Commentary 3427 Junctions and vesicular trafficking during Drosophila cellularization

Thomas Lecuit Laboratoire de Génétique et de Physiologie du Développement, UMR 6545 CNRS-Université de la Méditerranée, Institut de Biologie du Développement de Marseille (IBDM), Campus de Luminy, case 907, 13288 Marseille CEDEX 09, France (e-mail: [email protected])

Journal of Cell Science 117, 3427-3433 Published by The Company of Biologists 2004 doi:10.1242/jcs.01312

Summary The cellularization that converts the syncytial Drosophila defined sites of the plasma membrane underlies membrane embryo into thousands of distinct cells is a hybrid form of growth and surface polarization. Genetic approaches have cleavage. It derives from cytokinesis and has acquired identified conserved core cellular pathways required for specific features required for epithelial biogenesis. these processes, such as vesicular trafficking along the Cellularization generates an epithelial layer in which biosynthetic and endocytic routes, and vesicular insertion adjacent cells are connected by apical adherens junctions. into the plasma membrane. The novel proteins Nullo and If this process goes awry, subsequent development is Slam, which are specifically induced during cellularization, dramatically affected, in particular tissue remodelling represent developmental regulators of membrane growth during gastrulation. Cellularization is associated with the during cellularization. invagination of the plasma membrane between adjacent nuclei at the cell cortex, the formation of a basal-lateral surface and the assembly of apical adherens junctions. The Key words: Drosophila, Cellularization, Apical adherens junctions, regulated mobilization of intracellular pools of vesicles at Basal adherens junctions

Introduction cell in the newly formed epithelium. This hybrid nature makes Early embryonic development in most organisms involves the cellularization a particularly interesting system for studies formation of a sheet of cells that then undergoes dramatic of cell polarization during development. Although some rearrangements during gastrulation. The pathways that yield mechanisms may be specific to cellularization, others might the first embryonic tissues can be quite different. They always share more features with other cellular processes than is comprise a phase of cell division, and the end result is often superficially apparent. Recent reports show that several widely the production of an epithelial tissue that is ready to undergo conserved proteins required for the formation of junctions and extensive remodelling. In the mouse embryo, cell divisions for vesicular traffic are indeed important during cellularization precede compaction, the process whereby un-polarized cells (Lee et al., 2003; Pelissier et al., 2003; Riggs et al., 2003). together form an epithelial sphere. In amphibians such as These studies confirm the idea that cellularization requires the Xenopus, mitosis produces new epithelial cells at each round regulated insertion of vesicles into the plasma membrane. of division. The polarity of a given cell is thus transmitted to When the fertilized Drosophila egg is laid, it is a large (0.5 its descendents. In Drosophila, and many other arthropods, the mm long), ellipsoid cell. Within about 2 hours, 13 synchronous nuclei first divide in a syncytium and, later on, thousands of cycles of nuclear division in a single cytoplasm (the syncytium) epithelial cells are produced simultaneously around the yield about 5000 nuclei localized at the cortex of the syncytial circumference of the embryo by invagination of the plasma embryo (Fig. 1A). During mitosis, the plasma membrane membrane (Fig. 1) (Foe et al., 1993; Schejter and Wieschaus, invaginates slightly and partially isolates each dividing nucleus, 1993a). Cellularization is a remarkable process that seems to forming a so-called metaphase or pseudo-cleavage furrow. have appeared several times in evolution. For instance, the When nuclei enter interphase 14, mitoses are blocked, the nuclei gametophytes in plants, such as in Arabidopsis thaliana, also stay in interphase, and membrane invagination becomes much form by cellularization of a large syncytium (Sorensen et al., more significant: within about 1 hour of development from the 2002). entry into interphase 14, the invaginating membrane packages Drosophila cellularization is a hybrid process. It is akin to single nuclei, organelles and cytoskeletal elements into 5000 embryonic cell division and is based on cytokinesis. The cells and forms a columnar epithelium 35 µm tall (Fig. 1B-D). membrane invaginates between nuclei localized at the cortex Cellularization proceeds first in a slow phase (0.3 µm/minute of the embryo, and several genes required during standard for 40 minutes) and then in a fast phase (0.8 µm/minute for 20 cytokinesis are also important for cellularization. However, minutes). At the onset of cellularization, the plasma membrane the main product of cellularization is an epithelium. forms a dome that is rich in villous projections (VPs), the Cellularization has thus acquired mechanisms important for the somatic bud above each nucleus (Fig. 2A,B). Adjacent somatic polarization of the invaginating plasma membrane and the buds are separated by shallow invagination of the plasma assembly of apical adherens junctions (AAJs) between each membrane (Fig. 2A,B, arrowhead). These fold to form donut- 3428 Journal of Cell Science 117 (16)

