Exocytosis: A New Exocyst Movie

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a new neurological mutation in the mouse. Proc. Natl. Acad. Sci. USA 73, 208–212.

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14. Berezniuk, I., Sironi, J., Callaway, M.B., Castro, L.M., Hirata, I.Y., Ferro, E.S., and Fricker, L.D. (2010). CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813– 1823.

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Exocytosis: A New Exocyst Movie Kunrong Mei and Wei Guo* Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cub.2018.11.022

Using genome editing and advanced light microscopy, a recent study has offered new insights into the dynamic assembly and disassembly of the exocyst complex during vesicle tethering and membrane fusion. Exocytosis — the transport of secretory vesicles to the plasma membrane for fusion — plays important roles in cell morphogenesis, growth, division, migration and signaling. The exocyst is an evolutionarily conserved heterooctameric complex consisting of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 subunits [1–3]. The primary function of the exocyst is to mediate the tethering of secretory vesicles to the plasma membrane before SNAREmediated fusion [4]. Using CRISPR– Cas9-mediated gene editing, light microscopic imaging and correlation spectroscopy, Ahmed and colleagues [5] have now provided us with new insight into the dynamics of the exocyst complex during exocytosis. Since the early purifications from yeast in 1995 [1,2], intensive effort has been made to understand the architecture and assembly of the exocyst complex [6]. Two interaction studies in yeast and mammalian cells have suggested the existence of two subcomplexes consisting of Sec3–Sec5–Sec6–Sec8 (‘subcomplex I’), and Sec10–Sec15–Exo70–Exo84 (‘subcomplex II’), respectively [7,8]. Most recently, cryo-electron microscopy studies revealed that the formation of the

two subcomplexes is mediated by fourhelix bundles consisting of the conserved CorEx (‘core of exocyst’) motif from each of the subunits [9]. The two subcomplexes then, like two hands, clasp each other to form the octameric holo-complex [9]. While the structural studies have advanced our understanding of the fully assembled exocyst complex, it is also important to elucidate how the exocyst assembles during vesicle transport, tethering and fusion. To understand the dynamics of exocyst assembly in exocytosis, earlier work by Boyd and colleagues [10] elegantly combined fluorescence recovery after photobleaching (FRAP) and immunogold electron microscopy to monitor each of the eight subunits of the complex in yeast cells. They demonstrated that a subset of exocyst subunits (Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and some of Exo70) ride the vesicles along the actin cables to the daughter cell (‘bud’), where they assemble with Sec3 at the plasma membrane. Also using yeast, Donovan and Bretscher [11] reported that the secretory vesicles are tethered to the plasma membrane for around 18 seconds until membrane fusion, and that Sec3, Sec5 and Sec15 leave the vesicles coincident with

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fusion. These studies have provided important information on the dynamics of exocyst action during exocytosis in yeast. However, some questions remained open: how are the exocyst subunits organized on the vesicles? Is there pre-assembly of the subunits before recruitment onto the vesicle? Moreover, how is the exocyst assembled during exocytosis in mammalian cells? A major challenge to the tracking of the exocyst in mammalian cells is that overexpression of individual exocyst subunits may cause their mislocalization, aggregation and degradation [12]. By silencing endogenous Sec8 and replacing it with Sec8–RFP at a low expression level, Rivera-Molina and Toomre [12] showed that, in HeLa cells, Sec8 arrives at the plasma membrane on vesicles around 7.5 seconds prior to vesicle fusion and leaves 2 seconds after fusion. In the new study, Ahmed et al. [5] used CRISPR–Cas9-mediated gene editing to generate an array of cell lines expressing exocyst subunits tagged with fluorescent proteins. Immunoprecipitation experiments revealed the interactions of all the exocyst subunits and their known binding partners such as small GTPases in these cells, confirming the functionality of the tagged exocyst subunits. When Sec8,

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Figure 1. A model of exocyst assembly during exocytosis. (A) The assembly of the exocyst complex in mammalian cells (based on [5]). Before being loaded onto the secretory vesicles, Sec3, Sec5, Sec6 and Sec8 (subcomplex I components) and Sec10, Sec15, Exo70 and Exo84 (subcomplex II components) begin to assemble into subcomplexes. The two subcomplexes are recruited to the vesicle independently. Upon arrival at the plasma membrane, the two subcomplexes assemble into the holo-exocyst complex to tether the vesicle onto the plasma membrane. Right before fusion, Sec3 is released from the exocyst complex. After fusion has begun, the remaining exocyst subunits leave the fusion site. (B) The assembly of the exocyst in yeast (based on [10,11]). Sec3, and maybe some of the Exo70 proteins, are located at the plasma membrane, while the other seven exocyst subunits are carried by the vesicle. When the vesicle arrives at the plasma membrane, Sec3 interacts with other subunits to form the holo-exocyst complex, which tethers the vesicle to the plasma membrane. Colored ovals represent the individual exocyst subunits; V, secretory vesicle; PM, plasma membrane; SN, SNARE proteins.

