Protein Interactions

It has been reported that MKLP1 can be detected in an immunoprecipitate of murine plk (Lee, 1995). This association of the two proteins would be consistent with a role for plks in cytokinesis in metazoans, as has been shown in fission yeast (Ohkura, 1995). Immunoprecipitation experiments were carried out on extracts of Drosophila embryos using antibodies against Polo protein kinase. Western blot analysis indicates that Pav is present in the Polo immunoprecipitates obtained with either of three different anti-polo monoclonal antibodies. Immunostaining experiments were undertaken to determine the extent to which these proteins colocalize throughout the mitotic cycle. Using the mAb 294 anti-Polo antibody in cells of wild-type embryos, it was observed that Polo protein kinase localizes at centrosomes early in mitosis and, simultaneously, some staining is detected associated with the metaphase chromosomes. The immunostaining of the centrosomes by the anti-Polo antibody is progressively lost during anaphase-telophase concurrently with an increase in the staining of the midbody. By the end of telophase Polo protein seems to be associated only with the midbody. There is extensive colocalization of Pav to the centrosome and subsequently to the midbody during mitosis. However, in pav mutant embryos, no centrosome staining is observed either with the anti-Pav or the anti-Polo antibodies. The midbody staining is also dramatically changed in the mutant embryos, being completely lost with the anti-Pav antibody and dramatically reduced with the anti-Polo antibody. Thus it would appear that the localization of Polo protein kinase at the spindle poles and in the central spindle requires the function of Pav (Adams, 1998).

A biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, Inner centromere protein (INCENP) and Aurora B kinase, was performed in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibits the ability of cells to achieve a normal metaphase chromosome alignment. Cells are not blocked in mitosis, however, and enter an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appears to be normal, however cytokinesis often fails. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis (Adams, 2001).

A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis

Cytokinesis, the final step in cell division, involves the formation and constriction of an actomyosin-based contractile ring. The mechanism that positions the contractile ring is unknown, but derives from the spindle midzone. An interaction between Pebble [a Rho GTP exchange factor (GEF)], and the Rho family GTPase-activating protein, RacGAP50C, has been shown to connect the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. It is proposed that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis (Somers, 2003).

In Drosophila melanogaster embryonic epithelial cells, constriction occurs during anaphase B and telophase to generate two daughter cells, each containing one set of the recently separated sister chromatids. Constriction of the cleavage furrow proceeds through the activity of the myosin II motor protein acting on an F-actin network. Members of the Rho subfamily of small G proteins are potent regulators of the actin cytoskeleton in a variety of contexts. Like all small G proteins, Rho1 is active when GTP is bound and inactive when GDP is bound. Activation is mediated by guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP and the uptake of GTP, whereas inactivation is regulated by GTPase-activating proteins (GAPs) that stimulate the intrinsic GTPase activity of the G protein (Somers, 2003).

Molecular and genetic studies have shown that the D. melanogaster RhoGEF, Pebble (PBL), and its mammalian ortholog, the protooncogene ECT2, are required for cytokinesis. pbl mutant embryos proceed normally through the first 13 syncytial mitotic cycles following fertilization and cellularize normally during G2 phase of cycle 14, but they fail to undergo cytokinesis during the fourteenth and subsequent division cycles. Pbl binds to Rho1, but not Rac1 or Cdc42, and sensitized pbl mutant alleles show strong genetic interactions with Rho1 but not Rac1 or Cdc42 alleles. During cytokinesis in epithelial cells of the embryo, Pbl accumulates in the contractile ring during furrowing, where it appears to stimulate Rho1-mediated organization and activity of the actomyosin contractile ring (Somers, 2003).

Reorganization of the actomyosin contractile apparatus occurs coincident with reorganization of the microtubule network. During anaphase, the mitotic spindle is remodeled to form a midzone bundled microtubule structure referred to as the central spindle, which is further compacted into a late cytokinetic structure termed the midbody. Curiously, another regulator of Rho family G protein activity, the Caenorhabditis elegans CYK-4 GAP, is required for microtubule bundling, because microtubule reorganization fails in cyk-4 mutant embryos. It also fails in embryos mutant for the zen-4/CeMKLP1 gene, which encodes a kinesin-like protein that forms a complex with CYK-4. This complex has been shown to bundle microtubules in vitro. The CYK-4 and ZEN-4 proteins and their respective mammalian orthologs localize to the central spindle and are all essential for cytokinesis, as is Pavarotti (Pav), the D. melanogaster ortholog of ZEN-4 (Somers, 2003).

It is not known how remodeling of the microtubule and F-actin networks is coordinated during cytokinesis. Although initial studies focused on their role in F-actin remodeling, recent studies have now linked Rho family members to microtubule organization. For example, depolymerization of microtubules results in an increase in the amount of active RhoA and the formation of contractile actin bundles, while microtubule polymerization results in an increase in the amount of active Rac1 and the formation of lamellipodia. RhoA can also mediate selective microtubule stabilization, while the Rac1/Cdc42 effector PAK is capable of activating the microtubule destabilizer Stathmin. It is possible, therefore, that Rho family members play roles in both F-actin and microtubule organization during dynamic processes such as cytokinesis. An important but poorly understood aspect of the relationship between the microtubule and F-actin networks is the nature of the signal that positions the contractile ring and initiates furrowing. It is now generally accepted that the signal originates from the midzone of the anaphase microtubule network, although the nature of the stimulus is unknown (Somers, 2003).

A complex has been identified between two Rho family regulators, the RhoGEF Pbl and RacGAP50C, the D. melanogaster ortholog of the CYK-4 Rho family GAP. A ring of RacGAP50C and Pav, associated with cortical microtubules, colocalizes with Pbl in dividing embryonic epithelial cells, forming a link between the actomyosin and microtubule networks. These observations suggest a molecular model for contractile ring positioning and function whereby RacGAP50C-Pav complexes, positioned at the equatorial cortex of the cell by their association with microtubules, interact with cortical Pbl to activate Rho1, initiate formation of the contractile ring, and coordinate F-actin and microtubule dynamics during furrowing (Somers, 2003).

Thus, in Drosophila embryonic epithelial cells at the onset of cytokinesis, the two Rho family regulators are part of a cortical double-ring structure at the site of cleavage furrowing. The RacGAP50C ring is associated with cortical microtubules, presumably through its interaction with the Pavarotti kinesin-like protein. Pav colocalizes with RacGAP50C, and coimmunoprecipitation experiments have shown that they form a complex in vivo. The RacGAP50C-Pav ring appears to abut or overlap the Pbl-containing contractile ring. The Pav kinesin-like protein, RacGAP50C and Pbl RhoGEF trimolecular complex simultaneously associates with, and has the capacity to control, both the actin and microtubule cytoskeletons as they are remodeled during cytokinesis. Furthermore, this complex appears to be a conserved feature of animal cytokinesis, since the mammalian Pbl and RacGAP50C orthologs, the protooncogene ECT2 and MgcRacGAP, bind to each other in a yeast two-hybrid assay and colocalize during mitosis (Somers, 2003).

The interaction between Pbl and RacGAP50C occurs through an extended BRCT domain of Pbl and an N-terminal coiled-coil domain of RacGAP50C. RacGAP50C binds Pav through sequences adjacent to the Pbl-interacting domain, indicating the presence, in Drosophila, of the so-called centralspindlin complex (Mishima, 2002) first identified from analysis of CYK-4 and ZEN-4, the C. elegans RacGAP50C and Pav orthologs (Somers, 2003).

RacGAP50C-Pav complexes were found to be cytoplasmic at prophase, associated with mitotic spindles during metaphase, concentrated in the spindle midzone during anaphase, and localized to the midbody at cytokinesis and to the nucleus during interphase. During late anaphase and early telophase in Drosophila epithelial cells, RacGAP50C-Pav complexes not only localize to the overlapping microtubules of the centrally located anaphase spindle, but also to distinct cortical microtubules. Cortical microtubules have been reported in dividing Drosophila neuroblasts (Savoian, 2002), and they can be seen in all D. melanogaster anaphase cells examined. Localization of the RacGAP50C-Pav complexes to the microtubule midzone is independent of its interaction with Pbl, since RacGAP50C is found to localize appropriately in Pbl mutant cells. However, localization of RacGAP50C is dependent on the Pav kinesin-like protein. The affinity of the RacGAP-KLP complex for microtubules, the cortical localization of the microtubules, and the plus end-directed nature of the Pav kinesin-like motor protein appear sufficient to account for localization of the complex to an equatorial cortical ring (Somers, 2003).

The molecular signal that positions the contractile ring and initiates furrowing remains to be elucidated. A number of studies have shown that the signal derives from the overlapping midzone microtubules that form during anaphase. One of the most striking aspects of the formation of the Pbl-RacGAP50C ring is that it is present in the earliest examples of furrowing that were observed. The existence of this ring at the onset of cytokinesis suggests a molecular model for the positioning and regulation of the contractile ring. Specifically, it is proposed that the initiation signal corresponds to the microtubule-mediated arrival of the RacGAP50C-Pav kinesin-like protein complex at its equatorial ring and establishment of the interaction with the Pbl RhoGEF. It is proposed that this interaction results in activation of RhoGEF activity. Rho1 would then be activated to initiate contractile ring formation and furrowing through activation of factors such as Diaphanous and myosin. This model accounts for the role of microtubules in positioning the contractile ring, because microtubules deliver the RacGAP50C-Pav complexes to their interaction site with Pbl. It also accounts for the conclusion, made by Gatti and colleagues from their studies of cytokinesis, that there is a requirement for both the central spindle and a cortical Pbl-containing apparatus for the onset of cytokinesis (see Somma, 2002; Somers, 2003).

There is strong support for this model beyond the evidence described here. Importantly, consistent with the observation that DRacGAP50C RNAi-treated S2 cells show no furrowing, pav and pbl mutant cells fail to form a contractile ring and do not undergo furrow ingression. However, contradictory evidence has come from C. elegans, where cyk-4 and zen-4 mutant cells initiate but fail to complete furrowing. Two possible explanations are suggested for these contradictory observations. The first is that D. melanogaster epithelial cells may use a different cytokinesis mechanism than that used in the early C. elegans embryo. In support of this, the cell types are very different in size and exhibit differences in microtubule organization during anaphase and telophase. Alternatively, it is possible that the C. elegans phenotypes do not represent the true null phenotype. The cyk-4 allele used to determine the phenotype is a temperature-sensitive allele, which may not abolish all activity at the restrictive temperature. The zen-4 allele used to generate germline mutant clones is a premature truncation that would eliminate all function. However, to observe the phenotype, germline clones were generated, perhaps requiring zen-4 activity to undergo the previous division. Some of the product may therefore have persisted to produce the partial furrowing observed in the mutant embryos. It therefore remains to be seen whether the model proposed here is applicable to cytokinesis in all animal cells (Somers, 2003 and references therein).