Fig. 1. Cellularization links embryonic cleavage to the formation of an epithelial layer. The plasma membrane (PM, green) invaginates between the nuclei (N, black) located at the cortex of the syncytial embryo shown at the onset (A) and at the end (B) of cellularization. When cellularization is completed, 5000 columnar epithelial cells are formed. The apical adherens junctions (AAJ, red in B) have assembled and ensure the cohesion of the epithelial layer. Cellularization is thus associated with the growth and polarization of the cell surface. (C,D) Confocal sections of a cellularizing embryo during slow phase (C) and at the end of fast phase (D) corresponding to the boxed areas shown in A and B. The embryos are stained with antibodies to the integral membrane protein neurotactin (Nrt, green) that marks the entire cell surface, to the junctional protein PaTJ (turquoise) that marks the membrane front called the furrow canal (FC), to the small GTPase Rab11 (red), a marker of apical recycling endosomes (see Fig. 2), and with the DNA dye Hoechst (Hoe, dark blue) to show the nuclei. Bar, 5 µm. shaped structures called furrow canals (FCs), which are The prevalent model of cellularization ten years ago was separated from the contiguous plasma membrane by basal largely influenced by the understanding of cytokinesis at that adherens junctions (BAJs) (Fig. 2C,C’,D) (Hunter et al., 2002; time, and proposed that the plasma membrane invaginates Hunter and Wieschaus, 2000; Muller and Wieschaus, 1996). following specific rearrangements of cytoskeletal elements The FC forms the membrane front as it invaginates (Fig. 2C,C’). (Foe and Alberts, 1983; Foe et al., 1993; Schejter and The FC is analogous in composition to the contractile ring of Wieschaus, 1993a; Schejter and Wieschaus, 1993b). In this standard cytokinesis and contains F-actin (Warn and Robert- context, the plasma membrane was believed to follow actin and Nicoud, 1990), myosin II (Lecuit et al., 2002; Royou et al., microtubule (MT) rearrangements passively. For instance, the 2003; Sisson et al., 2000; Young et al., 1991), anillin (Field requirement for intact actin filaments during cellularization and Alberts, 1995), cofilin, spectrins (Thomas and Williams, (Foe and Alberts, 1983), and the similar biochemical 1999), septins (Adam et al., 2000; Fares et al., 1995) and compositions of the FC and of the cytokinesis front, in formins/diaphanous (Afshar et al., 2000). FCs form adjacent to particular the accumulation of myosin II in this membrane the BAJs, which contain complexes of the cell-cell adhesion domain, reinforced the idea that a contractile actin-myosin molecule E-cadherin and β-catenin (termed Armadillo in network at the membrane front might drive membrane Drosophila). The FC also contains junctional proteins such as invagination. In addition, Foe showed that MTs are essential PATJ (originally called Discs lost, DLT) (Bhat et al., 1999; for cellularization and that, at the onset of cellularization, astral Pielage et al., 2003). The hybrid nature of cellularization MTs dramatically extend their + ends basally towards the dense (combining cytokinesis and epithelial polarization) is thus yolk granules present about 30-40 microns inside the syncytial already reflected in the organization of the membrane front embryo (Foe and Alberts, 1983). Foe proposed that growth of during the slow phase of cellularization. astral MTs might push the membrane inward (Foe et al., 1993). The fast phase, which produces more than half of the total Since about five years ago, several studies of cellularization lateral membrane surface area, is also associated with the have progressively changed this perception in parallel with new formation of AAJs, which connect adjacent cells in the newly insights into the mechanisms of standard cytokinesis. The idea formed epithelium and are required for the integrity of the that vesicular insertion in the plasma membrane is an active tissue. During the fast phase, E-cadherin/β-catenin complexes process that is essential during cellularization (Lecuit and first appear in punctate structures in the apico-lateral region of Wieschaus, 2000) and more generally during cytokinesis the plasma membrane and form the spotted-adherens junctions (Drechsel et al., 1997; Jantsch-Plunger and Glotzer, 1999), (SAJs). At the end of cellularization, E-cadherin/β-catenin which was first proposed several decades ago (Bluemink and complexes form a more continuous belt that will progressively de Laat, 1973; Fullilove and Jacobson, 1971), became more constitute the proper AAJs as it matures and stabilizes. prevalent (reviewed by Glotzer, 2001; O’Halloran, 2000) and Additional proteins important for the stabilization of the E- changed the model of cellularization. cadherin/β-catenin complexes are recruited at AAJs at the end The apparent increase in the plasma membrane surface area of cellularization, including F-actin, atypical protein kinase C during cellularization indicates two possible scenarios: (aPKC) (Wodarz et al., 2000), and the PDZ-domain-containing cellularization could either deploy apical membrane stored in proteins PAR-3 (termed Bazooka in Drosophila) (Kuchinke et the VPs (Fullilove and Jacobson, 1971; Turner and Mahowald, al., 1998), PAR-6 (Petronczki and Knoblich, 2001) and PATJ 1976), or involve the insertion of intracellular membrane (Bhat et al., 1999). vesicles into the plasma membrane (Fullilove and Jacobson, Drosophila cellularization 3429

