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).

GENE STRUCTURE

cDNA clone length - 2392

Bases in 5' UTR - 260

Exons - 10 (CG7867-RA), 7 (CG7867-RB), and 9 (CG7867-RC)

Bases in 3' UTR - 1114

PROTEIN STRUCTURE

Amino Acids - 502

Structural Domains

Mapping and reversion studies demonstrate that the nuf mutation is caused by the insertion of a modified transposable P-element (Sullivan, 1993). This facilitated the isolation of flanking genomic DNA that was used to isolate three cDNA clones, all of which contained an insert of approximately 2.3 kb. One of these cDNAs was used as a probe for northern analysis; a major 2.4 kb and a minor 3.5 kb transcript present in RNA preparations from 0-3 hour wild-type embryos. Both of these transcripts are greatly reduced in RNA preparations from similar collections of nuf-derived embryos (Rothwell, 1998).

nuf encodes a highly phosphorylated novel protein with coiled-coil domains at its C-terminal end. Sequence analysis of the 2.3 kb nuf cDNA reveals a putative open reading frame of 1506 bp immediately preceded by the sequence AAAC(ATG), which agrees well with the consensus translation start sequence in Drosophila. The predicted Nuf protein contains 502 amino acids with a predicted molecular mass of 57 kDa. Further analysis reveals three regions near the Nuf C terminus strongly predicted to form coiled-coils. The probability of forming coiled-coils is greater than 95% for residues 231-382 and 389-431 and greater than 90% for residues 442-472. The first two coiled-coil domains are separated by a potential 6 amino acid 'hinge segment' containing proline (Rothwell, 1998).

Arf proteins are members of a large family of small GTPases involved in the regulation of membrane-trafficking pathways. Arfo2 and Nuf show significant similarities at the COOH terminus (300-aa region). This region is predicted to form extensive coiled-coils and is 28% identical and 54% conserved between these two proteins. A striking feature of these proteins is that they contain a previously identified 20-aa Rab11-binding domain at their extreme COOH termini (Hales, 2001; Prekeris, 2001; Riggs, 2003 and references therein).

date revised: 22 May 2004

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