourne J, Doxsey SJ. Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission. Cell. 2005;123:75–87. PubMed

Guo W, Grant A, Novick P. Exo84p is an exocyst protein essential for secretion. J Biol Chem. 1999a;274:23558–23564. PubMed

Guo W, Roth D, Walch-Solimena C, Novick P. The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 1999b;18:1071–1080. PubMed PMC

Guo W, Tamanoi F, Novick P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nat Cell Biol. 2001;3:353–360. PubMed

Gutierrez R, Lindeboom JJ, Paredez AR, Emons AMC, Ehrhardt DW. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nat Cell Biol. 2009;11:797–806. PubMed

Hála M, et al. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell. 2008;20:1330–1345. PubMed PMC

He B, Xi F, Zhang X, Zhang J, Guo W. Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. EMBO J. 2007;26:4053–4065. PubMed PMC

Heider M, Munson M. Exorcising the exocyst complex. Traffic. 2012;13:898–907. PubMed PMC

Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron. 1998;20:1111–1122. PubMed

Karimi M, Inzé D, Depicker A. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002;7:193–195. PubMed

Kato N, Fujikawa Y, Fuselier T, Adamou-Dodo R, Nishitani A, Sato MH. Luminescence detection of SNARE–SNARE interaction in Arabidopsis protoplasts. Plant Mol Biol. 2010;72:433–444. PubMed

Ketelaar T, Galway ME, Mulder BM, Emons AMC. Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes. J Microsc. 2008;231:265–273. PubMed

Konopka CA, Backues SK, Bednarek SY. Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane. Plant Cell. 2008;20:1363–1380. PubMed PMC

Konopka CA, Bednarek SY. Comparison of the dynamics and functional redundancy of the Arabidopsis dynamin-related isoforms DRP1A and DRP1C during plant development. Plant Physiol. 2008a;147:1590–1602. PubMed PMC

Konopka Ca, Bednarek SY. Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex. Plant J. 2008b;53:186–196. PubMed

Kulich I, Cole R, Drdová E, Cvrčková F, Soukup A, Fowler J, Žárský V. Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin. New Phytol. 2010;188:615–625. PubMed

Langowski L, Růžička K, Naramoto S, Kleine-Vehn J, Friml J. Trafficking to the outer polar domain defines the root-soil interface. Curr Biol. 2010;20:904–908. PubMed

Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S. A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol. 2007;17:947–952. PubMed

Lipka V, Kwon C, Panstruga R. SNARE-ware: the role of SNARE-domain proteins in plant biology. Annu Rev Cell Dev Biol. 2007;23:147–174. PubMed

Lipschutz JH, Guo W, O'Brien LE, Nguyen YH, Novick P, Mostov KE. Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins. Mol Biol Cell. 2000;11:4259–4275. PubMed PMC

Marc J, Granger C, Brincat J, Fisher D, Kao T, McCubbin A, Cyr R. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell. 1998;10:1927–1940. PubMed PMC

Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng. 2007;104:34–41. PubMed

Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980;21:205–215. PubMed

Pecˇenková T, Hála M, Kulich I, Kocourková D, Drdová E, Fendrych M, Toupalová H, Žárský V. The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant–pathogen interaction. J Exp Bot. 2011;62:2107–2116. PubMed PMC

Riedl J, et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods. 2008;5:605–607. PubMed PMC

Robinson NGG, Guo L, Imai J, Toh-e A, Matsui Y, Tamanoi F. Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol Cell Biol. 1999;19:3580–3587. PubMed PMC

Seguí-Simarro JM, Austin JR, 2nd, White EA, Staehelin LA. Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell. 2004;16:836–856. PubMed PMC

Sivaram MVS, Saporita JA, Furgason MLM, Boettcher AJ, Munson M. Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p. Biochemistry. 2005;44:6302–6311. PubMed

Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993;362:318–324. PubMed

Songer JA, Munson M. Sec6p anchors the assembled exocyst complex at sites of secretion. Mol Biol Cell. 2009;20:973–982. PubMed PMC

Synek L, Schlager N, Eliáš M, Quentin M, Hauser MT, Žárský V. AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J. 2006;48:54–72. PubMed PMC

Teh O-K, Moore I. An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature. 2007;448:493–496. PubMed

Tian GW, et al. High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol. 2004;135:25–38. PubMed PMC

Toyooka K, Goto Y, Asatsuma S, Koizumi M, Mitsui T, Matsuoka K. A mobile secretory vesicle cluster involved in mass transport from the Golgi to the plant cell exterior. Plant Cell. 2009;21:1212–1229. PubMed PMC

Tsuboi T, Ravier Ma, Xie H, Ewart M-A, Gould GW, Baldwin Sa, Rutter GA. Mammalian exocyst complex is required for the docking step of insulin vesicle exocytosis. J Biol Chem. 2005;280:25565–25570. PubMed

Uemura T, Ueda T, Ohniwa RL, Nakano A, Takeyasu K, Sato MH. Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells. Cell Struct Funct. 2004;29:49–65. PubMed

Vega IE, Hsu SC. The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J Neurosci. 2001;21:3839–3848. PubMed PMC

Wang J, Ding Y, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L. EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell. 2010;22:4009–4030. PubMed PMC

Wang S, Liu Y, Adamson CL, Valdez G, Guo W, Hsu SC. The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization. J Biol Chem. 2004;279:35958–35966. PubMed

Willats WG, McCartney L, Knox JP. In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana. Planta. 2001;213:37–44. PubMed

Wu H, Rossi G, Brennwald P. The ghost in the machine: small GTPases as spatial regulators of exocytosis. Trends Cell Biol. 2008;18:397–404. PubMed PMC

Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 2002;296:913–916. PubMed

Žárský V, Cvrcˇková F, Potocký M, Hála M. Exocytosis and cell polarity in plants— exocyst and recycling domains. New Phytol. 2009;183:255–272. PubMed

Zhang X, Bi E, Novick P, Du L, Kozminski KG, Lipschutz JH, Guo W. Cdc42 interacts with the exocyst and regulates polarized secretion. J Biol Chem. 2001;276:46745–46750. PubMed

Zhang X, Orlando K, He B, Xi F, Zhang J, Zajac A, Guo W. Membrane association and functional regulation of Sec3 by phospholipids and Cdc42. J Cell Biol. 2008;180:145–158. PubMed PMC

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