Fig. 2. Cell-surface remodelling and vesicular transport pathways underlying cellularization. Transmission electron micrographs (A,C,C’) and schematic representations of embryos at the onset (A,B,E) and 20 minutes after the beginning (C,C’,D,F) of cellularization. (A) Above each nucleus (N), the plasma membrane (PM) forms a dome called the somatic bud that is rich in villous projections (VP). Adjacent somatic buds are separated by a flat depression of the plasma membrane (arrowhead). (B) This membrane domain contains specific sets of junctional proteins such as E-cadherin/β-catenin complexes, myosin II, PaTJ and Slam, as well as DLG/SAP97 and the multi-PDZ-containing protein Scribbles (Scb). (C,C’) As the plasma membrane folds back and forms the furrow canal (FC), (D) three distinct and adjacent membrane domains assemble containing MyoII/PaTJ/Slam in the FC (red), E-cadherin/β-catenin (green) at the basal adherens junction (BAJ) and DLG/Scb (blue) just apical to the BAJ. The two apposed cell surfaces (C, black arrowheads) grow progressively during the slow phase. Several vesicular organelles can be detected in the vicinity of the cell surface (C, white arrowheads). The formation of the BAJ as shown in D is dependent on Slam (Lecuit et al., 2002). Junctional stabilization requires Nullo, which localizes more broadly in the FC, at the BAJ and apical to the BAJ (Hunter and Wieschaus, 2000). Failure to form or stabilize the BAJ is always associated with an inhibition of membrane invagination in slow phase. (E,F) Trafficking through sub-apical perinuclear recycling endosomes (RE, pink) is required for membrane invagination. This process is controlled by the small GTPase Rab11 and Dynamin/Shibire. Vesicular transport from the Golgi apparatus to RE as well as from the apical plasma membrane to RE by Rab5 might also contribute to this trafficking pathway. Bar, 2 µm.

1971; Loncar and Singer, 1995). Various studies support the the Golgi-associated protein Lava-lamp results in similar notion that intracellular membrane pools are indeed used as defects (Sisson et al., 2000). In fact, the requirement for MTs a raw material during cellularization and that vesicular during cellularization was also shown to stem largely from trafficking is essential for this process. First, embryos their implication in secretory trafficking (Lecuit and containing mutations in the SNARE protein syntaxin 1 fail to Wieschaus, 2000). The depolymerization of MTs at the onset cellularize (Burgess et al., 1997). Syntaxin 1 is required for of cellularization blocks the post-Golgi trafficking to the insertion of vesicles into the plasma membrane in a variety of plasma membrane of the transmembrane cell adhesion protein organisms. Pulse-labelling experiments in living embryos have neurotactin (Nrt) (Lecuit and Wieschaus, 2000) and causes probed the dynamics of the plasma membrane during major defects in ER-to-Golgi vesicular transport (T. Lecuit, cellularization (Lecuit and Wieschaus, 2000). These show that, unpublished) in conjunction with the inhibition of membrane after labelling of the plasma membrane during cellularization, invagination. specific regions of the cell surface become devoid of labelling This different interpretation of the requirement for intact first apically then apico-laterally. These regions could either MTs during cellularization is characteristic of the recent define sites of vesicle insertion or sites of vesicle conceptual change in our understanding of cellularization. internalization. Further support for the role for vesicle Together, these studies have substantiated the notion that exocytosis comes from studies showing that injection of cellularization relies on the mobilization of vesicles into the brefeldin A, which inhibits endoplasmic reticulum (ER)-to- plasma membrane, in part from the secretory pathway. Golgi transport, reduces the speed of membrane invagination Moreover, the pulse-labelling experiments during (Sisson et al., 2000); injection of antibodies directed against cellularization show that membrane growth is a polarized 3430 Journal of Cell Science 117 (16) process. The polarized remodelling of the cell surface, and in centrosomes during cellularization and at earlier stages. They particular the polarized targeting of pools of vesicles, might be reside in tubulo-vesicular structures around the MT-organizing important for the growth of the plasma membrane. In addition, centers (MTOCs) and physically interact in Drosophila such a polarized targeting might underlie the polarization of (Hickson et al., 2003; Riggs et al., 