InteractiveFly: GeneBrief

nuclear fallout: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition via vesicle fusion. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two remodeling processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homolog of arfophilin-2, an ADP ribosylation factor (ARF; see InterPro's ADP-ribosylation factor) effector that binds Rab-protein 11 (and Arf5) and influences recycling endosome (RE) organization. Nuf has been shown to be an important component of the RE; these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component implicated in vesicle targeting. Tests for protein interaction and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation. Membrane addition is mediated via endosomal-mediated membrane delivery to the site of furrow formation (Riggs, 2003).

It has been proposed that endosomes are organized into distinct domains defined by combinations of Rab proteins (Zerial, 2001). These provide a platform for regulatory/effector proteins to create a distinct fusion-competent domain. The proteins are thought to act cooperatively, and loss of one may destabilize the domain. Nuf and Rab11 may be mutually required for the stable formation of such a domain at the RE of the Drosophila embryo (Riggs, 2003).

The production of two daughter cells at the end of mitosis is accomplished through a dramatic constriction of the plasma membrane. This is known as cytokinesis and involves the formation of an actin/myosin-based contractile ring that forms perpendicular to and midway between the anaphase spindle. In animal cells, the position of the mitotic spindle largely determines the position and orientation of the contractile ring. Actin, myosin II, and other furrow components (such as anillin and the septins) are recruited to this site and form the contractile ring. Once the contractile ring forms, constriction of the plasma membrane occurs (Riggs, 2003 and references therein).

Although the mechanism of constriction is contractile, recent reports have begun to define the role of membrane addition in this process. A cell undergoing cytokinesis requires significant additional membrane to accommodate the increased surface area of producing two daughter cells. Work in Xenopus relying on a variety of surface-marking techniques indicates that the additional membrane has a different composition from the original membrane. This suggests that the membrane is not derived from the expansion of preexisting surface membrane, but instead forms through insertion of membrane from internal stores. In plant cells, it is well established that the additional membrane necessary for cytokinesis is provided through a Golgi-based delivery system. In Caenorhabditis elegans ovaries, RNA interference inhibition of Rab11, the small GTPase required for vesicle transport through the recycling endosome, causes cytokinesis defects, including furrow regression and scission (Skop, 2001). Mutation and RNA interference analyses demonstrate that the t-SNARE syntaxin 1 is required for cytokinesis during early embryogenesis. Lamellar bodies, the ER, and internal lipid stores may also prove important in providing membrane for cytokinesis furrows (Riggs, 2003 and references therein).

The rapid and simultaneous formation of thousands of furrows during early Drosophila embryogenesis makes this system particularly valuable for studying the recruitment of membrane and other furrow components during cytokinesis. Drosophila development begins with 13 synchronous, rapid, syncytial nuclear divisions. After nine divisions in the interior of the embryo, divisions 10-13 occur in the actin-rich cortex, just beneath the plasma membrane. The nuclei and their associated centrosomes induce a dramatic redistribution of the cortical actin. During interphase, actin concentrates into caps centered above each cortical nucleus and its apically positioned centrosomes. As the nuclei progress into prophase, the centrosomes migrate toward opposite poles and the actin caps undergo a dramatic redistribution to form an oblong ring outlining each nucleus and its associated separated centrosome pair. These rings are equivalent in composition to conventional cytokinesis contractile rings and include actin, myosin II, spectrins, cofilin, ARP, anillin, septins, and formins. In addition, these components are closely associated with the plasma membrane and are required for the invagination of these rings around the spindles. These rings are referred to as metaphase or pseudocleavage furrows. At metaphase, the furrows invaginate to a depth of ~8 microm to form a half shell that encompasses each spindle. During late anaphase and telophase, the metaphase furrows rapidly regress. Centrosome duplication occurs during late anaphase, and the newly formed centrosome pairs locate apically. The actin caps reform directly above the centrosome pairs in the next interphase. This alternation between interphase actin caps and metaphase furrows occurs until interphase of nuclear cycle 14. At this point, the nuclei remain in interphase and an inverted microtubule basket, which originates from an apically positioned centrosome pair, guides invagination of the cellularization furrows. At a depth of micro5 µm, the furrows pinch off at their base to form individual mononucleate cells (Riggs, 2003 and references therein).

Genetic and biochemical analyses indicate that vesicle fusion plays an important role in furrow formation in early Drosophila embryogenesis. Mutations in dynamin, a GTPase involved in endocytic vesicle formation, disrupt cellular furrow formation and result in an abnormal accumulation of vesicles in the cytoplasm. Unconventional myosin VI has been shown to be involved in the transport of cytoplasmic particles in the Drosophila embryo, and mutations in this gene cause defects in formation of the metaphase furrows. alpha-Adaptin, a coated vesicle component necessary for receptor-mediated endocytosis, is concentrated apically and laterally around the metaphase and cellularization furrows. Syntaxin 1, a t-SNARE involved in vesicle targeting, is also required for cellularization in Drosophila. Inhibition of Golgi-based vesicle transport inhibits progression of the cellularization furrow front. In addition, a major source of this membrane necessary for the cellularization furrows is derived internally rather than from the plasma membrane (Riggs, 2003 and references therein).

Activities associated with the centrosome are also important for vesicle-mediated metaphase and cellular furrow formation. Insights into the centrosome-associated activities directing these rearrangements have come from the analysis of the maternal effect mutation, nuclear fallout (nuf). Nuf encodes a pericentrosomal protein that is essential for normal metaphase and cellularization furrow formation. Nuf concentrates at the centrosomes during prophase, when metaphase furrows are forming (Rothwell, 1998). In the nuf mutation, microtubule dynamics and distribution appear normal, but remodeling and recruitment of actin to the furrows is disrupted and actin remains abnormally concentrated around the centrosomes. Vesicle-based membrane recruitment to the furrows is also disrupted in nuf-derived embryos (Rothwell, 1999; Zhang, 2000). These phenotypes lead to the intriguing suggestion that a common mechanism mediates actin remodeling and membrane addition during cytokinesis (Riggs, 2003 and references therein).

Additional insight into these two processes is provided by demonstrating that Nuf is a component of the RE, and nuf phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a member of the Rab family of small GTPases specific to the RE. In addition, Rab11 and Nuf exhibit a mutual dependence for their normal localization to the RE. Rab11-deficient embryos produce metaphase and cellular furrow defects strikingly similar to those observed in nuf-derived embryos. In accord with these results, recent reports demonstrate that Nuf is a homolog of arfophilin-2 (Arfo2), an ADP ribosylation factor (Arf) effector that also binds Rab11 and influences RE organization (Hickson, 2003). Together, these reports suggest that actin remodeling during the initial stages of cytokinesis may in part rely on endosomal-mediated membrane delivery to the site of furrow formation (Riggs, 2003).

Nuf and Arfo2 are functionally as well as structurally related. In HeLa cells, Arfo2 localizes to the perinuclear TGN with staining also observed at the centrosomes and focal adhesions (Hickson, 2003). In Drosophila, Nuf has a similar localization at the centrosomes (Rothwell, 1998). Overexpression of either Drosophila Nuf or human Arfo2 in mammalian cells results in a collapse of the late RE to a pericentrosomal region (Hickson, 2003). These observations suggest that Nuf and Arfo2 are functionally similar and play a role in maintaining the integrity of the RE (Riggs, 2003).

The fact that both Nuf and Arfo2 contain a conserved Rab11-binding domain provides additional support for a common function at the RE. Similar to Arfs, Rabs are members of a large family of small GTPases involved in the regulation of vesicle-trafficking pathways (Segev, 2001). However, unlike Arfs, Rabs are thought to be involved in vesicle targeting rather than vesicle biogenesis. Rab11 is primarily localized at the RE and plays an essential role in receptor-mediated recycling to the plasma membrane (Ullrich, 1996; Sheff, 2002). In addition, the Rab11 GTPase cycle is essential for normal RE organization and function (Ullrich, 1996). Sequence analysis of Arfo2 and Nuf reveals a common conserved 20-aa Rab11-binding domain originally identified among members of the Rab11-interacting protein family (Hales, 2001; Prekeris, 2001). In accord with this observation, Arfo2 and Nuf physically interact with Rab11 (Riggs, 2003).

Nuf is primarily associated with the RE in the early Drosophila embryo. Nuf shows extensive colocalization with Rab11. The most significant difference between the distribution of Rab11 and Nuf in the early embryo is that the former maintains a constant level of pericentriolar staining, whereas levels of the latter oscillate with the cell cycle. During the cortical syncytial divisions, pericentriolar Nuf staining is at its highest levels at prophase and negligible during metaphase and anaphase. It is not known whether this is a result of cycling of Nuf levels, subcellular location, or both. At nuclear cycle 14, Nuf levels are highest during interphase as the cellularization furrows are forming. Thus, maximal pericentriolar levels of Nuf are correlated with metaphase and cellular furrow formation and invagination. Nuf is highly phosphorylated (Rothwell, 1998), raising the possibility that its localization and/or levels may be regulated by cell cycle–dependent kinases (Riggs, 2003).