Midzone microtubule bundles have been shown to be required continuously for cytokinesis in cultured cells. The cortical Pbl-RacGAP50C-Pav ring, which persists and narrows as cytokinesis proceeds, is ideally positioned to coordinate actomyosin contraction and the bundling of microtubules. Actin filament activity is regulated by Pbl, which is required for establishment and/or maintenance of the contractile ring through activation of the Rho1 GTPase. Microtubule bundling activity has been demonstrated for CYK-4 and ZEN-4. It is likely, therefore, that the complex between the Pbl RhoGEF, RacGAP50C and the Pav kinesin-like protein functions to coordinate F-actin and microtubule remodeling during contractile ring constriction (Somers, 2003).

While actomyosin regulation and microtubule bundling may be the primary regulatory roles of these factors, there are additional ways that the Pbl RhoGEF and RacGAP50C could influence both the actin and microtubule-based cytoskeleton. Rho downstream effectors have been shown to regulate both cytoskeletal systems. For example, the Rho1 target, Diaphanous, mediates actin reorganization but also affects the stability of microtubules (Somers, 2003).

The CYK-4 and ZEN-4 microtubule bundling activity does not require the presence of any of the small G proteins, but the site-directed mutant analysis described in this study suggests a requirement for the GTPase-activating domain of RacGAP50C. Consistent with this, a GAP domain-defective form of MgcRacGAP appears to act as a dominant-negative protein, inducing cytokinetic defects. If such a target of RacGAP50C GAP activity exists, it has still not been identified. However, the evidence is inconsistent with RacGAP50C acting as the Rho1 GAP that opposes Pbl, based on the synergistic nature of pbl and RacGAP50C genetic interactions and on the absence of genetic interactions between RacGAP50C and Rho1. Consistent with this, in vitro assays show that the CYK-4 and MgcRacGAP homologs target Rac and Cdc42 with far greater efficiency than Rho1 (Somers, 2003 and references therein).

Ths study has identified complexes between the RhoGEF Pbl and the Rho family GAP, RacGAP50C, and between RacGAP50C and the kinesin-like protein, Pav, that connect the contractile ring to cortical microtubules during cytokinesis. During late stages in anaphase and during telophase, these proteins localize to a cortical ring where furrowing is initiated, constricting as furrowing proceeds. These observations suggest a model for the molecular control of cytokinesis in animal cells, whereby microtubule-dependent cortical equatorial localization of RacGAP50C-Pav kinesin-like protein complexes is the positioning signal generated by the central spindle microtubules, and formation of complexes with the Pbl RhoGEF allows coordination of F-actin and microtubule remodeling (Somers, 2003).

Function and regulation of Tumbleweed (RacGAP50C) in neuroblast proliferation and neuronal morphogenesis

RacGAP50C corresponds to the tumbleweed (tum) gene previously identified based on its defects in dendrite development of sensory neurons (Gao, 1999). Using mushroom body neurogenesis and morphogenesis as a model, Tumbleweed (Tum), Pavarotti (Pav), and their association are shown to be required for neuroblast proliferation. Tum with a mutation predicted to disrupt the GTPase-activating protein (GAP) activity still largely retains its activity in regulating cell division but is impaired in its activity to limit axon growth. Tum and Pavarotti regulate the subcellular localization of one another in postmitotic neurons, and cytoplasmic accumulation of both proteins disrupts axon development in a GAP-dependent manner. Taken together with previous studies of RacGAP50C in regulating cytokinesis, it is proposed that Tum serves as a scaffolding protein in regulating cell division but acts as a GAP to limit axon growth in postmitotic neurons (Goldstein, 2005).

A role for Tumbleweed/RacGAP50C, and potentially Pav, in postmitotic neurons would predict that these proteins should be expressed there. No endogenous Tum or Pav was detected in the intact CNS by using existing or newly generated antibodies. Therefore epitope-tagged transgenes of Tum and Pav, alone and in combination, were expressed to examine their subcellular localization in MB neurons. A series of GFP-tagged Pav WT (GFP::PavWT) and mutant transgenes have previously been used to study their localization in Drosophila egg chambers and live embryos (Minestrini, 2002 and 2003). It was found that GFP::PavWT not only rescues pav mutants but has similar localization to endogenous Pav, the ring canals (remnants of incomplete cytokinesis rings), and the oocyte nuclei. When expressed in MB neurons, GFP::PavWT is detected in the nuclei and dendrites and is highly concentrated in the axons of the MB neurons (Goldstein, 2005).

Surprisingly, UAS-MYC::TumWT is enriched in the nuclei when overexpressed in MB neurons. Expression of TumWT tagged with yellow fluorescent protein at its carboxyl terminus revealed a similar nuclear localization, indicating that the nuclear localization is not an artifact of its epitope tag. MYC::TumWT localization was always predominantly nuclear in MB neurons examined in larval and pupal stages (Goldstein, 2005).

Coexpression of UAS-GFP::PavWT and UAS-MYC::Tum alters the subcellular localization of both proteins. These proteins are colocalized in MB neurons, and their localization pattern is a combination of the subcellular distributions of MYC::Tum alone and GFP::PavWT alone. GFP::PavWT is more concentrated in nuclei and less concentrated in axons compared with its expression alone; however, MYC::Tum is now weakly detectable in axons. These observations are consistent with the notion that Tum and Pav bind to each other and regulate each other's subcellular localization in postmitotic neurons (Goldstein, 2005).

Expression of UAS-GFP::PavWT alone or UAS-MYC::TumWT alone does not affect the gross MB axon projection as judged by coexpression with mCD8::GFP. Expression of both UAS-MYC::TumWT and UAS-GFP::PavWT leads to a high occurrence of axon misguidance (14 of 28 MBs examined). The majority of FasII-positive axons form a ball-like structure at their initial trajectory, and only a small subset of axons is able to extend beyond this ball-like structure into the medial lobe. In other cases, axons fail to enter their correct path at the branch point for the dorsal and medial lobe. One possible explanation is that cytoplasmic accumulation of Tum, by means of Pav binding, disrupts axon development (Goldstein, 2005).

To further test this hypothesis, use was made of a Pav mutant transgene. Mutations in three of the nuclear localization signals of Pav (PavNLS) blocks the nuclear localization of Pav protein. As predicted, the nuclear localization of GFP::PavWT, but not its dendritic or axonal localization, is disrupted in GFP::PavNLS. GFP::PavNLS does not lead to any gross morphological changes when expressed alone. As with UAS-GFP::PavWT, coexpression of GFP::PavNLS and MYC::Tum results in altered subcellular localization of both proteins: GFP::PavNLS is localized to the nucleus, and more MYC::Tum is 'dragged' out of the nucleus. Interestingly, almost all MB axons are misrouted under this condition. In addition to forming a cluster of axons in a ball-like form as with GFP::PavWT, axons bypass their normal path via the peduncle and instead project medially toward the front of the brain just below the normal medial lobe and then project dorsally to innervate the medial lobe. The gain-of-function phenotype seen with coexpression of Tum and Pav suggests that they form a complex that disrupts the normal development of axons. The occurrence and severity of this axon misrouting is increased when the Pav transgene has a decreased drive to enter the nucleus, indicating that Pav may act as a transporter of Tum and that local concentrations of Tum in the axon directly influences its capacity to affect axonal development (Goldstein, 2005).

To test whether the GAP activity is necessary for the axon misrouting, a point mutation (TumR417L) of the conserved arginine residue critical for GAP activity was introduced into the UAS construct. As with the WT form, UAS-MYC::TumR417L localizes to the nuclei of MB neurons when expressed in MB neurons and does not lead to morphological phenotypes. When coexpressed with UAS-GFP::PavNLS, the subcellular localization of MYC::TumR417L and GFP::PavNLS reflects a pattern similar to that seen with WT Tum coexpression, with MYC::TumR417L detected in both axons and in the nuclei of MB neurons. However, with the loss of the conserved arginine in the GAP domain, axon disruption capability of Tum is almost completely abolished, suggesting that the GAP activity of Tum mediates the gain-of-function phenotype seen with Tum and Pav coexpression (Goldstein, 2005).

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. A collection of genes was examined that causes defects in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. These findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex (Dean, 2005).

This study has discovered three steps in the myosin II localization/activation process that involve distinct groups of genes: (1) an initial recruitment of myosin II to the equatorial cortex that is independent of F-actin and centralspindlin but requires rho1 signaling; (2) a secondary stabilization of myosin II at the midzone that requires F-actin and a second set of genes that are likely involved in building a specific type of actin network, and (3) the activation of furrowing once myosin II is localized that depends on centralspindlin (Dean, 2005).

Rho1, its activating guanidine nucleotide exchange factor pebble, and rho kinase are each required for the initial recruitment of myosin II to the equatorial cortex. Rho1 has been implicated in two pathways that are important for cytokinesis. In the first pathway, rho1 signals to F-actin through the formin diaphanous. However, proteins on this F-actin pathway, including F-actin itself, are not essential for the initial myosin II recruitment to the equatorial cortex. However, rho kinase, another downstream target of rho1, is essential. Because rho kinase phosphorylates the myosin II RLC, it is possible that phosphorylation of the RLC is essential for myosin II recruitment to the furrow. This hypothesis could not be directly tested, because the myosin II heavy chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).

Phosphorylation of the RLC both activates the motor domain and, in some myosins, increases bipolar thick filament formation. Because F-actin is not required for myosin II recruitment, activation of the motor is unlikely to be the mechanism by which phosphorylation of the RLC would cause recruitment of myosin II to the equatorial cortex. It is quite possible, however, that the rho kinase-mediated myosin II phosphorylation leads to thick filament assembly and that this assembly is important for localization of myosin to the equatorial cortex. Indeed, in Dictyostelium, it is clear that bipolar thick filament formation is sufficient for myosin II localization to the midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains unknown (Dean, 2005).

In contrast to the lack of F-actin involvement in the early recruitment of myosin II to the equatorial cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain myosin II in the equatorial region. Interestingly, the downstream rho1 effectors diaphanous/formin and chickadee/profilin are also necessary for myosin II maintenance at the equatorial midzone. Although the loss of these genes could deplete F-actin, phalloidin staining has shown that F-actin is still present in all of the RNAi-treated cells. In addition, these RNAi-treated cells still contract, unlike when F-actin is completely disrupted with LatA. Thus, myosin II appears to be interacting with F-actin in the cortex as it disperses in dynamic patches throughout the cortex of these diaphanous- or chickadee-depleted cells (Dean, 2005).

It is suggested that the role of diaphanous/formin and chickadee/profilin in maintaining the myosin II contractile ring is through the creation of specific F-actin structures. In particular, formin- and profilin-mediated nucleation results in unbranched actin filaments because profilin promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled filaments. These parallel filaments contrast with Arp2/3-mediated nucleation, which creates a highly branched actin filament network. Indeed, Arp2/3, although essential for lamellipodia formation, is not required for cytokinesis in Drosophila cells. The hypothesis here is that once myosin II is recruited to the equatorial cortex of the cell by a rho kinase-dependent mechanism, possibly localized activation of RLC phosphorylation, it is retained there because of its higher affinity for parallel, unbranched actin filaments than to branched actin networks. Consistent with this hypothesis, myosin II is depleted from the lamellipodia in migrating cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are more likely to be aligned in parallel bundles. Thus, it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow maintains a higher concentration of parallel actin filaments in this region compared with the rest of the cortex, and these parallel filaments serve to selectively retain myosin II at the equator to form a stable contractile ring. In the absence of these parallel actin filaments, myosin II can bind branched F-actin throughout the cortex, perhaps occasionally organizing them into parallel bundles that cause increased myosin recruitment corresponding to the flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).