a component of subcomplex I, was silenced, subcomplex I was disrupted while subcomplex II remained intact. Conversely, silencing of Sec10 led to the loss of subcomplex II components while subcomplex I remained intact [5]. This observation suggests that the connectivity of mammalian exocyst subunits is similar to that in yeast [8,9] and is consistent with a recent finding that the cryo-EM structure of human exocyst is very close to the yeast counterpart [13]. Structure-based sequence alignment suggests that the CorEx motifs, which likely mediate the core assembly of the complex, are conserved in mammals and yeast [9]. When Sec10 was silenced, Sec3 and Sec5 remained associated with the plasma membrane, while Exo70 lost association with the vesicle and plasma membrane. Conversely, silencing Sec8 or Sec3 caused disassociation of Sec5 but not Exo70 from the plasma membrane [5]. These data suggested that each of the two subcomplexes could associate with the vesicles and plasma membrane in the absence of the other [5]. However, either one of the

subcomplexes alone was not sufficient to promote membrane fusion. When and where are these exocyst subcomplexes formed? When do they assemble into the holo-exocyst complex? Ahmed and colleagues [5] addressed these questions by combining time-lapse total internal reflection fluorescence microscopy (TIRFM) and fluorescence cross-correlation spectroscopy (FCCS). Similar to the arrival of vesicles marked by Rab11, GFP-tagged Sec3, Sec5, Sec6 and Exo70 all arrive at the plasma membrane 10–14 seconds prior to fusion, suggesting that the exocyst arrives at the plasma membrane with the secretory vesicles [5]. This arrival time is similar to previous reports in both mammals and yeast [11,12]. Strikingly, while Sec8 and Sec5 arrive together near the plasma membrane, Exo70 arrives 77 milliseconds later than Sec5 [5], suggesting that, even though subcomplex I and subcomplex II arrive at the plasma membrane together with the vesicles, they can arrive independently and then assemble into the holo-exocyst complex at the plasma membrane. Furthermore, FCCS revealed that 70% of

Sec8 was coupled to Sec5, and 50% of Sec8 was associated with vesicles, whereas the other half was freely diffusing, suggesting that a fraction of the exocyst subcomplexes is pre-assembled in the cytoplasm before being loaded onto the vesicles [5]. Interestingly, while Sec5, Sec6 and Exo70 depart at a median time of 1.3 seconds after fusion, Sec3 departs 0.4 seconds prior to fusion, implying a distinct role of Sec3 in vesicle tethering and membrane fusion [5]. Consistent with its earlier departure from the fusion site, Sec3 also showed higher mobility on the plasma membrane and lower abundance at the intercellular junctions of epithelial cells than other exocyst subunits [5]. In yeast, Sec3 binds to the t-SNARE protein Sso1/2 (the yeast homologs of syntaxin) to promote the formation of the initial binary t-SNARE complex (Sso1/2–Sec9) before the subsequent v-SNARE protein joins the t-SNAREs for fusion; Sec9 then displaces Sec3 from Sso1/2 as Sec9 binds to Sso1/2 to form the binary complex [14]. Although Sec3 is a subcomplex I subunit, its binding to the