2003). the cell surface. This idea is supported by the observation that Rab11 and NUF thus probably co-operate in the exocytosis the formation of SAJs (marked by the localization of E- of vesicles required for the invagination of the plasma cadherin) coincides with the insertion of vesicles in the apico- membrane during cellularization, as well as at earlier stages. lateral region of the cell surface during the fast phase (inferred Riggs et al. have emphasized the remodelling of the actin- from pulse-labelling experiments) (Lecuit and Wieschaus, myosin cytoskeleton at the cell surface over the need for vesicle 2000). These observations suggest a link between membrane insertion into the plasma membrane during furrow formation growth and surface polarization. However, they leave open the (Riggs et al., 2003). By contrast, Pelissier et al. favour a role questions of the identity of the specific organelles through for membrane insertion to explain the phenotypes. In light of which vesicular trafficking occurs during cellularization and the fact that the actin-myosin II cytoskeleton appears non- the precise location of where vesicular insertion finally occurs. essential for cellularization (Royou et al., 2003), Rab11 is more likely to provide membrane material necessary for invagination. Regardless of its precise function, it clearly plays Trafficking through recycling endosomes is required a role in trafficking through recycling endosomes during for furrowing membrane invagination (Fig. 2E,F). The transport of vesicles along the endocytic pathway proceeds Dynamin/Shibire also functions during cellularization. through distinct organelles. Internalized vesicles at the plasma Experiments two decades ago indicated first that shibire plays membrane are targeted to early endosomes, also called sorting a role in the early Drosophila embryo (Swanson, 1981). It is endosomes. Vesicle endocytosis requires dynamin (termed possible to block shibire function at defined time points with Shibire in Drosophila) (Chen et al., 1991; van der Bliek and a temperature-sensitive mutant (shibire-ts). Pelissier et al. Meyerowitz, 1991), which is a GTPase that regulates the shifted shibire-ts embryos to the restrictive temperature (RT) fission of endocytic coated vesicles (McNiven, 1998), and at the onset of cellularization and found that membrane trafficking to early endosomes is dependent on the small invagination is blocked (Pelissier et al., 2003). However, when GTPase Rab5 (Bucci et al., 1992; Horiuchi et al., 1997). the shift is performed at the end of the slow phase, Endocytosed vesicles are then either transported back to the cellularization is only moderately affected, which shows that plasma membrane, to late endosomes and lysosomes for shibire is more critically required during slow phase. In degradation or to the peri-centriolar recycling endosome, from addition to controlling endocytosis from the plasma membrane, which material is also recycled to the plasma membrane. In shibire appears to control vesicular trafficking through Rab11- polarized epithelial cells, the apical recycling endosome is positive endosomes. Pelissier et al. shifted shibire-ts mutant required for cell polarization and the transfer of vesicles from embryos to the RT at the onset of cellularization, at a time when the apical to the basolateral surface (or vice versa) by the cell adhesion transmembrane protein Nrt, which is transcytosis (Apodaca et al., 1994; Mostov et al., 2000). synthesized de novo during slow phase (Lecuit and Wieschaus, Trafficking through recycling endosomes is controlled in part 2000), is not synthesized yet (Pelissier et al., 2003). by the small GTPase Rab11 both in mammals and in Surprisingly, after 20 minutes at the RT, the newly synthesized Drosophila (Dollar et al., 2002; Ullrich et al., 1996). Rab11 Nrt, which traffics normally through the ER and the Golgi, is localizes to this organelle in vertebrates and in invertebrates not properly inserted in the plasma membrane in shibire-ts (Dollar et al., 2002; Sonnichsen et al., 2000). Dynamin also mutant embryos. Instead, Nrt accumulates in the sub-apical controls budding of vesicles from recycling endosomes in Rab11-positive recycling endosomes. Shibire is detected in mammalian cells (van Dam and Stoorvogel, 2002). Rab11-positive endosomes during the slow phase, which In rab11-hypomorphic mutant embryos, metaphase furrows suggests that it also controls the budding of vesicles from form randomly around nuclei, and cellularization is not recycling endosomes, as has been reported in some mammalian uniform, resulting in multinucleate cells. Injection of cells (van Dam and Stoorvogel, 2002). Electron microscopy dominant-negative Rab11 (Rab11S25N) a few minutes before has revealed a large accumulation of coated pits on sub-apical cellularization strongly inhibits membrane invagination during endosome-like compartments in shibire mutant embryos cellularization, in both the slow and fast phases. Such a defect (Pelissier et al., 2003), further supporting this conclusion. is not seen when a wild-type form of Rab11 or a dominant- Rab11-containing sub-apical recycling endosomes act as negative