Further evidence that Nuf is intimately associated with pericentriolar endosomal material comes from live analysis of Nuf dynamics in the early embryo. This analysis reveals a dynamic punctate distribution of Nuf rapidly moving to and from the centrosome. Dual imaging reveals that these puncta are closely associated with astral microtubules, and disruption of the microtubule network severely disrupts GFP-Nuf distribution and movement (unpublished data). This colocalization and dependency of the microtubule network has also been demonstrated for Rab11 and GFP-Arfo2 (Mammoto, 1999; Hickson, 2003). In comparison with live fluorescent analysis of GFP-Rab11 in mammalian systems (Sonnichsen, 2000), GFP-Nuf shows a similar localization, distribution, and movement pattern. This supports the view that Nuf localizes to the RE and that these images reflect RE dynamics in the Drosophila embryo (Riggs, 2003).

The results also demonstrate a mutual dependence of Nuf and Rab11 for their localization to the RE. In nuf-derived embryos, the robust Rab11 pericentriolar distribution is completely disrupted. Whether Nuf is specifically disrupting Rab11 localization to the RE or more globally disrupting RE integrity is not known. However, the effect of Nuf is believed to be specific to the RE, since Golgi morphology and distribution is normal in nuf-derived embryos. The effect of nuf mutations on Rab11 localization is consistent with reports (Hickson, 2003) demonstrating that overexpression of GFP-Arfo2 alters the organization of Rab11 in mammalian cells. Conversely, Nuf pericentriolar localization fails in embryos with reduced levels of Rab11 (Riggs, 2003).

Analysis of nuclear and cortical cytoskeletal defects in nuf- and rab11-derived embryos supports the idea that Nuf and Rab11 are involved in a similar function at the RE. As observed in the nuf mutation, embryos with reduced levels of Rab11 disrupt the syncytial nuclear divisions only after the nuclei reach the cortex. This phenotype indicates that Rab11 is involved in a process specific to the cortical divisions such as cytoskeletal rearrangements or furrow formation. Also like nuf, rab11-derived embryos exhibit fusions between nonsister nuclei, a hallmark of defective furrow formation (Riggs, 2003).

Previous analysis of nuf-derived embryos has revealed normal actin organization during interphase, but gaps occur in the actin network early in the process of furrow formation (Rothwell, 1998). Analysis of rab11-derived embryos reveals an equivalent phenotype with respect to actin; the interphase actin caps form normally, but the actin-based metaphase furrows are disrupted. Previous analysis of actin dynamics in the nuf-derived embryos revealed that actin recruitment during the initial stages of furrow formation is compromised (Rothwell, 1999). Fixed analysis of actin defects in rab11-derived embryos reveals actin gaps at the initial stages of furrow formation. Therefore, the rab11 furrow defects are likely the result of defects in the initial recruitment of actin to the furrows (Riggs, 2003).

Although the nuf mutation only partially disrupts actin recruitment to the invaginating furrows, it has a much more severe effect on membrane recruitment. The Drosophila homolog of the dystrobrevins, Dah, was used as a marker for furrow membrane (Zhang, 1996). Biochemical analysis demonstrates that this protein associates tightly with actin and membrane, suggesting it is involved in linking the cortical cytoskeleton and the plasma membrane (Zhang, 2000). Immunofluorescent analysis reveals that it localizes to the plasma membrane and invaginating furrows, as well as vesicles that accumulate at furrow formation sites (Rothwell, 1999). These vesicles are often associated with actin, suggesting that they incorporate as a unit into the growing furrow. In nuf-derived embryos, there is some localization of Dah at the furrows; however, most remain in vesicles widely dispersed throughout the cortex (Riggs, 2003; Rothwell, 1999). The effect of the rab11 mutation on Dah localization is even more severe. There is no Dah localization at the furrows, and few Dah-containing vesicles are seen throughout the cortex (Riggs, 2003).

nuf and rab11 mutations disrupt membrane recruitment and actin remodeling during the early stages of furrow formation, supporting the argument that these proteins function in a common process at the RE. Analysis of Rab11 function in C. elegans reveals that it also is important for normal furrow progression during cytokinesis (Skop, 2001). However, this analysis showed varying degrees of defects during furrow invagination, suggesting a role for Rab11 during either the initial stages or latter stages (or both) of cytokinesis. In the Drosophila embryo, Rab11 appears to be involved in the initial stages of furrow formation when actin is being recruited to the invaginating furrow (Riggs, 2003).

These analyses indicate that activities of Nuf and Rab11 at the RE influence cortical actin dynamics. Specifically, they direct the recruitment of actin to the sites of metaphase furrow formation. One explanation for this linkage between the endosome and cortical actin dynamics is that membrane and actin are recruited as a unit to the metaphase furrows (Rothwell, 1999). Immunofluorescent analysis reveals that Dah-containing vesicles are often tightly associated with actin at the leading edge of the invaginating furrows. Therefore, disrupting membrane recruitment would also disrupt actin recruitment (Riggs, 2003).

An intriguing alternative explanation for trafficking activities at the RE influencing actin recruitment during the initial stages of furrow formation comes from reports that Rac GTPases are positioned in the cell through the endosomal recycling pathway. For example, Arf6 GTPase regulates an endosomal recycling pathway and cortical actin remodeling at the plasma membrane. In HeLa cells, ARF6 and Rac1, a potent actin organizer, colocalize at the plasma membrane as well as the RE. Mutational analysis and drug analyses indicate that ARF6 influences actin dynamics by regulating the trafficking of Rac1 to the plasma membrane. This latter model readily explains the effects of Rab11 and Nuf mutations on both actin recruitment and membrane delivery. These proteins are not only required at the RE for membrane delivery to the metaphase and cellularization furrows, but they are also required for the delivery of actin-remodeling proteins, such as Rac, to the plasma membrane (Riggs, 2003 and references therein).

Cortical actin remodeling and localized plasma membrane expansion not only mediate cytokinetic furrow formation, but also are involved in cell motility, lamellipodia formation, and phagocytosis. Phagocytosis is particularly interesting because recent work has shown that it occurs through targeted delivery of vesicles from the RE. Accumulation of RE-derived VAMP3-containing vesicles occurs at the site of phagosome formation, and disruption of VAMP3 with tetanus toxin prevents phagosome formation. As has been demonstrated for metaphase and cellular furrow formation, activity at the RE may also mediate cortical actin cytoskeletal remodeling during phagocytosis (Riggs, 2003 and references therein).

REGULATION

Protein Interactions

Nuf is a structural and functional homolog of Arfo2 (Hickson, 2003) and contains a highly conserved 20-aa Rab11-binding site. This binding domain was first identified by Prekeris (2001) and Hales (2001) as important for the interaction between Rab11 and a novel family of putative Rab11 effector proteins. Within this domain, Nuf and Arfo2 contain eight identical and six conserved amino acids. Nuf and hRip11, a mammalian Rab11 effector protein, contain ten identical and three conserved amino acids. This sequence conservation, combined with the colocalization results, prompted an examination of whether Nuf and Rab11 physically interact. Bacterially expressed GST-Rab11 was mixed with CHO cells transiently expressing GFP-Nuf. GTPgammaS and GDPßS were added to the buffer to test the nucleotide specificity of the interaction. GFP-Nuf is effectively pulled down by both GST-Rab11+GTPgammaS and GST-Rab11+GDPßS, indicating that the interaction is not tightly linked to the state of the nucleotide. GST-Rab11+GDPßS pulls down Nuf to a lesser extent than GST-Rab11+GTPgammaS. Nucleotide-independent binding has also been observed with other Rab11 effectors, Rab11-FIP2 (Hales, 2001) and Arfo2, the mammalian homolog of Nuf (Hickson, 2003). To test the specificity of the interaction, similar pull-down experiments were performed with Rab5, a component of the early endosome. Unlike the results with GST-Rab11, GFP-Nuf is not pulled down by GST-Rab5 in either the activated or unactivated form (Riggs, 2003).

To determine if Nuf is required for pericentriolar Rab11 localization, Rab11 (Rab-protein 11) localization was examined in nuf-derived embryos. Rab11 exhibits a concentrated punctate distribution around the centrosome during prophase. In nuf-derived embryos, both the punctate distribution and concentration of Rab11 around the centrosomes is completely abolished. Although levels of Nuf at the centrosome are greatly reduced during metaphase, Nuf is required for Rab11 centrosome localization at this stage as well. Nuf is also required for Rab11 localization during cellularization. The robust tight localization of Rab11 around the centrosome during cellularization is absent in nuf-derived embryos. It is believed that mislocalization of Rab11 in nuf is not a result of a general disruption of the intracellular transport pathway, since staining with Golgi marker Lava-lamp revealed normal Golgi distribution throughout the cell cycle in wild-type and nuf-derived embryos. From this analysis, it cannot be determined whether levels of Rab11 protein are reduced in nuf-derived embryos (Riggs, 2003).