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Dispatches other members of this subcomplex seems weaker than the association between these other subcomplex I subunits. However, Sec3 is indispensable for the assembly of this subcomplex because its silencing significantly reduced the interactions between Sec5 and Sec8 [5]. It is also intriguing that, even though mammalian and yeast Sec3 are predicted to be very similar in structure, both containing an amino-terminal pleckstrin homology (PH) domain, the long coiledcoil CorEx motif and an extended helical rod at the carboxyl terminus, their characteristics appear to be quite different. Mammalian Sec3 is found on the vesicle prior to fusion, whereas in yeast Sec3 is located at the plasma membrane before the arrival of the vesicles [10]. The current study by Ahmed et al. [5] describes the dynamics of exocyst assembly in mammalian cells, from which a model of exocyst assembly and disassembly during exocytosis can be proposed (Figure 1). Before being loaded onto the secretory vesicles, the exocyst subunits begin to assemble into their subcomplexes. Then subcomplex I and subcomplex II are independently recruited to the vesicles. Once arriving at the plasma membrane, the two subcomplexes assemble into the holoexocyst complex to tether the vesicle to the plasma membrane and promote the subsequent membrane fusion. Right before fusion happens, the exocyst complex disassembles to release Sec3. Then the other subunits leave the fusion site 2 seconds after fusion. This new exocyst movie raises a number of further questions. When are subcomplex I and subcomplex II loaded onto the vesicles? Is there an inhibitory mechanism that prevents the two subcomplexes from assembling into the holo-exocyst complex before the arrival of the secretory vesicles at the plasma membrane? If so, how is the inhibition removed when the vesicles arrive at the sites of secretion? Also, why do the vesicles, the tethering machinery, and some tethering regulators [5,11,12] remain at the plasma membrane for 12–18 seconds, and what happens during this period of time? Last but not least, how is the assembly of the exocyst regulated in different cellular contexts and under different pathophysiological conditions? No doubt more exocyst sequels will follow.

REFERENCES 1. TerBush, D.R., and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130, 299–312. 2. TerBush, D.R., Maurice, T., Roth, D., and Novick, P. (1996). The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494. 3. Hsu, S.C., Ting, A.E., Hazuka, C.D., Davanger, S., Kenny, J.W., Kee, Y., and Scheller, R.H. (1996). The mammalian brain rsec6/8 complex. Neuron 17, 1209–1219. 4. Mei, K., and Guo, W. (2018). The exocyst complex. Curr. Biol. 28, R922–R925. 5. Ahmed, S.M., Nishida-Fukuda, H., Li, Y., McDonald, W.H., Gradinaru, C., and Macara, I. (2018). Exocyst dynamics during vesicle tethering and fusion. Nat. Commun. 9, 5140. 6. Lepore, D.M., Martinez-Nunez, L., and Munson, M. (2018). Exposing the elusive exocyst structure. Trends Biochem. Sci. 43, 714–725. 7. Katoh, Y., Nozaki, S., Hartanto, D., Miyano, R., and Nakayama, K. (2015). Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci. 128, 2351–2362.

8. Heider, M.R., Gu, M., Duffy, C.M., Mirza, A.M., Marcotte, L.L., Walls, A.C., Farrall, N., Hakhverdyan, Z., Field, M.C., Rout, M.P., et al. (2016). Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat. Struct. Mol. Biol. 23, 59–66. 9. Mei, K., Li, Y., Wang, S., Shao, G., Wang, J., Ding, Y., Luo, G., Yue, P., Liu, J.-J., Wang, X., et al. (2018). Cryo-EM structure of the exocyst complex. Nat. Struct. Mol. Biol. 25, 139–146. 10. Boyd, C., Hughes, T., Pypaert, M., and Novick, P. (2004). Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol. 167, 889–901. 11. Donovan, K.W., and Bretscher, A. (2015). Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process. J. Cell Biol. 210, 181–189. 12. Rivera-Molina, F., and Toomre, D. (2013). Livecell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion. J. Cell Biol. 201, 673–680. 13. Zhai, Y., Zhang, D., Yu, L., Sun, F., and Sun, F. Large protein complex production using the SmartBac system - strategies and applications. bioRxiv 219246; https://doi.org/ 10.1101/219246. 14. Yue, P., Zhang, Y., Mei, K., Wang, S., Lesigang, J., Zhu, Y., Dong, G., and Guo, W. (2017). Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion. Nat. Commun. 8, 14236.

Larval Development: Making Ants into Soldiers H. Frederik Nijhout Department of Biology, Duke University, Durham, NC 27708, USA Correspondence: [email protected] https://doi.org/10.1016/j.cub.2018.11.019

Many ant species have complex caste systems, with reproductive queens and sterile workers, which often play distinct roles in the maintenance and defense of the colony. A new study sheds light on how these worker caste systems evolved and the mechanisms by which totipotent larvae give rise to the alternative adult castes. The ant genus Pheidole contains more than a thousand species [1,2] that produce colonies with two distinct nonreproductive castes: small-bodied workers and large-bodied soldiers, the latter of which often have exceptionally large heads and mandibles. Any larva can

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develop into either a worker or a soldier, governed largely by the nutritive and pheromonal signals it receives. But soldiers are not just large-bodied workers. When a larva is set on a soldier-bound trajectory, it not only grows to be larger, but the scaling relationship among its