Live-cell imaging shows that when pavarotti or racGAP50c are depleted, the cells do not display significant contractions despite recruiting myosin II to the equatorial cortex. Although there is some modest membrane contractile activity in these cells, it is clear that significant contraction or furrowing requires both components of the centralspindlin complex. It is surprising that only these proteins were found to be necessary for cortical contraction at sites of myosin II localization. Data from fixed cells, as well as earlier studies, indicated that Drosophila cells do not undergo equatorial contractions during mitosis when Diaphanous or Chickadee is depleted. However, live-cell imaging shows that when either of these two genes is depleted in S2 cells, not only is myosin II transiently localized to the equatorial cortex before dispersing, but cells do indeed display transient equatorial contraction. It is difficult to recognize these events in fixed cells because of their transient nature and the somewhat irregular shapes of cells depleted of these proteins. This work highlights the importance of live-cell imaging in the study of dynamic processes such as cytokinesis (Dean, 2005).

In addition to the suppression of furrowing, depletion of centralspindlin also leads to an inability to retain F-actin exclusively at the equatorial cortex during cytokinesis. This similar phenotype of the centralspindlin complex and the F-actin affecting proteins suggests that centralspindlin may be an upstream regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti have been shown to accumulate at the spindle poles and are associated with an abnormal accumulation of F-actin near the centrosomes. Centralspindlin may be acting indirectly by helping to localize an important actin-affecting protein at the central spindle, or it may act more directly on the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been hypothesized that centralspindlin affects the F-actin cortex through rho1 signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion, and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of centralspindlin may itself be important for signaling to the F-actin cortex. Finally, centralspindlin cannot be the major actomyosin ring positioning signal because myosin II is properly recruited in its absence (Dean, 2005).

Tum/RacGAP50C provides a critical link between anaphase microtubules and the assembly of the contractile ring in Drosophila melanogaster

A central question in understanding cytokinesis is how the cleavage plane is positioned. Although the positioning signal is likely to be transmitted via the anaphase microtubule array to the cell cortex, exactly how the microtubule array determines the site of contractile ring formation remains unresolved. By analysing tum/RacGAP50C mutant Drosophila embryos it has been shown that cells lacking Tumbleweed (Tum) do not form furrows and fail to localise the key cytokinetic components Pebble (a RhoGEF), Aurora B kinase, Diaphanous, Pav-KLP and Anillin. The GAP activity of Tum is required for cytokinesis: in its absence cytokinesis fails early even though Tum is present on microtubules at the cell equator where the furrow should form. Disruption of the Pebble-interacting domain leaves Tum localised to the cell equator on cortically associated microtubules, again with no evidence of furrowing. These data support a model in which Tum/RacGAP, via its interaction with Pbl, provides a critical link between the anaphase microtubule spindle and cytokinetic furrow formation in Drosophila cells (Zavortink, 2005).

Cells lacking detectable Tum progress through the mitotic cycle, successfully assembling a metaphase spindle and undergoing anaphase A and B. However, they fail to form or maintain a distinct central spindle or establish a cytokinetic furrow. Telophase cells possessed variable numbers of microtubule bundles, but these were rarely organised into a central spindle. This phenotype is similar to that seen in cells depleted of the Tum binding partner, Pav-KLP (Zavortink, 2005).

Significantly, and consistent with the absence of furrowing, none of the other cytokinetic components that have been checked to date (AurB, Dia, Anillin, Pbl and Pav-KLP) localise correctly in the absence of Tum. These observations show that Tum is required at the very earliest stages of furrow formation, consistent with suggestions that the central spindle and/or bundled midzone microtubules direct the earliest events of cytokinesis (Zavortink, 2005).

It was initially proposed that Tum interacts with Pbl at the cell equator and initiates changes in Rho activity, culminating in formation of the furrow. When the Pbl-interaction-domain deletion protein, TumDeltaPbl, is the only Tum protein present, cells proceed to a point in cytokinesis where assembled centralspindlin complexes are found at the cortex, bridging bundles of microtubules from opposite poles, but they do not proceed beyond this stage, suggesting that the Pbl-Tum interaction becomes critical at this time. This observation satisfies one important prediction of the model, that disruption of the Tum-Pebble interaction prevents cytokinesis but does not affect earlier centralspindlin-based microtubule localisation, supporting the proposal that the interaction between Tum and Pbl is the bridge between the anaphase mitotic spindle and contractile ring assembly (Zavortink, 2005).

Microtubule function in tum mutant cells is perturbed at anaphase, leading to the loss or disruption of the central spindle. It has been suggested that central spindle microtubules are unstable in the absence of bundling so the concomitant loss of Pav-KLP with Tum could explain this loss of microtubule organisation. However cases in cells overexpressing mutant forms of Tum were observed in which central spindle organisation is disrupted even in the presence of apparently stable centralspindlin complexes. If Tum does not interact with Pbl, or if GAP activity is compromised, central spindles fail to form or are unstable, even though the modified Tum is still delivered to the ends of microtubules associated with the cell cortex. Equatorial-cortex-associated microtubule bundles and misdirected non-cortical bundles form, but normal central spindles are rarely seen in these cells even though TumDeltaPbl apparently can bind Pav-KLP. Loss of the central spindle has also been observed in S2 cells depleted of Pbl by RNAi and in spermatocyte-specific pbl alleles (Zavortink, 2005).

Pbl and Tum may exert these effects on central spindle microtubule organisation by directly affecting the bundling activity and higher order structure of centralspindlin. Alternatively, they may act indirectly, influencing centralspindlin function by regulating the local concentration of active GTPases, a possibility that is supported by the failure of GAP-specific mutations to form or maintain a central spindle. However, the possibility cannot be ruled out that the TumDeltaPbl, TumDeltaEIE and TumDeltaYRL (the latter two with a deletions in the gap domain) constructs delete critical amino acid residues required for centralspindlin functions that are unrelated to GTPase regulation (Zavortink, 2005).

Although the central spindle is perturbed in the absence of a Tum-Pbl interaction or if Tum GAP activity is altered, a subpopulation of microtubules still contacts the cell cortex at the equator and both TumDeltaPbl and TumDeltaYRL can accumulate there, highlighting one difference between this microtubule population and the central spindle. Specific subpopulations of microtubules are beginning to be identified during anaphase and there is some evidence that a subpopulation of microtubules is stabilised at the equatorial cortex. Although a number of studies have provided links between Rho family GTPases and the stabilisation of microtubule cortex and microtubule-kinetochore interactions in mammalian cells, the presence of TumDeltaYRL at the ends of microtubules at the cell equator indicates that if Tum plays a role in the stabilisation of these microtubules at the furrow site it can do so in the absence of GAP activity (Zavortink, 2005).

Experiments with two different GAP deletions demonstrate that GAP activity is required for cytokinesis in the ectoderm. The DeltaEIE deletion removes three amino acids at the end of the 'A helix', which positions the catalytic, or finger loop containing the essential arginine (Arg417 in Tum). In vitro analysis of a similar deletion in n-chimaerin eliminates all GAP activity but results in a protein with higher affinity for GTPase than a wild-type protein. An equivalent deletion in Cdc42GAP also lacks GAP activity but, by several biochemical measures, has similar stability and structure to the wild-type protein. The YRL deletion removes the essential catalytic arginine and the two amino acids around it; in n-chimaerin a similar deletion eliminates all GAP activity. The localisation of these two proteins during mitosis in tum mutant cells differs. The EIE deletion seems to affect the behaviour of Tum more severely, resulting in diffuse localisation of the protein during anaphase, whereas TumDeltaYRL protein reaches the cell equator at anaphase but no cytokinetic constriction occurs. Irrespective of these differences, neither GAP-deficient form of Tum is capable of rescuing the tum cytokinetic defect (Zavortink, 2005).

These results differ from a recent analysis of Tum function in the larval nervous system, where Arg417 substituted Tum was able to rescue cytokinesis in larval neuroblasts. A different balance of cytokinetic mechanisms that contributes to cytokinesis may have evolved to meet the special requirements for asymmetric cell divisions in the CNS or cell divisions in an epithelial sheet. It is interesting to note, however, that in vitro studies in which the GAP DeltaEIE or DeltaYRL triplet mutations were compared to single arginine substitution mutants, the single arginine mutations always have significant GTPase-activating ability remaining. Indeed structural and biochemical studies of several GAP domains have emphasised that stabilisation of the switch 1 and switch 2 loops of bound GTPase by the GAP protein contributes significantly to GAP activity, suggesting the possibility that Arg417-substituted Tum may still have sufficient GAP activity to support cytokinesis, at least in some cell types (Zavortink, 2005).

Understanding the role of Tum and its orthologs, Cyk-4 and MgcRacGAP, in cytokinesis is complicated by conflicting evidence from different experimental systems. In contrast to the current observations, C. elegans eggs and mammalian tissue culture cells in which centralspindlin members are depleted, initiate cytokinesis, but the furrow regresses. The predominant aberrant phenotype produced by expression of GAP deletion mutants in the current experiments was a cell arrested before a furrow was evident, demonstrating that Tum is needed early in furrow formation. Some apparent late-stage cytokinesis failures were observed in embryos expressing TumDeltaYRL like those seen in mammalian cells, indicating that there is a second critical phase for Tum GAP activity in cytokinesis. It is suggested that late-stage defects may reveal an ancestral function of Tum and its homologs, with an earlier cytokinetic function adopted in Drosophila. There is a growing consensus that some of the differing observations between cell types and species reflect real differences in the way cells perform cytokinesis, rather than differences in the efficacy of RNAi, the perdurance of proteins, redundancy of protein functions or other experimental variables, but this issue remains unresolved (Zavortink, 2005).

Mammalian Tum, MgcRacGAP, exhibits several functions not seen in the current study. Expression of a MgcRacGAP GAP-defective mutant protein in mammalian cells affects chromosome attachment to the spindle at prometaphase via regulation of Cdc42, generating cells arrested in prometaphase or cells with micronuclei. Micronucleation was not seen in the current in this study, but multipolar anaphase cells were seen in tum mutants, indicating that Tum-deficient cells that have failed cytokinesis can transit the cell cycle and successfully enter anaphase again, suggesting that Tum does not have a critical role in prometaphase in these cells (Zavortink, 2005).