Whether Rab11 is required for normal pericentriolar Nuf localization was also examined. Because Rab11 is an essential gene, a combination of hypomorphic rab11 alleles were used that permitted normal zygotic development (Jankovics, 2001). However, these transheterozygote females produced embryos with reduced levels of maternally supplied Rab11 and showed a reduced hatch rate. Wild-type and rab11-derived embryos were double stained for Nuf and DNA, and were examined during the syncytial divisions and cellularization. During prophase, while the pericentriolar localization of Nuf was robust in control embryos, pericentriolar Nuf levels were absent in rab11-derived embryos. The same result was obtained when cellularizing rab11-derived embryos were examined; the normal pericentriolar localization of Nuf is completely abolished. From this analysis, it cannot be determined whether levels of Nuf protein are reduced in rab11-derived embryos. These experiments demonstrate that Nuf and Rab11 are mutually dependent on one another for their localization to the RE (Riggs, 2003).

Asymmetric Rab11 endosomes regulate Delta recycling and specify cell fate in the Drosophila nervous system

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. This study describes a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. Trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, it was demonstrated that recycling endosomes are essential for Delta activity. These results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments (Emery, 2005).

To test whether Rab11 asymmetry is important for cell-fate specification, Rab11 accumulation in the pIIa cell was induced by nuf expression. Postorbital ES organs, which can easily be scored in fairly high numbers, were used. Cell-fate transformations upon nuf overexpression have been described (Abdelilah-Seyfried, 2000), but surprisingly, they do not occur at high frequency. Such transformations can, however, be observed upon coexpression of constitutively active Rab11. Upon expression of nonphosphorylatable lgl, Numb and Neuralized asymmetry are disrupted, but most ES organs still develop normally. When both pathways are disrupted by coexpression of lgl3A and nuf, however, a large fraction of ES organs shows cell-fate transformations that are consistent with a higher level of Delta activity in pIIa. Lineage analysis shows cell-fate transformations in 44% of postorbital ES organs, and in 18% of these, pIIb cells are transformed into pIIa cells (6% in ES organs expressing lgl3A alone). Twenty-five percent of the cell fate transformations affect the first (SOP) while 75% affect the second (pIIa) division, indicating that Rab11 asymmetry also plays a role in other divisions of the SOP lineage. Taken together, these results suggest that two partially redundant pathways exist to generate asymmetry in the SOP lineage: the Par proteins phosphorylate Lgl to direct Numb and Neuralized into the pIIb cell where they repress Notch or activate Delta, respectively. In the pIIa cell, inhibition of Nuf and Rab11 inhibits Delta by preventing its trafficking through the recycling endosome (Emery, 2005).

These results suggest that cells can also regulate signal transduction pathways by controlling the formation or distribution of whole endocytic compartments. After SOP division, Rab11-positive vesicles accumulate around the centrosome in this cell but not in pIIa. Rab11 plays a well-documented role in controlling vesicular protein transport through recycling endosomes to the plasma membrane (Zerial, 2001). Dominant-negative forms of Rab11 inhibit the recycling of endocytosed Transferrin receptors or recruitment of H⁺-K⁺-ATPase to the plasma membrane suggesting that Rab11 regulates trafficking of vesicular cargo through the recycling endosomal compartment. In SOP cells, the asymmetric localization of Rab11 reflects a different ability of pIIa and pIIb cells to recycle the Notch ligand Delta. Rab11 asymmetry is observed 3.5 min after cytokinesis but Delta is in recycling endosomes only 15 min after endocytosis. Thus, the protein is endocytosed before mitosis and recycles back to the plasma membrane in pIIb but not in pIIa. In pIIa, more Delta/Hrs double-positive vesicles are observed, indicating that the protein enters a late-endosomal pathway (Emery, 2005).

Several observations indicate that passage through recycling endosomes is essential for Delta to signal. In a marrow stromal cell line, OP9, inhibition of recycling endosomes dramatically reduces Delta signaling capacity. Similarly, blocking the recycling pathway by overexpression of a dominant-negative form of Rab11 in SOP cells causes relocalization of Delta into enlarged late endosomes. In Drosophila wing discs, Delta has been postulated to pass through a specific endocytic recycling pathway to acquire signaling capacity (Wang, 2004). Finally, Jafar-Nejad (2005) demonstrates that the Rab11 binding partner Sec15 is required both for Delta trafficking and Notch activation in the SOP lineage. Sec15 is a component of the exocyst and is a Rab11 effector (Zhang, 2004). Although Sec15 is not asymmetric itself, it is conceivable that the higher amounts of GTP bound Rab11 in pIIb increase its activity in delivering Delta to the plasma membrane. A difference between Delta trafficking in pIIa and pIIb has been observed previously (Le Borgne, 2003), but both Delta/Hrs vesicles and total number of Delta vesicles were actually higher in pIIb in these previous experiments. While these earlier experiments analyzed the whole two cell stage, this study focusses on the short time interval right after mitosis where Rab11 is asymmetric. This explains the different outcome and might in fact indicate that pIIb cells switch from an initial phase where Delta is recycled to a later phase where trafficking is regulated by neuralized-dependent endocytosis (Emery, 2005).

Although many cell types in different organisms undergo asymmetric cell division, only one mechanism has been identified so far that directs this important biological process in animals. This mechanism involves the Par proteins, which phosphorylate Lgl on one side and direct cell fate determinants to the opposite side of the cell cortex. Several results indicate that other pathways might exist: in dividing progenitor cells of the mammalian brain, Numb segregates into one of the two daughter cells and is required for lineage specification. However, some of these divisions are asymmetric, although their orientation predicts that Numb would be inherited by both daughter cells. In Drosophila SOP cells, lgl3A overexpression affects both Numb and Neuralized localization but has only a minor influence on the asymmetric outcome of the division. The results indicate that the asymmetric distribution of Rab11 is established through a distinct pathway: (1) Rab11 asymmetry is unaffected in SOP cells overexpressing lgl3A; (2) Rab11 is still asymmetric in dlg mutants where Par proteins do not localize and Numb and Neuralized segregate into both daughter cells; (3) Rab11 asymmetry can be uncoupled from Numb and Neuralized localization by the expression of inscuteable; (4) the events responsible for Rab11 asymmetry seem to occur in the pIIa cell, but none of the known determinants is inherited by this daughter cell. Although the observations could also be explained if Numb or Neuralized would relieve a general suppression of recycling endosome formation in the SOP lineage, this is unlikely since Rab11 asymmetry is unaffected in numb or neuralized mutants. More likely, an unknown factor could act on Nuf or the centrosome in the pIIa cell to prevent Rab11 accumulation. Nuf localization is cell cycle regulated, and a key regulatory component could be missing in pIIa. For example, Nuf is highly phosphorylated and differential activity of a kinase or phosphatase could prevent its pericentriolar localization in the pIIa cell. Homologs of Nuf exist and bind to Rab11 in vertebrates. Their expression pattern has not yet been described but it will be interesting to determine whether these homologs regulate Notch signaling in vertebrates and are responsible for asymmetric cell division in the mammalian brain (Emery, 2005).

The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation

Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, this study demonstrates that microtubules are required for proper metaphase furrow formation. It is proposed that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows (Riggs, 2007; full text of article).

Microtubule-based motility has been implicated in many steps in endocytosis, and there is increasing evidence that it influences the distribution and activity of endocytic organelles. The work presented in this study suggests that motor-based movement of Rab effectors may be another means of regulating endosomal activity. Previous studies have shown that the Drosophila Rab11 effector, Nuf, is required for stable Rab11 localization at the RE and thus RE activity. Nuf concentrates at the MTOC during interphase through prophase and disperses into the cytoplasm at metaphase. This study demonstrates that Nuf relies on microtubules and minus-end microtubule motor Dynein both for its accumulation and maintenance at the MTOC. This raises the possibility that the Dynein-dependent delivery of Nuf to the RE may play a role in regulating Rab11 activity at the RE. Significantly maximal localization of Nuf at the MTOC-associated RE occurs during late interphase and prophase. This is the time of the establishment and formation of the metaphase furrows, which rely on RE-based vesicle delivery (Riggs, 2007).