form of Rab1, which is implicated in ER-to-Golgi trafficking intermediates during cellularization, and vesicular transport, is injected. Both when dominant-negative Rab11 is exocytosis from this compartment might be required for injected and in rab11-mutant embryos, the nuclei fail to align membrane invagination. Rab11 (Low et al., 2003; Skop et al., correctly in an apical position and ‘fall’ from the cortex. This 2001) also functions in conventional cytokinesis. Two series of phenotype is reminiscent of nuclear-fallout mutant embryos. questions remain. Where do the vesicles upstream of Rab11- Nuclear fallout (NUF) is a member of the arfophilin family of containing endosomes come from? And where are the exocytic proteins (Hickson et al., 2003), arfophilin being an ADP vesicles finally inserted in the plasma membrane? ribosylation factor (Arf)-binding protein that binds the small The finding that newly synthesized Nrt accumulates in GTPases Arf5 and Rab11 in mammalian cells. NUF is required Rab11-containing endosomes in shibire mutants (Pelissier et for membrane invagination during the formation of metaphase al., 2003) suggests that the secretory pathway provides vesicles furrows as well as during cellularization (Rothwell et al., 1998; upstream of Rab11-positive endosomes and that vesicular Rothwell et al., 1999). Both NUF and Rab11 localize near the trafficking through the secretory pathway intersects to some Drosophila cellularization 3431 extent with recycling compartments. This can also be inferred another component of the exocyst, is required for membrane from studies of cell polarization in Mabin-Darby canine kidney invagination during Drosophila cellularization (T. Schwarz, (MDCK) cells (Ang et al., 2003; Folsch et al., 2003). The personal communication). clathrin adaptor complex AP1, which is required for secretory A recent report further supports the role of the BAJ in the trafficking, exists in two forms: (1) a ubiquitous form, AP1A, growth of the plasma membrane during cellularization (Lee et contains the µ1A subunit, and (2) an epithelial-cell-specific al., 2003), implicating the junctional protein Discs large (DLG) form, AP1B, contains the µ1B subunit required for basolateral and the transmembrane protein Strabismus (Stbm) in vesicular targeting (Folsch et al., 1999). Interestingly, whereas cellularization. Stbm is involved in planar cell polarity in AP1A is required for trans-Golgi network (TGN)-to-endosome epithelial cells (Fanto and McNeill, 2004). In stbm-mutant transport and localizes in the TGN, AP1B localizes to recycling embryos, cellularization appears inhibited in a way that is endosomes and recruits to this compartment Sec8 and Exo70 reminiscent of that in nullo-mutant embryos. The membrane (Ang et al., 2003; Folsch et al., 2003), two components of the does not invaginate uniformly and produces multinucleate exocyst complex required for basolateral targeting in epithelial cells. In addition, the junctional proteins DLG and PaTJ are not cells (Grindstaff et al., 1998; Yeaman et al., 2001). Since a properly localized at the BAJ, which suggests that junctions do dominant-negative mutant of Rab5, a regulator of vesicular not assemble or are not stabilized normally in the mutant, as trafficking from the plasma membrane to early endosomes, happens in nullo mutants. Stbm and DLG physically interact slows down membrane invagination, albeit to a lesser extent (through the first two PDZ domains of DLG). However, the in than dominant-negative Rab11, endocytosis from the plasma vivo localization data are not that simple and do not appear to membrane may also be important during cellularization support a straightforward functional interaction. In some (Pelissier et al., 2003). The direct visualization of vesicular epithelial tissues, DLG and Stbm partially colocalize at the cell trafficking during cellularization will be necessary to address surface or on uncharacterized intracellular organelles, but in these questions in the future. other tissues, including those that undergo cellularization, the proteins have distinct localizations. Stbm and DLG might interact in small vesicles that are difficult to observe by Junctions and membrane growth standard confocal microscopy, and this interaction could be The BAJ forms together with the FC in the first 10 minutes of linked to the formation or the stabilization of the BAJ. cellularization (Hunter and Wieschaus, 2000; Lecuit and Lee et al. have used mammalian cells to probe the Wieschaus, 2000). This process involves the re-organization, interaction between Drosophila Stbm and the human within the plane of the membrane, of many junctional proteins. orthologue of DLG, SAP97 (Lee et al., 2003). SAP97 and These are initially randomly distributed within the flat Stbm interact in vitro and display a better colocalization in invagination of the membrane (Fig. 2B) and subsequently intracellular organelles than do Stbm and DLG. In Drosophila segregate into distinct, adjacent membrane domains when the imaginal disc epithelia, ectopic expression of Stbm and BAJs and FCs have formed (Fig. 2D). SAP97 causes the formation of enlarged septate junctions Two genes, nullo