Expression of a MgcRacGAP GAP-defective protein also affects the cell cortex, causing blebbing during anaphase in mammalian cells. Blebbing in both Dictylostelium and mammalian cells is suppressed by substrate attachment and it is suggested that this phenotype, if it exists in Drosophila embryos, could be suppressed in cells in an epithelial sheet. All previous studies of Tum function in flies have noted non-cytokinetic functions revealed as aberrations of wingless signalling, EGFR signalling and axon migration, some of which might be indirect results of perturbation of cortical organisation (Zavortink, 2005).

Tum protein is required for the localisation of all cytokinetic components tested so far, including Pbl and Dia, two components that mark the earliest events in contractile ring formation. Disruption of the Pbl-interacting domain of Tum leaves centralspindlin at the cell equator, on cortically associated, bundled microtubules that are unable to induce furrowing, supporting the model for Tum-directed positioning of Pbl and, consequently, the cytokinetic furrow in Drosophila cells. A Tum protein with a defective GAP domain also arrives at the equatorial cortex at the ends of microtubules and remains there, but furrows do not form, demonstrating that Tum GAP activity is required at this early stage of cytokinesis. These results demonstrate the critical role Tum plays in initiation of cytokinetic furrowing in Drosophila cells and provides further evidence for the importance of the Tum-Pbl interaction in this process (Zavortink, 2005).

Role of Survivin in cytokinesis revealed by a separation-of-function allele

The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. This study identified a missense allele of Drosophila Survivin (FlyBase name: Deterin) that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this 'separation-of-function' allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems (Szafer-Glusman, 2011).

The chromosomal passenger complex (CPC), composed of the Ser-Thr kinase Aurora B and three partner proteins, plays several key roles in mitosis and meiosis, including regulation of attachment of kinetochores to microtubules, the spindle checkpoint that delays anaphase onset until all chromosomes are under tension on the spindle, regulation of sister chromatid cohesion, and cytokinesis. To accomplish these different tasks, the Aurora B kinase must be exquisitely localized in space and regulated in time. During mitosis, Aurora B associates with the microtubule-binding protein Inner Centromere Protein (INCENP), Borealin/DASRA/CSC-1, and the small, multifunctional BIR-motif protein Survivin to form the CPC (Szafer-Glusman, 2011).

Dissecting the role of individual CPC components has been hampered by the extraordinary interdependence of the four subunits; depletion of any single CPC protein by RNA interference knockdown in human cells affected the structural unit, localization, and function of the CPC. The structural basis of this interdependence is evident in the crystal structure of the Survivin-Borealin-INCENP core of the CPC complex, in which Borealin and INCENP associate with the C-terminal helical domain of Survivin to form a tight three-helix bundle (Szafer-Glusman, 2011 and references therein).

Strict localization of Aurora B by the CPC ensures that this kinase, which has multiple substrates, phosphorylates the correct targets at the proper points in cell cycle progression. Concentrated on chromosomes from G2, then at inner centromeres from prometaphase until the metaphase-to-anaphase transition, the CPC is required to regulate chromosome condensation, spindle formation and dynamics, kinetochore maturation, kinetochore-microtubule interaction, correct chromosome alignment, and control of the spindle checkpoint. At anaphase onset the CPC then translocates to the central spindle (CS) midzone and equatorial cortex and is involved in CS formation. At the CS Aurora B phosphorylates the centralspindlin components, Pav/mitotic kinesin-like protein 1 (MKLP1)/Zen-4 and the RacGAP50/MgcRacGAP/Cyc-4. However, the mechanisms that target the CPC to the spindle midzone and equatorial cortex after onset of anaphase and the mechanisms by which the CPC regulates central spindle formation and cytokinesis are not understood. In addition, the requirements for CPC function for critical events in metaphase and at the metaphase-anaphase transition have complicated analysis of how the CPC is localized and functions at later stages for cytokinesis (Szafer-Glusman, 2011).

This study characterized the role of Drosophila Survivin (dSurvivin), previously termed deterin and analyzed in its antiapoptotic activity, as a regulator of cell division, identifying a missense mutation (scapolo) in the Drosophila Survivin BIR domain that allows recruitment and function of the CPC in metaphase but disrupts CPC localization and function in anaphase and telophase. The findings reveal that Survivin plays a role in targeting the CPC and centralspindlin to the central spindle and the equatorial cell cortex during anaphase. In spermatocytes, Survivin function is also required to localize Polo and localize the small GTPase RhoA to set up the contractile ring machinery at the onset of cytokinesis. In larval neuroblasts undergoing mitotic division, however, the scapolo mutant did not block initial accumulation of Rho to a band at the equatorial cortex, although it did cause failure of cytokinesis. The different requirements for Survivin function for equatorial accumulation of Rho in spermatocytes versus neuroblasts may reflect a fundamental difference in the series of steps that lead to formation of the contractile ring in these two cell types (Szafer-Glusman, 2011).

A missense mutation leading to substitution of serine for the wild-type Pro-86 of Drosophila Survivin uncouples the function of Survivin in metaphase from function during anaphase and telophase, indicating a direct requirement for Survivin and the chromosomal passenger complex in orchestrating the profound reorganization of the cortical cytoskeleton at the cell equator at the onset of cytokinesis. This 'separation-of-function' allele allowed analysis of Survivin and CPC function during cytokinesis, which is normally obscured by the better-known roles of the CPC at centromeres during metaphase, when it facilitates alignment of chromosomes to the spindle equator and mediates the spindle checkpoint. The finding that a point mutation in the BIR domain disrupts activity of Survivin during cytokinesis challenges the model that the C-terminal domain of Survivin is sufficient for cytokinesis function (Lens, 2006) and indicates that residues in the BIR domain are important for localization and activity of Survivin at the central spindle (Szafer-Glusman, 2011).

Survivin associates with kinetochores and the central spindle with different dynamics, being highly mobile in prometaphase and metaphase and strongly immobile at the anaphase central spindle. This change in dynamics may underlie the largely normal localization and function of scapolo (scpo) (a missense allele of the Drosophila Survivin) at metaphase but the fully penetrant effect on assembly of the F-actin contractile ring and cytokinesis observed in scpo mutants (Giansanti, 2004; Szafer-Glusman, 2011).

Cytokinesis depends on the assembly of an equatorial actomyosin ring regulated by local activation of the small GTPase RhoA at the cortex, in turn catalyzed by the RhoGEF Ect2/Pebble. It has been proposed that association of RhoGEF/Pebble with centralspindlin promotes local RhoA activation at the cortex. In addition, the kinase polo (PLK1) has been implicated in RhoGEF localization and Rho activation, at least in part by phosphorylation of the centralspindlin component MgcRacGAP. The current observations that the Drosophila RhoA homologue, Rho1, failed to accumulate at the equatorial cortex in scpo mutant spermatocytes implicate Survivin and the CPC in the mechanism(s) that localize and activate RhoA at the equatorial cortex in these cells. This requirement may in part act through effects on Polo kinase. Failure to localize Polo to the central spindle in scpo mutant spermatocytes could prevent localization of RhoGEF by the centralspindlin complex and the consequent activation of Rho at the cortex. In this model, failure to localize Polo may contribute to the failure to form an equatorial ring of localized Rho1 and, in consequence, the inability to form a localized ring of myosin regulatory light chain and F-actin in scpo mutant male germ cells undergoing meiotic division. This mechanism may also explain the failure to maintain pole-to-equatorial microtubules observed in scpo mutant spermatocytes. It is likely that Rho-mediated activation of the Formin Dia helps stabilize microtubule arrays at the equatorial cortex of dividing cells, as active Rho and Formin (mDia) have been shown to regulate stabilization of microtubule arrays at the cortex of migrating fibroblasts. Consistent with this model, this study found that microtubules reached the plasma membrane at the equator of scpo dividing spermatocytes, but the bundles are transient and fail to form stable arrays at the cortex (Szafer-Glusman, 2011).

A striking finding of this work is the difference in requirement for Survivin function for localization of the Polo kinase and RhoA in anaphase neuroblasts versus spermatocytes. This difference raises two possibilities: either Survivin is not part of a universal signaling mechanism that directs cytokinesis, or different semiredundant mechanisms can drive cytokinesis, similar to redundancy between astral pulling and sliding of central spindle microtubules for anaphase B, and different cell types rely more strongly on one mechanism or the other. Indeed, consistent with the latter possibility, spermatocytes and neuroblasts display different cytoskeletal architectures during cytokinesis (Giansanti, 2006). In neuroblasts, actomyosin initially accumulates in a broad cortical band, presumably because this is the region of the cell cortex that escapes repression of Rho associated with the plus ends of astral microtubule. This initial wide band gradually narrows into a tight equatorial ring as the cell progresses into telophase. Thus assembly of the contractile apparatus in neuroblasts proceeds, as proposed for Caenorhabditis elegans embryos, in 'two genetically separable steps' in which localization of contractile machinery is initially independent of the central spindle. In support of this model, this study found that Rho1 accumulates in a broad cortical band in scpo mutant neuroblasts, suggesting that the first stage can occur independent of Survivin and CPC localization to the central spindle (Szafer-Glusman, 2011).

Spermatocytes, in contrast, do not form an initial wide equatorial band of contractile ring components. Instead, from their first appearance in early anaphase, the actomyosin rings in spermatocytes are tightly focused at the cell equator). It is speculated that this restricted initial localization of contractile ring components and the apparent lack of a preceding wide equatorial band may be a consequence of a more stringent global block to Rho1 activation at the cortex in spermatocytes than in neuroblasts. It is proposed that this global block is eventually overridden by positive regulation of Rho1 by local concentration of RhoGEF, in turn facilitated by CPC-dependent events associated with and/or localized by central spindle microtubules. Rho1 activation would then occur within a narrow peak exactly at the site where pole-to-equator microtubules interact to maximize RhoGEF deposition/concentration at the cortex. Indeed, F-actin ring assembly occurs locally and cytokinesis initiates immediately after the pole-to-equator microtubules contact the cortex in Drosophila spermatocytes. It is proposed that, according to this model, the defects in Survivin lead to lack of CPC activity and abnormal centralspindlin, resulting in absence of Rho1 and Polo kinase from the equator of scpo mutant spermatocytes (Szafer-Glusman, 2011).

In neuroblasts, where a more permissive cortex allows a broad belt of Rho1 activation at the cell equator, Survivin and CPC appear to promote gradual convergence of the initial broad band into a narrow ring centered at the maximum of RhoGEF activity at the cortex. In scpo mutants, which display irregular anaphase central spindles devoid of Pav, the broad Rho1 cortical band fails to narrow, the cells fail to form a focused, narrow ring of myosin, and cell division proceeds with inefficient and incomplete constriction (Szafer-Glusman, 2011).