Immunoprecipitation data demonstrates a physical interaction between Nuf and Dynein. This raises the possibility that the cell cycle-regulated localization of Nuf at the MTOC is mediated by a corresponding cell cycle-regulated interaction between Nuf and Dynein. Support for this idea comes from a study in vertebrate cells, demonstrating that Polo-like kinase (Plk) mediated phosphorylation of Ninein-like protein (Nlp), a microtubule-nucleating protein, directly determines its cell cycle-regulated localization at the centrosome. Like Nuf, Nlp localizes to the centrosome by associating with the minus-end-directed motor protein Dynein. As cells progress into metaphase, Plk is activated and phosphorylates Nlp on sites that are required for its association with Dynein. This disrupts Nlp ability to associate with Dynein and results in loss of Nlp from the centrosome (Riggs, 2007).

There is a strong correlation between maximal Nuf localization at the MTOC and furrow invagination. During the cortical divisions, furrow invagination and maximal Nuf concentration at the MTOC occurs during prophase. During cellularization, furrow invagination and maximal Nuf concentration at the MTOC occurs during interphase. Stable localization of Nuf and Rab11 at the MTOC during cellularization enabled a demonstration that microtubules are continuously required for maintaining Nuf and Rab11 at the MTOC. Colchicine-induced disruption of the interphase microtubules results in the rapid loss of Nuf from the MTOC. One interpretation of this result is that colchicine disrupts MTOC organization, which is required for maintaining Nuf at the MTOC. In contrast to the colchicine injections, injecting anti-Dynein antibody does not alter microtubule organization and results in a slow steady decrease of Nuf at the MTOC. This result suggests that the steady-state level of Nuf at the MTOC is maintained by continuous Dynein-dependent recruitment of Nuf to the MTOC. This also implies that Nuf is continuously released from the MTOC as well. The mechanism driving the release is unclear. Previous live analysis revealed vectorial movement of Nuf away from the centrosome, suggesting that it may rely on a kinesin, a plus-end-directed microtubule motor. If kinesin is involved, this implies that the balance between plus- and minus-end motor activities dictates whether Nuf is concentrated at the MTOC or dispersed in the cytoplasm. Recent work indicates that the positioning and activity of the early endosome is mediated through a balance of plus- and minus-end motor activities. In addition, investigations into cellular furrow elongation demonstrated that Lava lamp, a Golgi-associated protein, is complexed with Dynein and is responsible for Golgi-based movements necessary for latter half of furrow elongation (Riggs, 2007 and references therein).

The above studies demonstrate that microtubules are continuously required for proper Nuf localization at the MTOC. This raises the possibility that microtubule-based localization of Nuf at the MTOC is necessary for its association with the Rab11 and proper RE function. Because RE function is necessary for metaphase furrow formation, this predicts that microtubules are required for proper metaphase furrow formation. However previous studies did not observe defects in furrow formation when embryos were treated with microtubule inhibitors. It has been concluded that microtubules are dispensable for proper metaphase furrow formation in the early embryo. This issue was reexamined by injecting microtubule inhibitors at precise times throughout the cell cycle during the syncytial divisions. Because disrupting the microtubules at metaphase activates the spindle assembly checkpoint, the embryos were injected immediately after entry into anaphase. In these experiments, the nuclear cycle progressed normally but formation of the metaphase furrows were profoundly disrupted. Incorporation of GFP-tagged Moesin into the furrows that form at the next prophase completely fails. Thus these experiments define anaphase as a key time in which microtubules are required for recruiting actin to the furrows that form in the following prophase. The previous study failed to appreciate the role of microtubules in metaphase furrow formation because it was not possible to produce disruptions in the microtubule network at defined stages of the cell cycle (Riggs, 2007).

These studies also revealed that injecting colchicine at telophase produced no defects in actin recruitment. Similar injections at interphase through prophase also produced no defects in actin recruitment to the metaphase furrows. One interpretation of these results is that microtubules are specifically required during anaphase but not telophase or later for furrow formation in the next prophase. However it must be pointed the different classes of microtubules are differentially sensitive to microtubule inhibitors. Thus this differential sensitivity may contribute to the observed cell phase sensitivity of metaphase furrow formation to colchicine (Riggs, 2007).

That microtubules are required during anaphase for metaphase furrow formation in the following prophase is significant for a number of reasons. First, these studies support, although certainly do not prove, a model in which microtubule-based transport of Nuf to the MTOC is necessary for normal metaphase furrow formation. Second, anaphase/telophase is the point at which the metaphase furrows begin to regress. Thus the timing of furrow regression corresponds to the time at which microtubules are involved in establishing the next round of furrow formation. This indicates that the speed of the cortical divisions is not only achieved by an accelerated nuclear cycle but also by overlapping furrow regression with furrow formation. During anaphase, the replicated centrosomes possess robust astral arrays and the midbody has not yet fully formed. It is hypothesized that the plus ends of these overlapping arrays from neighboring centrosomes define the position of the metaphase furrow in the next cell cycle. This readily explains why furrows encompass the spindle and do not form at the midzone microtubules. Finally, although the furrows form at prophase, these studies identify anaphase as a critical time in which furrow is established. This also corresponds to the time at which microtubules are required during conventional furrow formation (Riggs, 2007).

Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization

Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila early embryo, Nuclear fallout, a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. In nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. RhoGEF2 does not properly localize at the furrow in nuf mutant embryos, and RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. A model is proposed in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway (Cao, 2008).

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. This study shows that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. These data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation (Le, 2021).

MTs have a crucial role in stabilizing apical myosin during epithelial morphogenesis both in early Drosophila embryos and in the Drosophila SG. In the SG, MTs interact with apicomedial myosin via Short stop, the Drosophila spectraplakin, emphasizing a direct interplay between the MT and the apical myosin networks. The data of this study reveal another key role of MTs in regulating protein trafficking to control the apical myosin networks during tissue invagination. During SG invagination, a network of longitudinal MT bundles is observed near the invagination pit. These data show apical enrichment of Rab11 in the same area is MT dependent and that this enrichment is important for forming the apicomedial myosin networks, suggesting a link between localized intracellular trafficking along MTs to apical myosin regulation (Le, 2021).

The dorsal/posterior region of the SG, where Rab11 is apically enriched in a MT-dependent manner, correlates with localized Fog signaling activity that promotes clustered apical constriction. Disruption of MTs or Rab11 knockdown reduces Fog signals in the apical domain of SG cells and causes dispersed Rok accumulation and defective apicomedial myosin formation. It is consistent with a previous study that the absence of Fog signal results in dispersed apical Rok and defects in apicomedial myosin formation. It is therefore proposed that MT- and Rab11-dependent apical trafficking regulates Fog signaling activity to control apical constriction during epithelial tube formation through transporting the Fog ligand. As recycling of membrane receptors to the cell surface plays an important role in regulating overall signaling activity, it is possible that Rab11 is involved in recycling the as yet unidentified SG receptor(s) of Fog to regulate Fog activity in the SG. Indeed, several GPCRs are recycled via Rab11. During epithelial invagination in early Drosophila embryogenesis, the concentration of the Fog ligand and receptor endocytosis by 'β-arrestin-2 have been shown as coupled processes to set the amplitude of apical Rho1 and myosin activation. It is possible that the movement of Fog receptor(s) that have internalized as a stable complex with 'β-arrestin is recycled back to the cell surface by Rab11. The Fog signaling pathway represents one of the best understood signaling cascades controlling epithelial morphogenesis. Although best studied in Drosophila, the pathway components have also been identified in other insects, suggesting a more widely conserved role for Fog signaling in development. Further work needs to be done to fully understand the regulatory mechanisms underlying the trafficking of Fog and its receptor(s) during epithelial morphogenesis (Le, 2021).

Analysis of apicomedial myosin shows that reduced Rab11 function not only causes a decrease of the myosin intensity but also causes myosin to be dispersed rather than forming proper myosin web structures in the apicomedial domain of SG cells. These data support the idea that Rab11 function is required for both concentration and spatial organization of apicomedial myosin. This can be explained by the combined effect of multiple cargos that are transported by Rab11, including Fog, Crb, and E-Cad. Time-lapse imaging of myosin will help determine how the dynamic behavior of apicomedial myosin is compromised when Rab11 function is disrupted (Le, 2021).

During branching morphogenesis in Drosophila trachea, MTs and dynein motors have a critical role in the proper localization of junctional proteins such as E-Cad. This is consistent with the observations with MT-dependent uniform distribution of E-Cad at adherens junctions in the invaginating SG, suggesting a conserved role of MT-dependent intracellular trafficking in junctional remodeling and stabilization during epithelial tube formation. The data further suggest that the MT networks and Rab11 have key roles in apical distribution of Crb and E-Cad in the SG and that proper levels of apical and junctional proteins are important for apical constriction during SG invagination. Based on these data, it is proposed that MT- and Rab11-dependent apical trafficking of Crb and E-Cad is critical for apical constriction during SG invagination. Alternatively, MTs have an additional role in assembling/anchoring these apical components through the regulation of unidentified molecules. Recent studies in Drosophila mesoderm invagination showed that MTs help establish actomyosin networks linked to cell junction to facilitate efficient force transmission to promote apical constriction. In Ko (2019), however, MT-interfering drugs and RNAi of CAMSAP end-binding protein were used to prevent MT functions and the effect cannot be directly compared with the current data where spastin was used to sever existing MTs. Direct monitoring of MT-dependent transport of Crb and E-Cad during SG invagination will help clarify the mechanism (Le, 2021).