and slam, that are required for the containing neurexin. Conversely, in dlg-mutant clones induced invagination of the plasma membrane during cellularization are in imaginal discs, the epithelial junctions are disorganized. also involved in the formation or the stabilization of the BAJ. Although Lee et al. interpret these results to mean that the two The product of the nullo gene controls the accumulation of proteins are essential for membrane formation, it is safer to E-cadherin/β-catenin complexes at BAJs. Nullo is a novel conclude that Stbm and DLG are simply required for the myristoylated protein that has a cluster of basic residues formation of junctions. Given the localization of DLG just required for its localization at the plasma membrane in the BAJ basal to the E-cadherin/β-catenin complex, DLG and Stbm and the FC (Hunter et al., 2002). Slam is a novel protein with might more specifically control the formation or the no apparent conserved domain. Slam colocalizes with myosin stabilization of the basal junctional complexes and thereby the II and is required for the recruitment of myosin II at the plasma formation of AJs. membrane in the FC (Lecuit et al., 2002). Both nullo and slam are induced specifically at the onset of cellularization, the protein synthesized being of zygotic rather than maternal origin Concluding remarks (Hunter and Wieschaus, 2000; Lecuit et al., 2002; Stein et al., Our picture of cellularization has considerably changed in two 2002). In slam-mutant embryos, vesicular insertion of Nrt into ways in the past five years. We have gradually moved from the the plasma membrane is compromised, which suggests that the idea that it is a Drosophila oddity to the realization that it is defect in membrane invagination might be caused by a defect one of several pathways leading to the formation of an in vesicle insertion at junctions. This proposal remains epithelium and that, as such, important cell biological hypothetical because vesicular insertion has never been questions about the process should be addressed (Lecuit and observed directly during cellularization. However, it is Wieschaus, 2002; Nelson, 2003). We have begun to identify supported by the fact that a conserved complex of proteins new regulators of membrane growth and invagination during involved in vesicular insertion from budding yeast to humans, cellularization. Some of the trafficking pathways that underlie the exocyst (Lipschutz and Mostov, 2002), is recruited to AJs vesicular transport during cellularization are also now known. and is involved in the formation of the basolateral cell surface The re-organization of the cell surface that accompanies and is during the polarization of epithelial MDCK cells (Grindstaff et required for membrane growth has brought to light the al., 1998). The Sec6 and Sec8 components of the exocyst in importance of the BAJs during cellularization. As a result, the particular localize at mammalian AJs (Grindstaff et al., 1998; picture has also enlarged, leaving many important questions Yeaman et al., 2001). Interestingly, sec5, a gene that encodes open. Answering them will first require better tools to follow 3432 Journal of Cell Science 117 (16) vesicular trafficking and refine further the analysis of mutants The AP-1A and AP-1B clathrin adaptor complexes define biochemically and in trafficking pathways and junction assembly. It is also functionally distinct membrane domains. J. Cell Biol. 163, 351-362. important to keep in mind an essential product of Fullilove, S. L. and Jacobson, A. G. (1971). Nuclear elongation and cytokinesis in Drosophila montana. Dev. Biol. 26, 560-577. cellularization that remains largely mysterious, namely the Glotzer, M. (2001). Animal cell cytokinesis. Annu. Rev. Cell Dev. Biol. 17, AAJs, during the fast phase. The picture will thus certainly 351-386. incorporate more features and become even larger in the near Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., future. Rodriguez-Boulan, E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93, I thank Anne Pelissier for the staining of embryos shown in Fig. 731-740. 1C,D and Jean-Paul Chauvin for his help with obtaining the electron Hickson, G. R., Matheson, J., Riggs, B., Maier, V. H., Fielding, A. B., micrographs shown in Fig. 2. Research in our laboratory his supported Prekeris, R., Sullivan, W., Barr, F. A. and Gould, G. W. (2003). by an ATIP grant from the CNRS, a ‘subvention libre’ from the Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling Association pour la Recherche contre le Cancer, the Fondation pour endosome distribution and are related to Drosophila nuclear fallout. Mol. la Recherche Médicale, and the EMBO Young Investigator Biol. Cell 14, 2908-2920. Programme. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Woodman, P., Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, M. et al. (1997). A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149- References 1159. Adam, J. C., Pringle, J. R. and Peifer, M. (2000). Evidence for functional Hunter, C. and Wieschaus, E. (2000). Regulated expression of nullo is differentiation among Drosophila septins in cytokinesis and cellularization. required for the formation of distinct apical and basal adherens junctions in Mol. Biol. Cell 11, 3123-3135. the Drosophila blastoderm. J. Cell Biol. 150, 391-401. Afshar, K., Stuart, B. and Wasserman, S. A. (2000). Functional analysis of Hunter, C., Sung, P., Schejter, E. D. and Wieschaus, E. (2002). Conserved the Drosophila diaphanous FH protein in early embryonic development. domains of the Nullo protein required for cell-surface localization and Development 127, 1887-1897. formation of adherens junctions. Mol. Biol. Cell 13, 146-157. Ang, A. L., Folsch, H., Koivisto, U. M., Pypaert, M. and Mellman, I. (2003). Jantsch-Plunger, V. and Glotzer, M. (1999). Depletion of syntaxins in the The Rab8 GTPase selectively regulates AP-1B-dependent basolateral early Caenorhabditis elegans embryo reveals a role for membrane fusion transport in polarized Madin-Darby canine kidney cells. J. Cell Biol. 163, events in cytokinesis. Curr. Biol. 9, 738-745. 339-350. Kuchinke, U., Grawe, F. and Knust, E. (1998). Control of spindle orientation Apodaca, G., Katz, L. A. and Mostov, K. E. (1994). Receptor-mediated in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. transcytosis of IgA in MDCK cells is via apical recycling endosomes. J. Cell 8, 1357-1365. Biol. 125, 67-86. Lecuit, T. and Wieschaus, E. (2000). Polarized insertion of new membrane Bhat, M. A., Izaddoost, S., Lu, Y., Cho, K. O., Choi, K. W. and Bellen, H. from a cytoplasmic reservoir during cleavage of the Drosophila embryo. J. J. (1999). Discs Lost, a novel multi-PDZ domain protein, establishes and Cell Biol. 150, 849-860. maintains epithelial polarity. Cell 96, 833-845. Lecuit, T. and Wieschaus, E. (2002). Junctions as organizing centers in Bluemink, J. G. and de Laat, S. W. (1973). New membrane formation during epithelial cells? A fly perspective. Traffic 3, 92-97. cytokinesis in normal and cytochalasin B-treated eggs of Xenopus laevis. I. Lecuit, T., Samanta, R. and Wieschaus, E. (2002). Slam encodes a Electron microscope observations. J. Cell Biol. 59, 89-108. developmental regulator of polarized membrane growth during cleavage of Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., the Drosophila embryo. Dev. Cell 2, 425-436. Hoflack, B. and Zerial, M. (1992). The small GTPase rab5 functions as a Lee, O. K., Frese, K. K., James, J. S., Chadda, D., Chen, Z. H., Javier, R. regulatory factor in the early endocytic pathway. Cell 70, 715-728. T. and Cho, K. O. (2003). Discs-Large and Strabismus are functionally Burgess, R. W., Deitcher, D. L. and Schwarz, T. L. (1997). The synaptic linked to plasma membrane formation. Nat. Cell. Biol. 5, 987-993. protein syntaxin1 is required for cellularization of Drosophila embryos. J. Lipschutz, J. H. and Mostov, K. E. (2002). Exocytosis, the many masters of Cell Biol. 138, 861-875. the exocyst. Curr. Biol. 12, R212-R214. Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W., Poodry, C. A., Loncar, D. and Singer, S. J. (1995). Cell membrane formation during the Wadsworth, S. C. and Vallee, R. B. (1991). Multiple forms of dynamin cellularization of the syncytial blastoderm of Drosophila. Proc. Natl. Acad. are encoded by shibire, a Drosophila gene involved in endocytosis. Nature Sci. USA 92, 2199-2203. 351, 583-586. Low, S. H., Li, X., Miura, M., Kudo, N., Quinones, B. and Weimbs, T. Dollar, G., Struckhoff, E., Michaud, J. and Cohen, R. S. (2002). Rab11 (2003). Syntaxin 2 and endobrevin are required for the terminal step of polarization of the Drosophila oocyte, a novel link between membrane cytokinesis in mammalian cells. Dev. Cell 4, 753-759. trafficking, microtubule organization, and oskar mRNA localization and McNiven, M. A. (1998). Dynamin, a molecular motor with pinchase action. translation. Development 129, 517-526. Cell 94, 151-154. Drechsel, D. N., Hyman, A. A., Hall, A. and Glotzer, M. (1997). A Mostov, K. E., Verges, M. and Altschuler, Y. (2000). Membrane traffic in requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. polarized epithelial cells. Curr. Opin. Cell Biol. 12, 483-490. Curr. Biol. 7, 12-23. Muller, H. A. and Wieschaus, E. (1996). Armadillo, bazooka, and stardust Fanto, M. and McNeill, H. (2004). Planar polarity from flies to vertebrates. are critical for early stages in formation of the zonula adherens and J. Cell Sci. 117, 527-533. maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Fares, H., Peifer, M. and Pringle, J. R. (1995). Localization and possible Biol. 134, 149-163. functions of Drosophila septins. Mol. Biol. Cell 6, 1843-1859. Nelson, W. J. (2003). Adaptation of core mechanisms to generate cell polarity. Field, C. M. and Alberts, B. M. (1995). Anillin, a contractile ring protein that Nature 422, 766-774. cycles from the nucleus to the cell cortex. J. Cell Biol. 131, 165-178. O’Halloran, T. J. (2000). Membrane traffic and cytokinesis. Traffic 1, 921- Foe, V. E. and Alberts, B. M. (1983). Studies of nuclear and cytoplasmic 926. behaviour during the five mitotic cycles that precede gastrulation in Pelissier, A., Chauvin, J. P. and Lecuit, T. (2003). Trafficking through Rab11 Drosophila embryogenesis. J. Cell Sci. 61, 31-70. endosomes is required for cellularization during Drosophila embryogenesis. Foe, V. E., Odell, G. M. and Edgar, B. A. (1993). Mitosis and morphogenesis Curr. Biol. 13, 1848-1857. in the Drosophila embryo, point and counterpoint. In The Development of Petronczki, M. and Knoblich, J. A. (2001). DmPAR-6 directs epithelial Drosophila melanogaster, Vol. 1 (ed. M. Bate and A. Martinez-Arias) pp. polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. 