A key difference between neuroblasts and spermatocytes that may account, at least in part, for the differences in behavior of Rho1 and myosin complex proteins is in the relationship between Polo kinase and the CPC observed in scpo mutant mitotic versus male meiotic cells. In spermatocytes, Polo and the CPC are interdependent and Polo colocalizes with the CPC along its full journey from metaphase through anaphase and telophase. In neuroblasts, in contrast, Polo localization during cytokinesis appears to be independent of the CPC and centralspindlin, at least at early stages of cell division, but Polo appears to colocalize with Feo, the Drosophila homolog of PRC1, that required for central-spindle formation and cytokinesis. A second difference between neuroblasts and spermatocytes may be the recently described, spindle-independent backup system that can localize myosin to a broad band at the cell cortex near the future cleavage plane under control of the neuroblast cell polarity system. The broad localization of myosin to the cell cortex observed in ana/telophase neuroblasts in scpo mutants may be in part due to these redundant mechanisms (Szafer-Glusman, 2011).



The subcellular distribution of the Pav protein was examined throughout the cell cycle and its potential association with other molecules was studied. An antibody was raised against a 450-amino-acid peptide from the central region of the protein (residues 208-686) expressed in Escherichia coli. The antibody recognizes not only the bacterially expressed protein but also one predominant band at ~100 kD in Western blots of Drosophila embryo extracts. It immunostains wild type and pavB200+/ and embryos at cycle 16 to give distinct punctate nuclear staining in interphase cells in the ventral epidermis. In mitotic cells, bright bands of staining are seen equidistant from separating telophase nuclei, presumably at the site of the spindle interzone (see the Glover lab website for information about the distribution of Pav in mitotic cells). Bright spots are also seen that appear to correspond to midbodies persisting from earlier divisions. In contrast, pavB200 homozygous mutant embryos lack all nuclear and central spindle staining at cycle 16, except for some punctate spots likely to correspond to maternally contributed protein in remnants of putative midbodies from earlier cycles (Adams, 1998).

A closer examination of the localization of Pav reveals that in wild-type syncytial embryos, the protein is diffuse during interphase, but during prophase and prometaphase it becomes localized in the actin-containing furrows that separate nuclei and maintain nuclear spacing. During anaphase, the central, interzonal region of the mitotic spindle is stained. Anaphases showing staining at both sites are very rarely seen, even though the slight asynchrony between dividing nuclei in the blastoderm embryo allows all stages of anaphase to be observed in a single embryo. At telophase, staining intensifies at the spindle interzone, and as nuclei enter the next interphase the staining becomes diffuse once again (Adams, 1998).

In cellularized cycle 14 embryos, staining is nuclear during interphase. It is then possible to detect centrosomal staining in cells at metaphase and anaphase, and at late anaphase, the protein begins to concentrate at the central region of the spindle. As telophase progresses, staining remains associated with the spindle during its constriction by the contractile apparatus (Adams, 1998).

Since the septin homolog Peanut has been proposed to connect the central spindle with the contractile ring, Peanut distribution was studied with respect to that of Pav. At late anaphase, Peanut becomes concentrated at the spindle interzone, and colocalizes with Pav. At telophase, Pav remains associated with microtubules at the midbody, whereas Peanut staining extends further and is concentrated at either side of the midbody closer to the plasma membrane. Thus, Pav and Peanut do not fully colocalize at this stage. However, they do colocalize in the previously identified small punctate regions of staining. Since Peanut is known to be expelled from the cell at the end of mitosis, the presence of Pav in the same location supports the notion that these spots are remains of midbodies from previous divisions (Adams, 1998).

The Drosophila gene DmMKLP1 has a high similarity to members of the mitotic kinesin-like subfamily of kinesin proteins. DmMKLP1 has no known close relatives in the Drosophila genome and can therefore be assumed to be the ortholog of human MKLP1 and hamster CHOI kinesin-like proteins. In situ hybridization reveals a homogeneous maternal expression in the early embryo and a terminally restricted expression pattern at blastoderm stage. Later, the expression becomes increasingly restricted to the developing central nervous system, where it remains expressed at least until the end of embryogenesis (Schmid, 1998).

Localization of Pavarotti in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation (Minestrini, 2003).

These findings extend those from a study of the phenotype of pavarotti mutant embryos where a role for the gene product in cytokinesis was indicated by the generation of binucleate cells (Adams, 1998). Although spindle elongation seems to take place during anaphase, a properly organized central spindle does not form. Moreover, pavorotti mutants fail to initiate the formation of an actomyosin contractile ring after anaphase. These observations added to growing evidence that formation of the midzone region of the central spindle is a necessary precondition to organize the cleavage ring for cytokinesis. The immunolocalization of Pav-KLP to the spindle midzone is in keeping with such a role. The present study of the localization of GFP-tagged Pav-KLP in living embryos shows the motor protein also to be present at known sites of interaction between putative plus ends of microtubules and the actomyosin cytoskeleton in both synctial and cellularized embryos (Minestrini, 2003).

For the first 10 division cycles of the syncytial embryo, Pav-KLP seems not to interact with the actomyosin cytoskeleton. Before blastoderm most of the protein associates with the spindle. During this period, the molecule concentrates at the midzone during anaphase, much as it does in mononucleate cells, consistent with a plus end-directed motor activity. It also shows particularly strong labeling of astral microtubules. In this latter respect, localization of GFP-Pav-KLP differs from the previous observations by immunostaining that detected the endogenous protein on centrosomes rather than asters. Although either of these experimental approaches could lead to artifacts, it seems most likely that the particularly sensitive astral microtubules were insufficiently well preserved in the previous immunolocalization studies and so collapsed back to the centrosome during fixation. Its presence on astral microtubules may indicate a role in sliding antiparallel astral overlap microtubules against each other. Such a function is needed to keep the correct spacing between neighboring nuclei and for cortical migration of the nuclei during cycles 7-9 (Minestrini, 2003).

The characteristic colocalization of Pav-KLP with microtubules and actomyosin throughout subsequent successive stages of embryogenesis suggest Pav-KLP could mediate a crucial link between these components of the cytoskeleton. Once nuclei have migrated to the cortex, Pav-KLP begins a dynamic pattern of colocalization with putative plus ends of spindle microtubules and the cortical actomyosin cytoskeleton. During metaphase it associates with pseudocleavage furrows and then in anaphase and telophase, spindle associated GFP-Pav-KLP molecules move toward the central spindle and concentrate in the midbody (Minestrini, 2003).

During cycle 14 membranes extend toward the embryo's interior to compartmentalize the 6000 or so cortical nuclei. At this time, GFP-Pav-KLP is present both in the nucleus and at the tips of invaginating membranes where F-actin, myosin II, Peanut, and anillin are also known to localize. The movement of the actomyosin network and its associated proteins to invaginate membranes around the newly forming cells has been postulated to track along microtubules with a plus end-directed motor providing the motile force. Pav-KLP is certainly one candidate for such a task (Minestrini, 2003).

The dynamics of GFP-Pav-KLP localization in mononucleate cells undergoing cell division from cycle 14 onwards is consistent with the motor having at least two roles in facilitating the reorganization of the actomyosin cytoskeleton for cell division: one in the organization of or the transport of molecules along the central spindle during anaphase and telophase, and the other in reorganizing cortical molecules along astral microtubules. GFP-Pav-KLP is present in the cell nucleus until prophase and is redistributed into two populations after breakdown of the nuclear membrane. The first is represented by a low level of GFP signal occurring on the mitotic spindle around the time of metaphase and that will move to the spindle midzone from anaphase onward. The second population of GFP-Pav-KLP seems to constitute most of the GFP fluorescence. It is uniformly distributed at the cell cortex throughout metaphase. Such cortical association of the endogenous Pav-KLP is also apparent by immunostaining and can be seen in the original article by Adams (1998). From anaphase to telophase, cortical GFP-Pav-KLP moves from the polar regions of the cortex toward the equatorial region where the cleavage furrow will form. Here, it becomes incorporated into the cleavage ring. This dynamic relocalization of the protein is consistent with Pav-KLP providing the motor protein functions to transport components of the actomyosin cytoskeleton toward the plus ends of cortical microtubules to concentrate in a cylinder whose perimeter will become the contractile ring. Similar ideas have been previously suggested for plus end-directed motors. At the end of the division, when the ring is fully contracted, all GFP fluorescence accumulates in the midbody, and thus the majority of Pav-KLP seems to be discarded from the daughter cells with the loss of this structure (Minestrini, 2003).

Surprisingly, neither the formation and function of the mitotic spindle nor cytokinesis is visibly affected in embryonic cells expressing GFP-PavNLS(4-7)*, a mutant form of the protein lacking nuclear localization sequences and unable to localize to the nucleus during interphase. Cell division of otherwise wild-type embryos seems not to be affected by expression of this mutant molecule that is still able to localize to the cell cortex and at later stages of the division to the cleavage ring. Moreover expression of GFP-PavNLS(4-7)* mutant in a pav background is able to rescue the embryonic lethality of this stock, allowing survival to late pupal and adult stages. In contrast, expression of the putatively immotile GFP-PavDEAD molecule with a mutation in its ATP-binding site results in dominant lethality. This mutant form of Pav-KLP not only fails to enter the nucleus but also shows significantly reduced association with the cell cortex. This suggests that the plus end-directed motor activity of Pav-KLP is essential for its cortical localization and equatorial concentration. The rigor-like association of GFP-PavDEAD with microtubules is similar to that seen with an equivalent point mutation in the ATP-binding domain of the yeast motor protein Kar3p. Moreover, a similar mutant of the centromere-associated mitotic centromere-associated kinesin has also been observed to undergo rigor binding to microtubules, and fails to enter the nucleus. The GFP-PavDEAD protein seems not to be released from microtubules and is itself transported polewards. Two dominant mutant phenotypes are seen, presumably reflecting different degrees of expression of GFP-PavDEAD between cells. In the first, cells fail to enter cytokinesis and show no sign of formation of either a cleavage furrow or a central spindle. In this respect they resemble cells in pavarotti mutant embryos (Adams, 1998). In the second group of cells expressing the dominant mutant, daughter cells are produced that remain connected by stable microtubules, indicating defects at later stages of cytokinesis. This phenotype is very similar to that observed after the expression of the CHO1 isoform of mammalian MKLP1 with a mutation in its ATP-binding domain in CHO cells. This together with the finding that depletion of CHO1 by RNAi led to disorganization of midzone microtubules has led to the conclusion that this form of the mammalian homolog functions to ensure midbody formation for the completion of cytokinesis. Although it is possible that the homologous proteins have subtly different functions in different species or cell types, being required for the initiation of cytokinesis in some (Adams, 1998) and its completion in others, it is difficult to draw direct comparisons because one cannot be sure of the extent to which function is lost either in mutants or in RNAi experiments. However, expression of the 'motor-dead' protein in Drosophila does suggest both early and late roles for the molecule in cytokinesis (Minestrini, 2003).

A striking feature of cells in which the initiation of cytokinesis is prevented by expression of GFP-PavDEAD is the accumulation of the inactive motor protein on spindle pole microtubules. This may reflect either its transport by a minus end-directed motor or it may be the consequence of polar mircrotubule flux. A consequence of this is the accumulation of actin in the vicinity of the centrosome. A reorganization of the actomyosin cytoskeleton was also seen when this mutant form of the protein was expressed in Drosophila oogenesis (Minestrini, 2002). Although the molecular mechanism behind this reorganization of the actomyosin cytoskeleton is not known, it indicates that the motor protein is able to mediate associations between the actomyosin and microtubule cytoskeletons. Such interactions are central to cytokinesis in wild-type cells and in other processes that share aspects of this machinery at other stages in Drosophila development (Minestrini, 2003).