On knockdown of crb or E-Cad, less prominent apicomedial myosin web structures are observed in invaginating SGs, suggesting a requirement of Crb and E-Cad in proper organization of apical actomyosin networks during SG tube formation. Crb acts as a negative regulator of actomyosin dynamics during Drosophila dorsal closure and during SG invagination. It is possible that proper Crb levels are required for modulating myosin activity both in the apicomedial domain and at junctions during SG invagination. Both of which contribute to apical constriction and cell rearrangement, respectively. Anisotropic localization of Crb and myosin was observed at the SG placode boundary, where myosin accumulates at edges where Crb is lowest. Planar polarization of Rok at this boundary is modulated through phosphorylation by Pak1 downstream of Crb. A further test will help understand whether and how Crb might affect junctional myosin dynamics and SG invagination. As contractile actomyosin structures exert forces on adherens junction to drive apical constriction, it is speculated that apical constriction defects on E-Cad RNAi might be due to reduction of cell adhesion and/or of improper force transmission. It will be interesting to determine if the coordination of apical and junctional proteins and apical cytoskeletal networks through intracellular trafficking is conserved during tubular organ formation in general (Le, 2021).

Dhc is also apically enriched in the dorsal/posterior region of the invaginating SG. The data show that knockdown of Dhc64C not only affects Rok accumulation and apicomedial myosin formation but also disrupts MT organization in the SG. These data are consistent with previous findings that cytoplasmic dynein is associated with cellular structures and exerts tension on MTs. For example, dynein tethered at the cell cortex can apply a pulling force on the MT network by walking toward the minus end of a MT. Dynein also scaffolds the apical cell cortex to MTs to generate the forces that shape the tissue into a dome-like structure. In interphase cells, the force generated by dynein also regulates MT turnover and organization (Le, 2021).

In klar mutants, on the other hand, MT organization is not affected in the SG, suggesting that reduction of dynein-dependent trafficking by loss of klar does not cause changes in the MT networks. Notably, although the intensity of apicomedial myosin does not change on Dhc64C knockdown or in the klar mutant background, formation of apicomedial myosin web structures is affected. These data suggest a possible scenario that dynein function is not required for myosin concentration in the apical domain but is only needed for the spatial organization of apicomedial myosin. However, it cannot be ruled out that the zygotic knockdown of Dhc64C by RNAi is not strong enough to affect the intensity of apicomedial myosin. Dhc64C has strong maternal expression and is essential for oogenesis and early embryo development. Embryos with reduced maternal and zygotic pools of Dhc64C showed a range of morphological defects in the entire embryo, some of which were severely distorted. Precise roles for dynein and dynein-dependent trafficking in regulating apicomedial myosin formation remain to be elucidated (Le, 2021).

DEVELOPMENTAL BIOLOGY

Embryonic

Immunofluorescent analysis with this antibody reveals that, during the late syncytial divisions, Nuf is highly concentrated at the centrosomes during prophase and is cytoplasmic during the other stages of the nuclear cycle. Nuf is also concentrated at the centrosomes during interphase of nuclear cycle 14 and throughout cellularization. Double stains of wild-type embryos with Nuf and tubulin verify the centrosomal localization of Nuf. The Nuf antibody does not stain centrosomes in equivalently staged nuf-derived embryos. To show that this lack of staining was not due to an inability of the antibodies to access the centrosomes in the nuf-derived embryos, normal and nuf-derived embryos were stained with anti-centrosomin, a Drosophila centrosomal antibody. Both normal and nuf-derived embryos reacted strongly with this antibody. This demonstrates that nuf embryos contain centrosomes. Wild-type embryos were double stained with phalloidin and anti-Nuf antibodies to correlate actin dynamics with Nuf centrosomal localization. This analysis revealed that the localization of Nuf to the centrosomes during prophase of the syncytial divisions occurs during the most dramatic reorganization of actin from caps to furrows. Nuf centrosome staining during cellularization occurs as the actin enters cellularization furrows and as the furrows extend (Rothwell, 1998).

Nuf concentrates at the centrosomes during prophase and diffusely localizes throughout the cytoplasm during the remainder of the cell cycle (Rothwell, 1998). To visualize the cell cycle dynamics of Nuf in real time, a GFP-Nuf transgenic line was constructed. The GFP-Nuf construct completely rescues nuf-induced maternal lethality. Live analysis of a GFP-Nuf–expressing embryo indicates that during interphase, Nuf accumulates at each of the separating centrosomes. During prophase, immediately before nuclear envelope breakdown, Nuf accumulation peaks, concentrating around the base of the astral microtubules radiating away from the centrosomes. Nuf is absent on the side of the centrosome adjacent to the nuclear envelope. Significantly, maximal Nuf localization at prophase corresponds to the time of metaphase furrow invagination. Nuf localization is correlated with areas of high astral microtubule density. This is in accord with the finding that Nuf pericentriolar localization requires intact microtubules. Although the pericentriolar concentration of Nuf significantly diminishes during metaphase and anaphase, a small fraction of Nuf remains tightly associated with the centrosomes. During telophase, immediately after nuclear envelope reformation, Nuf begins accumulating at the newly duplicated centrosome pair. Low magnification images dramatically highlight the cell cycle regulation of Nuf subcellular localization. It is not known if Nuf is maintained in constant levels throughout the nuclear cycle and is simply cycling from the cytoplasm to the centrosomes, or if Nuf levels change throughout the cell cycle. The localization of Nuf during cellularization at nuclear cycle 14 differs significantly from its localization during the syncytial divisions. During the syncytial divisions, Nuf is present in lower levels at the centrosome during interphase, reaches its maximal concentration, and is highly dynamic during prophase. In contrast, during cellularization, Nuf reaches its maximal concentration during interphase and is relatively motionless, forming few flares and puncta. During the syncytial divisions, Nuf concentrates only in the region of the centrosome facing away from the nuclear envelope. However, during cellularization, Nuf is more evenly distributed around the centrosome, forming an intact ring. The differences between Nuf behavior during the syncytial divisions and cellularization may be a consequence of the more stable microtubule arrays that form during the prolonged interphase of nuclear cycle 14 (Riggs, 2003).

Nuf is extremely dynamic at the centrosome during prophase. Detailed imaging reveals that Nuf forms dynamic puncta and flares that rapidly migrate from the centrosomes. The puncta form, travel a short distance from the centrosome, then disappear. As described below, Nuf is associated with the recycling endosomal compartment. Therefore, this movement may reflect endosomal dynamics (Riggs, 2003).

The mammalian homolog of Nuf, Arfo2, physically associates and colocalizes with Rab11, a key component of the recycling endosome (Hickson, 2003). Rab11 is required for the integrity of the recycling endosome, and is believed to mediate transport of vesicles from the RE to the TGN, early endosome, and plasma membrane via a 'slow' recycling route (Ullrich, 1996; Ren, 1998). The pattern of Rab-protein 11 localization in the developing Drosophila oocyte has been characterized (Dollar 2002). Rab11 localizes at the posterior pole and is necessary for proper microtubule organization and Oskar mRNA localization. The pattern of Rab11 localization have been examined during the cortical divisions in the early Drosophila embryo. Rab11 exhibits a diffuse punctate localization that concentrates around the nuclei. As the embryos progress into prophase, Rab11 maintains its punctate morphology, but exhibits significantly increased concentration at the centrosomes. During metaphase, the centrosomal concentration of Rab11 decreases and there is a concomitant dispersal of Rab11 throughout the cytoplasm encompassing each chromosome-spindle complex. This trend continues as the nuclei enter anaphase. Even though the nuclear envelope is substantially broken down during metaphase and anaphase, Rab11 does not enter the interior nuclear space. During telophase, Rab11 puncta concentrate around the newly formed nuclear envelope. There is a slight increase in the concentration of Rab11 puncta at the centrosomes. Cellularization occurs during the prolonged interphase of nuclear cycle 14. At this time, Rab11 is highly concentrated around the pair of apically located sister centrosomes (Riggs, 2003).

The pericentriolar concentration of Rab11 in Drosophila embryos is equivalent to Rab11 localization observed in mammalian cells. In CHO cells, Rab11 is primarily localized to a discrete pericentriolar region with a lower concentration of puncta distributed throughout the cell (Ullrich, 1996). Colocalization experiments with internalized transferrin have indicated that Rab11 localizes to the pericentriolar RE (Ullrich, 1996; Sheff, 2002). GFP-Rab11 also exhibits a pericentriolar localization and colocalizes with the transferrin receptor (Sonnichsen, 2000). Given the equivalent staining patterns in Drosophila, it is concluded that Rab11 also localizes to the RE in syncytial and cellularized Drosophila embryos (Riggs, 2003).