149-300. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Cell. Biol. 3, 43-49. Folsch, H., Ohno, H., Bonifacino, J. S. and Mellman, I. (1999). A novel Pielage, J., Stork, T., Bunse, I. and Klämbt, C. (2003). The Drosophila cell clathrin adaptor complex mediates basolateral targeting in polarized survival gene discs lost encodes a cytoplasmic codanin-1-like protein, not a epithelial cells. Cell 99, 189-198. homolog of tight junction PDZ protein Patj. Dev. Cell 5, 841-851. Folsch, H., Pypaert, M., Maday, S., Pelletier, L. and Mellman, I. (2003). Riggs, B., Rothwell, W., Mische, S., Hickson, G. R., Matheson, J., Hays, Drosophila cellularization 3433

T. S., Gould, G. W. and Sullivan, W. (2003). Actin cytoskeleton mitosis and shares multiple components with cytokinesis. Development 129, remodeling during early Drosophila furrow formation requires recycling 5567-5576. endosomal components Nuclear-fallout and Rab11. J. Cell Biol. 163, 143- Stein, J. A., Broihier, H. T., Moore, L. A. and Lehmann, R. (2002). Slow 154. as molasses is required for polarized membrane growth and germ cell Rothwell, W. F., Fogarty, P., Field, C. M. and Sullivan, W. (1998). Nuclear- migration in Drosophila. Development 129, 3925-3934. fallout, a Drosophila protein that cycles from the cytoplasm to the Swanson, M. M. and Poodry, C. A. (1981). The shibire-ts mutant of centrosomes, regulates cortical microfilament organization. Development Drosophila, a probe for the study of embryonic development. Dev. Biol. 84, 125, 1295-1303. 465-470. Rothwell, W. F., Zhang, C. X., Zelano, C., Hsieh, T. S. and Sullivan, W. Thomas, G. H. and Williams, J. A. (1999). Dynamic rearrangement of the (1999). The Drosophila centrosomal protein Nuf is required for recruiting spectrin membrane skeleton during the generation of epithelial polarity in Dah, a membrane associated protein, to furrows in the early embryo. J. Cell Drosophila. J. Cell Sci. 112, 2843-2852. Sci. 112, 2885-2893. Turner, F. R. and Mahowald, A. P. (1976). Scanning electron microscopy of Royou, A., Field, C., Sisson, J. C., Sullivan, W. and Karess, R. (2003). Drosophila embryogenesis. 1. The structure of the egg envelopes and the Reassessing the role and dynamics of nonmuscle myosin II during furrow formation of the cellular blastoderm. Dev. Biol. 50, 95-108. formation in early Drosophila embryos. Mol. Biol. Cell 15, 838-850. Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. and Parton, R. G. (1996). Schejter, E. D. and Wieschaus, E. (1993a). Functional elements of the Rab11 regulates recycling through the pericentriolar recycling endosome. J. cytoskeleton in the early Drosophila embryo. Annu. Rev. Cell Biol. 9, 67- Cell Biol. 135, 913-924. 99. van Dam, E. M. and Stoorvogel, W. (2002). Dynamin-dependent transferrin Schejter, E. D. and Wieschaus, E. (1993b). Bottleneck acts as a regulator of receptor recycling by endosome-derived clathrin-coated vesicles. Mol. Biol. the microfilament network governing cellularization of the Drosophila Cell 13, 169-182. embryo. Cell 75, 373-385. van der Bliek, A. M. and Meyerowitz, E. M. (1991). Dynamin-like protein Sisson, J. C., Field, C., Ventura, R., Royou, A. and Sullivan, W. (2000). encoded by the Drosophila shibire gene associated with vesicular traffic. Lava lamp, a novel peripheral golgi protein, is required for Drosophila Nature 351, 411-414. melanogaster cellularization. J. Cell Biol. 151, 905-918. Warn, R. M. and Robert-Nicoud, M. (1990). F-actin organization during the Skop, A. R., Bergmann, D., Mohler, W. A. and White, J. G. (2001). cellularization of the Drosophila embryo as revealed with a confocal laser Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive scanning microscope. J. Cell Sci. 96, 35-42. membrane accumulation at the cleavage furrow apex. Curr. Biol. 11, 735- Wodarz, A., Ramrath, A., Grimm, A. and Knust, E. (2000). Drosophila 746. atypical protein kinase C associates with Bazooka and controls polarity of Sonnichsen, B., de Renzis, S., Nielsen, E., Rietdorf, J. and Zerial, M. epithelia and neuroblasts. J. Cell Biol. 150, 1361-1374. (2000). Distinct membrane domains on endosomes in the recycling pathway Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W. J. (2001). visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. Sec6/8 complexes on trans-Golgi network and plasma membrane regulate 149, 901-914. late stages of exocytosis in mammalian cells. J. Cell Biol. 155, 593-604. Sorensen, M. B., Mayer, U., Lukowitz, W., Robert, H., Chambrier, P., Young, P. E., Pesacreta, T. C. and Kiehart, D. P. (1991). Dynamic changes Jurgens, G., Somerville, C., Lepiniec, L. and Berger, F. (2002). in the distribution of cytoplasmic myosin during Drosophila embryogenesis. Cellularization in the endosperm of Arabidopsis thaliana is coupled to Development 111, 1-14.