The mammalian counterpart of Pav-KLP, CHO1, has also been shown to interact with actin. In this case, the interaction has been mapped to an exon that is not present in the splice variant form MKLP1. It is not known however, whether this interaction is direct or indirect. However, MKLP1 has been shown to undergo a direct interaction with a small G protein, Arf, that is required for membrane trafficking and may have a function in facilitating membrane fusion events at cytokinesis. Two members of the MKLP1 family have also been shown to interact directly with a conserved Rhofamily GTPase-activating protein. In C. elegans, this is encoded by cyk-4 and in Drosophila it corresponds to RacGAP50C (Somers, 2003). Somers has proposed that Pav-KLP transports RacGap50C along cortical microtubules to the cell equator. Once at the cell equator, it is proposed that RacGAP50C mediates another interaction with the Pebble Rho GEF to position contractile ring formation and coordinate F-actin remodeling. Ect2, a mammalian ortholog of Pebble, is also involved in cytokinesis. Many components of this emerging complex remain to be defined. It does not seem that the Pebble GEF opposes RacGAP50C, thus leaving the open question of what the respective opposing proteins might be. It is of considerable future interest to determine precisely how Pav-KLP interacts with other proteins and how they are loaded and unloaded during the highly dynamic process of cytokinesis (Minestrini, 2003).

Pavarotti function during spermatogenesis

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. These findings are discussed with respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis (Carmena, 1998).

To correlate the spindle defects in polo mutants with abnormal cytokinesis, the distribution of a number of proteins that associate with the contractile furrow was examined. The kinesin-like protein encoded by pavarotti can associate with Polo kinase. In mitotically dividing cells, both proteins can be found at spindle poles until anaphase, but become associated with the most central part of the spindle in the region of the cleavage furrow at telophase. This pattern of subcellular localization of both proteins is disrupted in pavarotti mutants. The distribution of Pav was examined in meiosis in both wild-type and polo1 mutant males. It was difficult to visualize Pav at the spindle poles in wild-type meiosis, although the protein could be observed in the cleavage furrows at the telophase of both meiotic divisions, and to the ring canals. polo1 mutant cysts contain ~30% of cells of mutant phenotype alongside cells of wild-type appearance. In such a mutant cyst, Pav can be seen associated with the cleavage furrow of the wild-type appearing cells. In contrast, the protein is not found between the telophase nuclei in cells showing a mutant phenotype, but instead appears to accumulate in the region of the poles (Carmena, 1998).

The other striking feature of cysts from polo1 mutant testes is the loss of synchrony of the meiotic divisions in comparison with wild-type meiosis. All of the cells in wild-type cysts progress through telophase. Pav is present in the rings of the cleavage furrows and in the ring canals. In addition, Pav is found associated with some (putative immature) axonemes, but not with others. It is speculated that this points to another function of this motor protein in transporting flagellar components to its growing distal tip, or to facilitate the elongation of organelles such as mitochondria in the sperm tail. However, the polo1 mutant cyst contains cells at a variety of late meiotic stages. Many cells have a wild-type appearance, in which case Pav may be seen in large cleavage rings at late anaphase-early telophase, or more compact rings at late telophase. In other cells such rings of Pav never seem to form even though the nuclei have moved to the poles of the spindle. This is frequently the case with multipolar cells. The polo1 mutation does not affect the association of Pav with the flagellar axonemes (Carmena, 1998).

Since septins are known to become incorporated into the contractile ring, attempts were made to localize the Drosophila septin Peanut in telophase cells from polo1 mutant cysts during meiosis. In wild-type cysts, Peanut appears in the cleavage furrow rings at anaphase, slightly ahead of Pav. The two proteins colocalize in the furrow at telophase, with Pav appearing more concentrated on the inner side of the ring. The ring canals appear to contain Pav but Peanut is less easily detected. In mutant cysts, there is a tendency for both proteins to accumulate in the larger cleavage furrow rings (Carmena, 1998).

The loss of synchrony of the meiotic divisions within a cyst is also apparent in late meiotic cysts. In the wild-type cysts, cells are uniformly at telophase, and show actin rings of comparable sizes, in contrast to the cells of mutant cysts that are at a variety of meiotic stages. Late telophase cells can be seen in which the spindle mid-body is well formed and is associated with a compact actin ring. Other bipolar cells in which the chromatin has migrated fully to the poles have no mid-zone structure to the spindle microtubules, and are lacking any actin ring. This directly links the defect in the central spindle to a failure to establish a contractile ring. A number of cells with tripolar spindles are also seen in this particular cyst. In some of these cells there is no indication of any actin ring formation, whereas in others a large somewhat misshapen actin ring forms. Such cells could arise as a result of cytokinesis failure in the first meiotic division, and failure of one of the centrosomes to separate in the second division (Carmena. 1998).

Intercellular protein movement in syncytial Drosophila follicle cells

Ring canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. This study shows by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, it was shown that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. Somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline (Airoldi, 2011).

Similarly to germline ring canals in Drosophila egg chambers, follicle cell ring canals are supported by filamentous actin lining the lumen. In germline ring canals, the highly dynamic actin cytoskeleton mediates ring canal expansion during egg chamber development. A 200 nm-thick mesh of F-actin bundles accumulates at the plasma membrane of germline ring canals, which reach an overall diameter of nearly 10 mm. By contrast, follicle cell ring canals do not expand during development, remaining about 250 nm in diameter with a monolayer of actin filaments (Airoldi, 2011).

The persistence of cleavage furrow proteins in ring canals is a common feature of both germline and somatic ring canals. Many of the follicle cell ring-canal components identified to date are proteins with known roles in cytokinesis. Anillin and Pav-KLP accumulate in cleavage furrows as they begin to constrict, and remain associated with ring canals, as was observed in images of live dividing follicle cells (Airoldi, 2011).

Mutations in scraps (the gene encoding Anillin) and pav cause defects in cytokinesis and result in multinucleate cells. In addition, an Anillin-binding protein called Cindr was recently identified as another component of both cleavage furrows and somatic ring canals (Airoldi, 2011).

In germline ring canals, Anillin, Pav-KLP and Cindr are present initially in ring canals, but only Pav-KLP persists throughout oogenesis. The recruitment of the robust actin cytoskeleton to ring canals in Drosophila female germline cells might displace cleavage furrow proteins (Airoldi, 2011).

The data helps refine the list of somatic ring canal proteins; Vsg can be added, and Nasrat and Polehole can be eliminate. GFP::Vsg was reported to be present in germline ring canals and follicle cell puncta. The localization of GFP::Vsg to follicle cell ring canals was confirmed using immunoEM and colocalization studies. Vsg is a predicted sialomucin protein with a single transmembrane domain and multiple predicted extracellular O-linked glycosylation sites. Its localization to somatic ring canals is highly reminiscent of Mucin-D localization; however, it was not possible to confirm this directly because Mucin-D antibodies are no longer available. Vsg (and Mucin-D) are also present in ring canals of larval imaginal disc and brains. Vsg protein bears predicted localization signals, including an N-terminal plasma membrane localization signal and a C-terminal vesicular trafficking sequence. Therefore, its specific localization to ring canals could indicate a role of vesicular trafficking in the formation of stable intercellular bridges (Airoldi, 2011).

Previous work on Vsg revealed roles in promoting cell proliferation and embryonic development, though its specific function is unclear. Its vertebrate homolog, endolyn, is targeted to endosomes and lysosomes, and functions in the maintenance of hematopoietic progenitors and myoblast fusion. The highest degree of similarity between Vsg and Endolyn is in the transmembrane and cytoplasmic domains, including the C-terminal lysosomal sorting signals, suggesting a common underlying role for this family of sialomucins (Airoldi, 2011).

Not all follicle cells have stable ring canals The data indicate that 70% of follicle cell divisions within the germarium result in a stable ring canal, and this increases to 89% of divisions outside the germarium. A possible explanation for the absence of ring canals is that a percentage of cell divisions complete cytokinesis and a ring canal is never formed. In light of the data, this would suggest that follicle cells within the germarium are more likely to complete abscission, possibly because these cells are still in the early stages of differentiation and still contain factors that promote normal cell division (Airoldi, 2011).

Alternatively, the absence of ring canals that wsd observed might be a result of instability or destruction of otherwise normal ring canals, or to the merging of existing ring canals. If so, the higher rate of ring canal loss in the germarium could be attributed to the mechanical stresses of migration as the follicle cells move to encapsulate the germline cyst and reposition for stalk formation. Once outside the germarium, the constant but smaller rate of ring canal loss could be a result of shuffling within the epithelium, cell death or eventual resolution of cytokinesis (Airoldi, 2011).

Specialized subpopulations of follicle cells lack ring canals. No ring canals were observed in mature stalk cells; however, ring canals were observed between cells in the region of the forming stalk as egg chambers exit the germarium. Loss of ring canals during stalk formation could be due to mechanical disruption as cells intercalate to form a single chain of cells. A model of mechanical disruption of existing ring canals is further supported by observations of border cells. Presumptive border cells in stage 8 egg chambers have normal ring canals, but migrating border cells contain fewer, smaller, and apparently fragmented GFP::Pav-KLP puncta (Airoldi, 2011).

No ring canals were observed in pairs of polar cells. This is not surprising, because the polar cell precursors are specified while in the germarium and do not continue to divide. Furthermore, the precursor population is reduced to only two cells through programmed cell death between the germarium and stage 5, which would further isolate the polar cells. However, additional investigation is necessary to determine whether the polar cell precursors have ring canals, either between themselves or with other main-body follicle cells (Airoldi, 2011).

Given the presence of persistent intercellular bridges within follicle cell lineages, whether follicle cell mitotic divisions are synchronized, as they are in male and female germline cells, was investigated. Our results clearly indicate that some level of synchrony exists between small groups of follicle cells because adjacent mitotic cells are significantly more frequent than predicted from a random distribution. The coordination of mitosis could be carried out by a cell nonautonomous mechanism in which mitosis-promoting factors pass through ring canals and initiate mitosis in the next cell. However, nearly all mitotic cells had ring canals connecting them to nonmitotic cells, so any propagation of mitosis-promoting signals would be limited. Furthermore, mitotic divisions within follicle cell lineages consisting of hundreds of cells are far from fully synchronized, suggesting that any influence of neighbors on entry into mitosis is weak. Alternatively, a cell-autonomous cell cycle 'timer' could be responsible for the apparent coordination of mitosis between sibling cells, regardless of a connecting ring canal. In this scenario, two sibling cells would enter mitosis together because their cell cycles were synchronized at the conclusion of the previous division. Of note, it was found that 47% of cells were in mitosis independently of all neighboring cells, which implies that if a cell-autonomous cell cycle timer controls entry into mitosis, the length of the overall cell cycle (9.6 hours) will vary by more than 24 minutes in nearly half of all cells. The data are insufficient to distinguish between cytoplasm sharing and timer mechanisms, because the existence of ring canals between neighboring mitotic cells fits both models (Airoldi, 2011).