Immunofluorescent analyses using anti-Nuf and anti-Rab11 antibodies reveal that during prophase, when both antigens are highly concentrated in the pericentriolar region, areas of maximal Nuf localization correspond to areas of maximal Rab11 localization. Almost without exception, Nuf colocalizes with Rab11. However, the converse is not true, and in regions more distal from the centrosome, Rab11, but not Nuf, is present. During cellularization at interphase of nuclear cycle 14, Nuf and Rab11 exhibit high pericentriolar concentrations and extensive colocalization. As observed for prophase of the cortical divisions, Nuf always colocalizes with Rab11, but there are regions of Rab11 localization in which Nuf is not present. Given that Rab11 is an excellent marker of the RE, these results support the notion that Nuf localizes to the RE during cortical syncytial divisions and during cellularization at interphase of nuclear cycle 14 (Riggs, 2003).

EFFECTS OF MUTATION

The nuf maternal-effect mutation specifically disrupts syncytial nuclear divisions only after the nuclei migrate to the cortex (Sullivan, 1993). These nuclear defects are a consequence of incomplete metaphase furrow formation, which allows inappropriate fusions between nonsister nuclei (Rothwell, 1998). Although the interphase actin caps form normally, large gaps are present in the metaphase and cellularization furrows. The gaps are observed in the earliest stages of furrow formation, suggesting that Nuf disrupts recruitment of actin to the furrows rather than in stabilization of actin once at the furrows. To determine if reduced maternal supplies of Rab11 produce cortical phenotypes similar to those observed in nuf mutations, rab11 transheterozygotes were used. The nuclear phenotype is equivalent to nuf. In rab11-derived embryos, nuclear distribution and morphology is normal in premigration and early cortical blastoderm embryos. However, during the late cortical divisions when the nuclei are more densely packed, the nuclear distribution and morphology is disrupted. In premigration and early cortical embryos, 8% (2/23) exhibit disrupted nuclear morphology. During the late cortical divisions, 65% (31/48) exhibit severely disrupted nuclear morphology. This is indicative of defects in the metaphase furrows that serve to separate neighboring nonsister nuclei (Riggs, 2003).

To examine the role of Rab11 in organizing the cortical cytoskeleton and metaphase furrows, wild-type, nuf-derived, and rab11-derived cortical nuclear cycle 12 embryos were double stained for DNA and actin. During interphase, actin organizes into caps apically positioned above each nucleus. In nuf- and rab11-derived embryos, actin cap formation occurs normally. As the embryos progress into prophase, the actin caps are dismantled and actin reorganizes into furrows encompassing each prophase nucleus and its developing spindle. As the nuclei progress into metaphase, these furrows become more pronounced and tightly focused. The actin-based furrow defects in rab11-derived embryos are strikingly similar to those observed in nuf-derived embryos. In both, the hexagonal furrow network is riddled with gaps. The gaps are present at prophase during the initial stages of furrow formation, suggesting defects in the initial actin recruitment. nuf and rab11 mutations also produce similar defects during cellularization at nuclear cycle 14, although defects in nuf-derived embryos are much more extensive than observed in rab11-derived embryos. This difference may be a result of partial zygotic rescue by the paternally supplied rab⁺ allele (Riggs, 2003).

nuf-derived embryos disrupt recruitment of membrane components during furrow invagination. The Drosophila protein Discontinuous actin hexagon (Dah) tightly associates with the plasma membrane as well as actin, and is thought to link cortical microfilaments to the plasma membrane (Zhang, 1996). In cortical Drosophila embryos, Dah localizes to the plasma membrane as well as to vesicles that concentrate at the leading edge of the invaginating furrows. Analysis of Dah mutations indicates that incorporation of these vesicles into the plasma membrane contributes to furrow invagination (Rothwell, 1999). To determine the role of Rab11 and Nuf in Dah-associated vesicle delivery, wild-type, nuf-derived, and rab11-derived embryos were double stained for actin and Dah. In nuf-derived embryos, incorporation of Dah into the metaphase furrows is dramatically reduced. Although Dah vesicles are observed, they are more randomly distributed throughout the cytoplasm. A similar defect is observed in rab11-derived embryos; incorporation of Dah into the invaginating metaphase furrows is disrupted. However, in contrast to nuf, Dah staining is not observed in the furrow regions and few Dah-staining vesicles are visible (Riggs, 2003).

Nuclear-fallout regulates cortical microfilament organization

nuclear fallout (nuf) is a maternal effect mutation that specifically disrupts the cortical syncytial divisions during Drosophila embryogenesis. The nuf gene encodes a highly phosphorylated novel protein of 502 amino acids with C-terminal regions predicted to form coiled-coils. During prophase of the late syncytial divisions, Nuf concentrates at the centrosomes and is generally cytoplasmic throughout the rest of the nuclear cycle. In nuf-derived embryos, the recruitment of actin from caps to furrows during prophase is disrupted. This results in incomplete metaphase furrows specifically in regions distant from the centrosomes. The nuf mutation does not disrupt Anillin or Peanut recruitment to the metaphase furrows, indicating that Nuf is not involved in the signaling of metaphase furrow formation. These results also suggest that Anillin and Peanut localization are independent of actin localization to the metaphase furrows. nuf also disrupts the initial stages of cellularization and produces disruptions in cellularization furrows similar to those observed in the metaphase furrows. The localization of Nuf to centrosomal regions throughout cellularization suggests that it plays a similar role in the initial formation of both metaphase and cellularization furrows. A model is presented in which Nuf provides a functional link between centrosomes and microfilaments (Rothwell, 1998).

Nuf is a member of a growing class of proteins that concentrate at the centrosome in a cell-cycle-specific manner. Immunofluorescent analysis demonstrates that Nuf is diffusely localized throughout the cortical cytoplasm from early metaphase through interphase. During prophase, Nuf concentrates at the centrosomes. Other Drosophila centrosomal proteins that exhibit a cell-cycle specific centrosomal localization include CP60 and CP190. These proteins localize to the nuclei during interphase and the centrosome at mitosis and are members of a larger protein complex. Separable domains within these proteins have been identified that are responsible for their nuclear and cytoplasmic localization (Rothwell, 1998 and references therein).

Given the difference in the cell cycle timing of the centrosomal localization between Nuf and the CP60/190 complex, it is likely that they are involved in different cellular functions. The recently characterized Drosophila Abnormal spindle protein localizes to regions near the centrosome during prophase through anaphase and concentrates at the midbody during telophase. Mutations in Asp disrupt microtubule organization. As with many proteins that localize to the centrosome, Asp and Nuf both include C-terminal regions with a high probability of forming coiled-coils (Rothwell, 1998).

In mutations that lack Centrosomin (Cnn), a protein that localizes to the centrosome throughout the Drosophila syncytial nuclear cycles, Nuf no longer localizes to the centrosome. Conversely, Cnn localization is not disrupted in nuf-derived embryos. These results suggest that Nuf is not a core component of the centrosome. In nuf-derived embryos, prior to migration, the nuclear divisions are normal. This phenotype is also in accord with the idea that Nuf is not a core component of the centrosome (Rothwell, 1998).

In nuf-derived embryos, there are no obvious defects in the interphase actin caps. During metaphase, however, the furrows are incomplete in regions near the metaphase plate. Through both live and fixed analysis, this defect is observed during the earliest stages of furrow formation at prophase. This suggests a defect in the recruitment of actin to these regions of the furrow rather than the stabilization of actin already present in these furrow regions. It is not known whether or not Nuf interacts directly with actin. Insight into the mechanism of Nuf action will require identifying interacting proteins and other components involved in the process. A potential interactor is Dah, a Drosophila protein with some homology to dystrophin. dah-derived embryos have syncytial and cellularization phenotypes similar to nuf, indicating that they may be involved in a common pathway (Rothwell, 1998).

Nuf is concentrated at the centrosomes during prophase and is cytoplasmic throughout the rest of the cell cycle. It is not known when, in the division cycle, Nuf is required for actin recruitment to the furrows. For example, cytoplasmic Nuf may regulate actin dynamics and the localization of Nuf to the centrosome may serve to sequester Nuf in an inactive state. Alternatively, Nuf may influence actin dynamics while it is localized to the centrosomes. Nuf localizes to the centrosomes during prophase, specifically when the most extensive reorganization of actin from caps to furrows is occurring. In addition, Nuf is concentrated at the centrosomes from early interphase of nuclear cycle 14 through to the completion of cellularization. nuf-derived embryos produce a dramatic cellularization phenotype with extensive gaps in the furrows and it is likely that, as found for the metaphase furrows, this is a result of failed actin recruitment. These results suggest that, at least during cellularization, the centrosomal localization of Nuf is important for its effect on actin localization (Rothwell, 1998).

The prophase centrosomal localization of Nuf coincides with the timing of actin reorganization and supports a model in which Nuf is acting at the centrosomes to organize cortical actin. Nuf may influence microfilament organization by organizing microtubules. Although previous analysis failed to reveal disruption in microtubule organization in the nuf mutant, it remains possible that subtle microtubule defects have been overlooked. Alternatively, Nuf may act more directly to influence microfilament organization. For example, centrosomal Nuf may facilitate the cap-to-furrow transition by stimulating severing or depolymerization of the microfilaments. Another possibility is that the cap-to-furrow transition involves loading and transport of components onto and along the microtubules and Nuf may be involved in this process (Rothwell, 1998).

Although nuf1 only partially disrupts the formation of metaphase furrows, molecular analysis indicates that it is a null allele. One explanation for the fact that the nuf1 phenotype is not more severe is that Nuf may be a member of a protein complex that is involved in the recruitment of actin to the metaphase furrows. The absence of Nuf compromises, but does not eliminate, function in this complex. During cellularization, the furrow defect is much more severe in nuf1-derived embryos and extensive regions of the embryo lack cellularization furrows. It may be that, during cellularization, greater demands are placed on the functioning of this complex. Alternatively, the partially disrupted metaphase furrows of nuf1-derived embryos may reflect the fact that this process involves redundant mechanisms. Null alleles of another maternal effect mutation, dah, also produce slight defects in the metaphase furrows and severe cellularization defects. In addition to actin, the effect was examined of the nuf mutation on Anillin (Drosophila name: Scrapes) and Peanut localization. Anillin, Peanut and many other proteins are common to both metaphase and more conventional cleavage furrows. Therefore, many of the lessons learned in one system will likely apply to the other. During the initial stages of furrow formation in nuf-derived embryos, Anillin and Peanut localize to furrow regions in which actin fails to localize. These results demonstrate that, during the initial stages of furrow formation, proper localization of Anillin and Peanut to the furrow is independent of the proper localization of actin to the furrow. These results also suggest that Nuf functions in a pathway that is downstream or independent of anillin and peanut localization. Because these initial events of furrow formation occur normally in nuf-derived embryos, the signals for positioning and timing of furrow formation are probably intact. Although during prophase the localization of Anillin and Peanut is independent of actin localization, as the embryos progress into metaphase, Anillin and Peanut fail to maintain their localization in regions of the furrows in which actin fails to localize (Rothwell, 1998). It is likely that one of the furrow components that fail to be recruited in nuf-derived embryos, possibly actin, is required for stabilization of the furrow during the prophase to metaphase transition (Rothwell, 1998).

nuf-derived embryos produce a dramatic cellularization phenotype in which the gaps in the furrows are so extensive that the furrows usually encompass multiple nuclei. It is likely that the origin of this phenotype is equivalent to that of the metaphase furrows. This phenotype is strikingly similar to that observed for the zygotic mutations nullo and serendipity alpha. Analysis of these genes demonstrates that they encode novel proteins that localize to the invaginating cellularization furrow. The nullo mutation disrupts the localization of Serendipity, but the serendipity mutation does not disrupt the localization of Nullo. This indicates that Serendipity functions downstream of Nullo. Since the cellularization furrows initiate normally in the nullo mutation, the Nullo protein apparently is not required for the initial stages of cellularization but is required for the stabilization of the growing cellularization furrow. Serendipity is also probably not required for the initial stages of cellularization, since it functions downstream of Nullo. In contrast, nuf disrupts the initial formation of the cellularization furrows. This indicates that the maternally supplied Nuf acts upstream to the zygotic genes nullo and serendipity in initiating and establishing the cellularization furrow (Rothwell, 1998).

Nuf is required for recruiting Dah, a membrane associated protein, to furrows in the early embryo

During mitosis of the Drosophila cortical syncytial divisions, actin-based membrane furrows separate adjacent spindles. Genetic analysis indicates that the centrosomal protein Nuf is specifically required for recruitment of components to the furrows and the membrane-associated protein Dah is primarily required for the inward invagination of the furrow membrane. Recruitment of actin, Anillin and Peanut to the furrows occurs normally in dah-derived embryos. However, subsequent invagination of the furrows fails in dah-derived embryos and the septins become dispersed throughout the cytoplasm. This indicates that stable septin localization requires Dah-mediated furrow invagination. Close examination of actin and Dah localization in wild-type embryos reveals that they associate in adjacent particles during interphase and co-localize in the invaginating furrows during prophase and metaphase. The Nuf centrosomal protein is required for recruiting the membrane-associated protein Dah to the furrows. In nuf-mutant embryos, much of the Dah does not reach the furrows and remains in a punctate distribution. This suggests that Dah is recruited to the furrows in vesicles and that the recruiting step is disrupted in nuf mutants. These studies lead to a model in which the centrosomes play an important role in the transport of membrane-associated proteins and other components to the developing furrows (Rothwell, 1999).

nuf and dah, identify distinct steps in the process of metaphase furrow formation in syncytial blastoderm embryos. During the initial stage, actin and other components are recruited to the furrow regions. Following this, furrow invagination occurs. Nuf is specifically involved in recruiting components to the furrow while Dah is required for furrow invagination. In nuf-mutants, actin recruitment to regions distant from the centrosomes often fails. This results in the inability of furrows to form in these regions (Rothwell, 1998). Alternatively, the partial formation of furrows in the nuf null mutation could be due to incomplete activity of a Nuf-containing multi-protein complex. Furrow regions closest to the centrosomes, in which actin and the other furrow components are properly recruited, undergo normal invagination. This indicates that Nuf is specifically required for recruitment of furrow components and is not involved in the subsequent stages of furrow formation. In contrast, recruitment of furrow components occurs normally in dah-derived embryos but furrow extension fails. Thus, Dah functions primarily in the invagination process. It is likely that other cortical components will fall into one of these classes or play roles in other distinct steps in furrow formation. For example, the unconventional myosin, 95F-myosin, appears to play a role in the invagination process; injection of antibodies directed against 95F myosin results in metaphase furrows that do not invaginate as deeply as those in normal embryos (Rothwell, 1999).

During cytokinesis, the cleavage furrow contracts through an actomyosin based mechanism. Actomyosin based contraction may also play a role in formation of the metaphase and cellularization furrows of the early Drosophila embryo. Myosin II is present at the tips of both the invaginating metaphase and cellularization furrows indicating that invagination may involve contractile processes. Myosin II localizes to metaphase furrows specifically during membrane invagination, leaving the furrow tips once they are fully formed. Injection of antimyosin II antibodies into syncytial embryos disrupts cortical nuclear organization and inhibits subsequent cellularization (Rothwell, 1999).

Several lines of evidence indicate that formation of cellularization furrows also requires membrane addition. The plasma membrane above each nucleus contains microvilli-like projections that increase in number during the initial, slow phase of cellularization and disappear in the later fast phase. Therefore, the early phase of cellularization may involve membrane recruitment at the cell surface for microvilli formation while the fast phase utilizes the excess membrane for invagination. However, calculations indicate that the membrane supplied by these microvilli-like projections is not sufficient to complete cellularization. Therefore, other mechanisms of membrane addition may be involved in the cellularization process. In support of this, coated pits and multilamellar bodies decorate the furrows. Some EM studies describe cellularization as a process involving vesicle alignment at the future furrow site followed by their fusion to form double membranes. It is suggested that that the slow and fast phases utilize different vesicle populations. Zygotic mutations that specifically effect the slow or fast phases support the idea that they occur through different mechanisms (Rothwell, 1999).

Genetic studies also support the idea that cellularization requires significant membrane addition. Syntaxins are a family of membrane proteins that are thought to provide specificity for targeting of vesicles to specific membrane compartments. Germline clones of Drosophila syntaxin produce extensive defects in cellularization. The Drosophila temperature-sensitive mutation shibire disrupts the gene encoding Dynamin, a protein required for endocytosis. In addition to neuronal defects, shibire also disrupts cellularization. At the restrictive temperature, cellularization furrows do not form and vesicles accumulate in the cytoplasm. These data suggest that processes related to endocytosis are required for cellularization (Rothwell, 1999).

Evidence is provided that metaphase furrows, a process very similar to cellularization, may also form through significant addition of membrane. Dah is a furrow component required for metaphase furrow formation and cellularization (Zhang, 1996). Diochemical analysis demonstrates that Dah is membrane-associated protein (Zhang, 2000). In the absence of Dah, although recruitment of furrow components occurs normally, furrow invagination fails. In addition, Dah localizes to membrane containing particles that often concentrate at the leading edge of the furrows. These results suggest that invagination of the metaphase furrows occurs through membrane addition and that Dah is a membrane-associated protein required for this process (Rothwell, 1999).