The pattern of connections between cells with ring canals is determined by spindle orientation with respect to ring canals from previous mitoses. In Drosophila germline mitoses, spindle orientation and thus ring canal inheritance is controlled by the fusome, a cytoplasmic organelle containing ER membranes that extends through each intercellular bridge. After four mitotic divisions, the 16 cells are maximally branched with the original two cells having four ring canals each and the youngest eight cells from the final division each having one ring canal. By contrast, the follicle cells, which do not have a fusome, appear to divide randomly with respect to pre-existing ring canals, and their pattern of connections is intermediate between fully branched and completely linear. Other mechanisms are likely to contribute to the pattern of follicle cell divisions, such as a limitation on how many direct connections an individual cell can have to hexagonally packed neighbors (Airoldi, 2011).

The presence in follicle cells of intercellular bridges large enough for the passage of small vesicles presents the possibility of extensive flow of information between these cells. However, many aspects of follicle cell biology strongly suggest a cell-autonomous system. For example, small subsets of follicle cells pattern the oocyte during development, an essential function that would be compromised by unrestricted spreading of localized signals to neighboring cells. Also, genetically mosaic patches of follicle cells appear to be reliably marked by expression of various reporters. Follicle cells are also known to have highly mosaic expression of many proteins, some of which clearly maintain very different levels of expression despite a connecting ring canal (Airoldi, 2011).

As a result, it was surprising to discover evidence for robust and rapid exchange of PA-GFP between cells, in patterns consistent with syncytia of follicle cells connected by ring canals. Importantly, this result was confirmed by observing movement of tagged endogenous proteins by FLIP. In both sets of experiments with fluorescent proteins, exchange of protein was observed in one or two immediate neighbors of the target cell. Not only does this argue against nonspecific activation or bleaching, it is also consistent with the number of ring canals per cell that were observed in other experiments. Co-expression of GFP::Oda and GFP::Pav-KLP confirmed these results by demonstrating that protein movement occurs only between cells connected by a ring canal (Airoldi, 2011).

Of note, FLIP data also show that some proteins are not exchanged through ring canals, whereas others pass freely. Components of large protein complexes, such as ribosomal proteins and those involved in mRNA biogenesis, appear restricted in their movement. By contrast, orinithine decarboxylase antizyme and calmodulin are relatively small (40-60 kDa), monomeric proteins, and exchange between cells rapidly. This suggests that large and/or highly complexed proteins do not exchange between cells at a high rate, but smaller proteins do. These results point to the possibility of extensive intercellular movement of protein among somatic cells connected by ring canals (Airoldi, 2011).

Emerging data now reveal that stable ring canals extensively interconnect follicle cell lineages. In addition, similar ring canals connect cells of the imaginal discs and larval brain. These somatic ring canals are over 100 times larger than gap junctions (250 nm for ring canals versus 1.5 nm for gap junctions) and thus provide a viable path for macromolecules to move between cells. Live-imaging data show that some proteins can freely exchange between connected follicle cells, which raises interesting questions about the true autonomy of these cells. A practical consideration concerns the use of GFP as a marker for genetic mosaic analysis, because unrestricted movement of GFP between cells would compromise it as a clonal marker (Airoldi, 2011).

For example, using GFP loss to identify a mitotic clone of homozygous mutant cells could be confounded by GFP movement from a twin-spot cell or an unrecombined heterozygous cell. However, the data suggest that for large clones, the movement of GFP is unlikely to affect the results. The majority (66%) of follicle cells have ring canals to only one or two other cells, which suggests that the number of potentially compromised cells would be small. Furthermore, GFP moving into a genetically mutant cell could be accompanied by wild-type gene product, potentially creating a wild-type phenotype. However, if small clones or single cells are being investigated, protein exchange through ring canals might pose a significant concern (Airoldi, 2011).

Further investigation to determine the extent and control of traffic through ring canals will reveal how follicle cells use and regulate their ability to directly communicate with neighboring cells (Airoldi, 2011).

Effects of Mutation or Deletion

Preliminary characterization of the phenotype of pav mutants by staining embryos with the neuronal marker mAb 22C10 indicates the formation of fewer and larger neurons than usual in the embryonic development of the PNS, and an absence of support cells. To investigate whether the large cells seen in pav mutant embryos arise from cell division defects, defects in pav mutants from cycle 14 (the first cycle under the control of the zygotic genome) onward through cycles 15 and 16 were sought. No differences were detected between wild-type and pav embryos in cycle 14 or 15, but in cycle 16, it was observed that although mitosis appeared to be normal within the ventral epidermis, interphase nuclei were frequently closely associated in pairs in both P-element and EMS-induced alleles of pav. The boundaries of the cells of the embryo were defined using an antibody against alpha-spectrin, a membrane-bound structural protein. The cell membrane does not invaginate during telophase in pav embryos as it does in wild type. Despite this failure of cytokinesis, nuclei appear to enter interphase 7 successfully, as nuclear laminae reform around both nuclei in binucleate cells. Such binucleate cells are never seen in wild type (Adams, 1998).

In addition to the cytokinesis defects that can be observed in essentially all cells at cycle 16, other mitotic defects have also been observed at a very low frequency in pavB200 and pavM187 embryos but not in the P-element alleles. The affected cells show multipolar spindles and multiple centrosomes that are always associated with what appear to be tetraploid masses of chromatin. This would suggest that in the pavM187 and pavB2004 alleles, binucleate cells are formed that can undergo another round of mitosis with double the usual number of centrosomes. This could occur if the maternal supply of pav gene product were to become exhausted in some cells during cycle 15. Such cells would fail to undertake cytokinesis but then proceed to attempt a sixteenth division, the last cell cycle for the majority of epidermal cells (Adams, 1998).

To address how pav mutations might affect cytokinesis, the structure of the mitotic spindle was examined in mutant embryos. The morphology of the spindle was indistinguishable from wild type in the mitotic stages up until late anaphase. No differences between wild-type and pav mutants were observed in the degree of elongation of the spindle as cells progress from metaphase to late anaphase. However, in pav mutants the distinctive morphology of the central spindle is substantially changed at telophase so that it appears to contain fewer bundles of microtubules. In addition, whereas peripheral spindle microtubules (i.e., those microtubules emanating from the centrosome that contact the cell cortex) are seen to become constricted in wild type, this appears not to occur in the pav mutant (Adams, 1998).

The localization of Peanut, Actin, and Anillin, proteins known to be required for cytokinesis, was also examined. The formation of the septin ring was examined using antibodies to the Drosophila septin, Peanut. The septin family of proteins was originally identified in Saccharomyces cerevisiae as required for bud neck formation and subsequently shown to be present in most eukaryotes. Although peanut has been shown to be required for cytokinesis in Drosophila, its exact role is unknown. Recent results suggest that it may mediate attachment between the actin network and the spindle. In wild-type cellularized embryos, Peanut protein is confined to the cell surfaces, with punctate spots of the protein at the site of the intercellular bridges remaining from the previous division. At late anaphase, Peanut accumulates in the equatorial region of the cell and appears at the contractile ring at telophase. In pav embryos, Peanut staining is generally weaker and more delocalized than in wild type, although some cells appear to have a near normal surface distribution of the protein. However, although areas of punctate Peanut staining can sometimes be seen in apparent association with the plasma membrane midway between the telophase nuclei, Peanut never appears at the site of the contractile ring (Adams, 1998).

pav mutants were examined to determine whether Actin or Anillin localizes at the contractile ring and if cleavage furrows can form. In wild-type cellularized embryos, Actin is present around the surface of the cell throughout the cell cycle and accumulates in the equatorial region of the cell during telophase before forming the contractile ring. The Actin binding protein Anillin is thought to be involved in regulating or organizing the contractile domains of the Actin cytoskeleton. It is found in the nucleus of wild-type cellularized embryos during interphase but is relocalized to the cortex during mitosis and to the cleavage furrow at cytokinesis. Anillin appears at the cleavage furrow slightly preceding Actin. In pav mutant embryos, there is no indication of the accumulation of either Actin or Anillin at the position that the contractile ring and cleavage furrow normally form. However, the distribution of these proteins at other stages of the cell cycle appears to be unaffected (Adams, 1998).

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

Double-stranded RNA-mediated interference (RNAi) was used to study Drosophila cytokinesis. Double-stranded RNAs for anillin, RacGAP50C, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. The phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and led to a proposal that the central spindle and the contractile ring are interdependent structures. Finally, these results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis (Somma, 2002).

The phenotypical analyses of RNAi-induced mutants in the RacGAP50C, rho1, and sqh genes provide the first description of the cytological defects that lead to cytokinesis failures when the function of these genes is ablated. Previous studies have shown that mutations in rho1 and sqh disrupt mitotic cytokinesis but have not defined the cytological phenotypes elicited by these mutations. In addition, pav and pbl (RNAi) cells have been characterized; the phenotypes of these (RNAi) cells are consistent with those previously observed in animals homozygous for mutations in these genes (Somma, 2002 and references therein).

Cells in which the RacGAP50C, pav, pbl, rho1, and sqh genes are ablated by RNAi normally undergo anaphase A, but they then fail to elongate and to undergo anaphase B. After anaphase A, mutant cells proceed toward telophase and decondense their chromosomes, forming typical telophase nuclei. However, these cells fail to develop a central spindle, to assemble an actomyosin contractile ring and to concentrate anillin in the cleavage furrow. This results in the formation of short, aberrant telophases that are unable to undergo cytokinesis and will thus give rise to binucleated cells (Somma, 2002).

The functional ablation of genes influencing either the actin or the microtubule cytoskeleton have similar effects on cytokinesis. The genes pbl, rho1, and sqh likely play primary roles in controlling the actin cytoskeleton. The sqh gene encodes a regulatory light chain of myosin II. Rho1 is a member of the Rho family GTPases that cycle from an inactive GDP-bound state to an active GTP-bound state under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs enhance the exchange of bound GDP for GTP, whereas GAPs increase the GTPase activity of Rho. Rho proteins and Rho GEFs, such as Drosophila Pbl and human ECT2, localize to the cleavage furrow and are required for contractile ring assembly. In contrast, the activities of RacGAP50C and pav are likely to primarily influence the function of the central spindle. The Pav kinesin-like protein, a homolog of the C. elegans ZEN-4, is localized in the central spindle, and is thought to mediate microtubule cross-linking at the central spindle midzone. The RacGAP50C gene encodes a Rho GAP, and it is orthologous to the cyk-4 gene of C. elegans. CYK-4 interacts with ZEN-4, and the two proteins are mutually dependent for their localization to the central spindle. The complete absence of Pav immunostaining in RacGAP50C (RNAi) telophases suggests a similar interaction between RacGAP50C and Pav, pointing to a role of RacGAP50C in central spindle assembly. In summary, the cytological phenotypes of pbl, rho1, and sqh (RNAi) cells indicate that a primary defect in acto-myosin ring formation results in a secondary defect in central spindle assembly. The phenotypes of RacGAP50C- and Pav-depleted cells suggest the converse: that a primary defect in the central spindle can secondarily disrupt contractile ring formation. Thus, taken together, these data indicate that the central spindle and the actomyosin ring are interrelated structures. Although the molecular mechanisms underlying the cross talk between these structures is not conpletely understood, two possibilities can be envisioned. The formation and maintenance of both the central spindle and the actomyosin ring could be mediated by physical interactions between interzonal microtubules and components of the contractile ring. Alternatively, the central spindle and the contractile ring could be coupled by a checkpoint-like regulatory mechanism, which would inhibit the formation of either of these structures when the other is not properly assembled (Somma, 2002).