95F-myosin (Jaguar) has also been hypothesized to play a role in recruiting particles necessary for furrow formation. The phenotype of embryos injected with antibodies against 95F-myosin is extremely similar to that of dah-mutant embryos and the protein products of these genes share similar localization patterns. Therefore, 95F-myosin and Dah may act together in the process of furrow extension. An attractive hypothesis is that 95F-myosin delivers vesicles containing Dah and other necessary furrow components to the furrow region (Rothwell, 1999).

nuf-derived embryos form incomplete furrows (Sullivan, 1993; Rothwell, 1998). To determine the role of contraction in metaphase furrow formation, invagination of the free ends of these incomplete furrows was examined; the free ends of metaphase furrows are stable and invaginate to the same extent as intact normal furrows. This result would not be expected if long range actomyosin-based contraction played a major role in furrow formation. This result is consistent with alternative mechanisms based on membrane addition. One potential mechanism is vesicle fusion (Rothwell, 1999).

The centrosome plays a key role in the formation of the actin caps and the metaphase furrows in the early Drosophila embryo. The centrosomal protein, Nuf, has been shown to be required for proper actin recruitment to the furrows (Rothwell, 1998). This study demonstrates that Nuf is also involved in recruiting membrane to the furrows. In wild-type embryos, membrane-bearing particles containing the Dah protein are recruited to the site of furrow formation as the furrows initiate formation during prophase. In similarly staged nuf-mutant embryos, many of these membrane containing particles are not properly recruited and remain in a perinuclear punctate distribution. Close examination of Dah and actin localization in wild-type embryos reveals that they localize to adjacent particles that lie between the nuclei and concentrate ahead of the furrow tips. At metaphase, the majority of Dah and actin co-localize in the furrows. Since Dah is not required for proper actin recruitment to the furrows, it is likely that Dah itself does not play a role in the transport mechanism. A model consistent with these observations is that Nuf acts at the centrosome to initiate the transport of vesicle-associated components along the microtubules to the furrow regions. This model accounts for the observation that furrow formation fails primarily in those regions most distant from the centrosomes in which the greatest demand would be placed on the transport process (Rothwell, 1998). Similar models have been proposed for membrane transport along the cytoskeleton in other systems. Long range transport of vesicles is thought to utilize kinesins and occur along microtubules; once at the cell periphery, the vesicles are transferred to actin filaments and actin-based motors carry them to their final destinations at the plasma membrane. The recent finding that a microtubule-based motor (conventional kinesin) and an actin motor (myosin V) directly interact is providing insight into the mechanism whereby the same vesicle can move along different cytoskeletal tracks. Transport of vesicles necessary for furrow formation in the early Drosophila embryo may also occur through coordinated microtubule and actin-based transport systems. In support of this model, drug studies show that disruption of microtubules by injection of colchicine into Drosophila embryos halts transport of particles to the cellularization furrows resulting in inhibition of membrane invagination. In addition, injection of antibodies against 95F-myosin disrupts invagination of the metaphase furrows that form during syncytial development (Rothwell, 1999).

Anillin and Peanut (a Drosophila septin) are conserved components of the metaphase and cytokinesis furrows and show a similar pattern of localization, both localizing early during the process of furrow formation. Mutational analysis of nuf derived embryos indicates that recruitment of these proteins to the developing furrows occurs independently of actin. Once the furrows are formed at metaphase, however, peanut and anillin are not maintained in regions where actin is not localized and furrow formation has failed (Rothwell, 1998). These results indicate that stable furrow localization of these proteins either requires actin or normally invaginated membranes (Rothwell, 1999).

Analysis of dah-derived embryos in which actin localization occurs normally but membrane invagination fails helps distinguish between these alternatives. In dah-derived embryos, prophase occurs relatively normally but membrane invagination during metaphase fails. Actin, Peanut and anillin localize normally during prophase in dah-derived embryos. However, during metaphase, Peanut localization specifically fails while actin and Anillin remain localized in the un-invaginated furrows. One interpretation of these results is that the septins require intact plasma membrane for stable localization. This interpretation is supported by co-localization and biochemical studies indicating that septins are intimately associated with membrane. Anillin, on the other hand, has a more diverse localization pattern in that it cycles between the nucleus and the cortex (Rothwell, 1999).

EVOLUTIONARY HOMOLOGS

Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, that was named arfophilin. Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle. Arfophilin bonds only to the active, GTP-bound form of ARF5 and does not bind to GTP-ARF3, which is a class I ARF. The N terminus of ARF5 (1-17 amino acids) is essential for binding to arfophilin. The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also binds to arfophilin, suggesting it is a target protein for GTP-bound forms of class II ARFs. The binding site for ARF on arfophilin is localized to the C terminus (residues 612-756), which contains putative coiled-coil structures. Recombinant arfophilin overexpressed in CHO-K1 cells is localized in the cytosol and translocates to a membrane fraction in association with GTP-bound ARF5. ARF5 containing the N terminus of ARF3 does not promote translocation, indicating that class II ARFs are specific carriers for arfophilin (Shin, 1999).

Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). Using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates it has been shown that, unexpectedly, ARF6 also binds to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further show that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, is essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bind to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 does not bind. In contrast, all three forms of ARF bind to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 is autoactivating. These findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector (Shin, 2001).

Rab11 (see Drosophila Rab-protein 11), a low molecular weight GTP-binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. A novel Rab11 effector, EF-hands-containing Rab11-interacting protein (Eferin), is described in this study. In addition, a 20-amino acid domain has been defined that is present at the C terminus of Eferin and other Rab11/25-interacting proteins, such as Rip11 and nRip11. Using biochemical techniques, this domain is shown to be necessary and sufficient for Rab11 binding in vitro and it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for binding to Rab11/25 possibly accounting for the diversity of Rab11 functions (Prekeris, 2001).

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling system. It has been hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. A family of four Rab11-interacting proteins is defined in this study: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associate with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacts with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a is dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalize with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalizes with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also co-enrich with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocate with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors (Hales, 2001).

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. A novel 80-kDa protein has been identified that interacts specifically with the GTP-bound conformation of Rab4, and it also interacts strongly with Rab11. This protein has been named Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homolog of Rip11 and therefore belongs to the recently described Rab11-FIP family (Lindsay, 2002a).

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells. The specificity of the interaction between Rab11-FIP2 and Rab11 has been characterized; it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. The COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself is insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, causes tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 is also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking (Lindsay, 2002b).

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Several of these proteins have been characterized with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. Two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4; several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end that includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. A new (sixth) member of the protein family has been identified, that has been named Rab11-FIP4; the family evolutionary complexity and chromosomal distribution is reported. Furthermore, it is proposed that the ability of these proteins to bind each other are important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity (Wallace, 2002a).

Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). The subcellular localization of Rab11-FIP4 in HeLa cells was investigated; it colocalizes extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. The carboxy-terminal region of Rab11-FIP4 [Rab11-FIP4(C-ter)] is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling (Wallace, 2002b).

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes (Cullis, 2002).

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and it binds both Arf5 and Rab11. A related protein, arfophilin-2, is described that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localizes to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment is selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions are localized around the centrosome in a region that is also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment is altered. The arfophilins belong to a conserved family that includes Drosophila Nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-Nuclear fallout in HeLa cells results in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. It is suggested that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment (Hickson, 2003).

The observation that GFP-Nuf overexpression in HeLa cells results in largely the same phenotype as that of GFP-arfophilin-2 overexpression suggests that these proteins are functionally related. This has several important implications. It suggests a potential role for the arfophilins in cell division, particularly cytokinesis, because Nuf is required for the formation of cellularization furrows in the Drosophila embryo. Because testis is a tissue undergoing a high degree of cell division, such a role may help to explain the high level of expression observed in this tissue. The findings also implicate the endosomal recycling compartment in cytokinesis. This is consistent with recent studies in C. elegans where RNA interference-induced suppression of Rab 11 leads to specific regression of the cleavage furrow at the final stage of abscission (Skop, 2001). The significance of the role of Rab 11 should not be overlooked, particularly given the interaction of arfophilins with this GTPase. Arfs have also been implicated in cellularization in Drosophila and cytokinesis in C. elegans. Such observations suggest the hypothesis that arfophilin-2, and by extension the endosomal recycling compartment, may be involved in traffic to the midbody during cytokinesis. Moreover, the identification of arfophilin-2 as a Rab 11 and Arf binding protein offers the tantalizing suggestion that this protein may integrate the Rab 11 and Arf signals to membrane traffic during cytokinesis (Hickson, 2003 and references therein).

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date revised: 17 December 2021

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