Although RacGAP50C, pav, pbl, rho1, and sqh (RNAi) cells display similar terminal phenotypes, the aberrant telophases observed in these cultures differ in both actin and anillin distribution. In rho1 telophases these proteins are excluded from the cell equator, in pbl they are uniformly distributed, and in RacGAP50C, pav, and sqh they concentrate in a wide equatorial band. This suggests that rho1 and pbl are required for actin and anillin accumulation in the equatorial region of the dividing cell. In contrast RacGAP50C, pav, and sqh seem to be required for the assembly of the contractile machinery from proteins already concentrated at the cell equator. In sqh (RNAi) cells the failure to assemble an actomyosin ring is likely to be a direct consequence of the depletion of an essential component of the ring. In RacGAP50C and pav cells this failure is instead likely to be a secondary effect of problems in central spindle assembly (Somma, 2002).

An interplay between the central spindle and the contractile ring has been suggested by studies on Drosophila male meiosis. Mutant spermatocytes in the chic, and dia loci, which encode products thought to be involved in contractile ring formation, and mutants in the kinesin-encoding gene klp3A, all display severe defects in both structures. Although all the extant results on Drosophila cells strongly suggest an interdependence of the central spindle and the contractile ring, it is currently unclear whether this is true in all animal cells. Studies on mammalian cells have shown that central spindle plays an essential role during cytokinesis. However, these experiments have provided limited information on whether perturbations in the actomyosin ring assembly disrupt the central spindle. The best evidence of an interplay between the central spindle and the contractile ring has been in rat kidney cells. By puncturing these cells with a blunt needle a physical barrier is created between the central spindle and the equatorial cortex. This barrier not only abrogates actomyosin ring assembly on the side of perforation facing the cortex, but also disrupts the organization of central spindle microtubules on the opposite side (Somma, 2002 and references therein).

In contrast, studies on C. elegans embryos indicate that, at least in the early stages of cytokinesis, the actomyosin ring and the central spindle can assemble independently. Why do Drosophila, and possibly mammalian cells, differ from C. elegans in the interactions between the central spindle and the contractile ring? It is believed that the answer to this question reflects differences in the distance between the central spindle and the equatorial cortex. In Drosophila and mammalian cells during central spindle assembly the equatorial cortex is very close to the interzonal microtubules. In contrast, in C. elegans embryos the central spindle assembles in the center of the cell when the cleavage furrow has just began to ingress, so that during their assembly the actomyosin ring and the central spindle lie a considerable distance apart. Only later in cell division, after substantial furrow ingression, can the actomyosin ring and the central spindle come into contact. It is thus hypothesized that in embryonic cells of C. elegans the cytokinetic process consists of two steps: an early step, where the central spindle and the contractile ring assemble independently in distant cellular regions, and a late step that begins when the central spindle and the contractile ring have come into contact. The early stage might be mediated by interactions between astral (rather than central spindle) microtubules and the contractile ring. The late step of C. elegans cytokinesis may then require that the contractile ring and the central spindle interact cooperatively to complete cytokinesis successfully. This two-step hypothesis also applies to other large cells, such as echinoderm eggs, where the central spindle and the cortex are separated by large masses of cytoplasmic material and seem to assemble independently (Somma, 2002 and references therein).

Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis

The kinesin-like protein encoded by pavarotti (Pav-KLP) is essential for cytokinesis and associates with the central part of the late mitotic spindle and interphase nuclei in somatic cells. Regions of the molecule that regulate its subcellular localization have been defined and the consequences of overexpressing mutant forms of the protein during oogenesis in Drosophila have been studied. Pav-KLP normally associates with the oocyte nucleus, but when over-expressed at moderate levels, its GFP tagged form also accumulates in nurse cell nuclei. At high expression levels this leads to loss of the microfilaments that tether these nuclei, so that they block the ring canals and prevent the 'dumping' of nurse cell cytoplasm into the oocyte, which results in sterility. Localization to these nuclei is prevented by mutations in either the conserved ATP-binding site of the motor domain or the nuclear localization sequences in the C-terminal domain. Both such mutations led to the formation of stable arrays of cytoplasmic microtubules and the progressive disruption of the actin cytoskeleton. The latter is evident by a breakdown of the cortical actin causing disruption of cell membranes; this breakdown ultimately results in the accumulation of cytoplasmic aggregates containing tubulin, actin and at least some of their binding proteins. Pav-KLP is also found associated with the ring canals, actin-rich structures built from remnants of the cytokinesis ring. The stalk domain alone is sufficient for the exclusive association of Pav-KLP to these structures, and this has no consequences for fertility. Whether disruption of actin structures by full-length cytoplasmic forms of Pav-KLP is a consequence of the resulting stabilized cytoplasmic microtubules per se or accumulation of the motor protein at ectopic cortical sites to sequester molecules that regulate actin behavior is discussed (Minestrini, 2002).

The extant pav mutant alleles result in embryonic lethality as a result of failure of cytokinesis in cycle 16, the second cycle after the protein is first required for this process following cellularization of the embryo. In mitotically dividing cells, Pav-KLP becomes incorporated into the mid-body which is discarded following cytokinesis, whereas in spermatogenesis it is retained in the remnants of the contractile ring that is used to build the ring canals. These structures form interconnections between the synchronously developing cells within a common cyst that will ultimately contain 64 spermatids. Pav-KLP becomes similarly incorporated into the analogous structures that connect the 15 nurse cells and oocyte in the developing egg chamber during oogenesis. Moreover this work defines the central stalk region of the molecule alone as being able to direct this localization. Since ring canals like mid-bodies are derived from the remnants of the cleavage furrow, it is possible that the end point of a similar series of localization events to those seen at the end of M-phase is being seen. However, as expression of GFP-PavSTALK only becomes maximal after the completion of the mitotic divisions in the germarium, most of the localization observed occurs after completion of this process most probably due to incorporation into the growing ring canals. The stalk domains of kinesin-like proteins are known to mediate intermolecular interactions leading to the formation of functional dimeric or trimeric molecules. Consequently, it is not possible to be certain that association of the stalk with ring canals can occur independently of the remaining portions of the molecule, since its localization was studied in flies that are still expressing wild-type protein. Nevertheless, it will be of future interest to search for direct interactions between this domain of the protein and other molecules that could participate in its localization to these subcellular structures of which actin associated proteins may be among the best candidates because of their abundance in ring canals (Minestrini, 2002).

A specific feature of the localization of Pav-KLP in wild-type oogenesis is its accumulation in the transcriptionally quiescent oocyte nucleus. It is likely that mRNA for the motor protein is actively transported from the nurse cells to the oocyte cytoplasm, where the protein is synthesized for transportation into the nucleus for storage. Subsequently the protein appears to be utilized during female meiosis as it is incorporated into the central spindle pole body that develops between the tandemly arranged spindles of meiosis II. When expressed at wild-type levels, no protein can be seen becoming incorporated into the nurse cell nuclei within the egg chamber. It is however, possible that it is simply easier to see the nuclear localization in the smaller oocyte nucleus. However, its incorporation into nurse cell nuclei is seen following its overexpression. This is consistent with the known properties of members of the MKLP-1 family, that is following completion of their function in cytokinesis they localize to nuclei during interphase. The only exception is sea urchin KRP110, which seems to have a perinuclear localization at that stage (Minestrini, 2002 and references therein).

The overexpression of wild-type Pav-KLP in the female germ line appears not to affect the four rounds of mitosis that take place in the germarium, since egg chambers contain the correct number of cells. It is believed that oocyte specification may be delayed however, because this is a microtubule-dependent process. The oocyte is specified but then fails to adopt its correct anterodorsal positioning during stage 2. It has been argued that the defective oocyte localization also seen in spindle C mutants is consequential to a delay in oocyte determination. The mipositioning of the oocyte in egg chambers overexpressing wild-type and mutant forms of Pav-KLP could also be explained as an indirect consequence of a delay to oocyte specification resulting from defects in microtubule-mediated transport. Mispositioning of the oocyte is also seen in mutants for spaghetti squash (sq), which encodes a regulatory myosin light chain; this points to the additional possibility of a role for the actin-myosin cytoskeleton in this process (Minestrini, 2002).

The major effect resulting from overexpression of GFP-Pav-KLP is a failure of nurse cells to 'dump' their cytoplasm into the oocyte. In wild-type egg chambers, thick actin filament bundles are required to keep the ring canals free from nurse cell nuclei to enable such a process to occur. Such structures are illuminated by GFP-Pav-KLP expressed at lower levels from the polyubiquitin promoter but are not observed following overexpression of the motor protein. These filaments are also missing in egg chambers of mutants for chickadee, singed and quail where fast cytoplasmic flow is also impaired. Similarly, sqh mutant egg chambers fail to efficiently transport their nurse cell cytoplasm into the oocyte. The overexpression of Pav-KLP may therefore similarly destabilize the actin cytoskeleton, leading to blockage of the ring canals by nurse cell nuclei and the failure of the nurse cell 'dumping' observed. It is suspected that the breakdown of the oocyte/nurse cell membrane and protrusion of nurse cell nuclei into the oocyte cytoplasm may result from an increase in intracellular pressure following the blockage of the ring canals (Minestrini, 2002).

The findings that a set of overlapping nuclear localization signals are present in the C-terminus of Pav-KLP confirms and extends similar observations made on the mammalian homolog. These sequences are not in themselves sufficient to mediate nuclear localization, which can also be prevented by a putative motor inactivating point mutation in the ATP-binding site of the motor protein. It is presumed that this is due to rigour binding of the putatively immotile protein to microtubules that consequently accumulate in the cytoplasm. It is formally possible that the functional ATP-binding site is a requirement for nuclear import of Pav-KLP, a possibility that could be tested in future by defining and mutating the microtubule-binding site. The rigour-like association of GFP-PavDEAD with cytoplasmic microtubules is, however, reminiscent of that seen with a yeast motor protein, Kar3p, carrying the equivalent G131E point mutation in its ATP-binding site. Moreover, when a similar mutant of the mitotic centromere-associated kinesin (MCAK) was expressed in CHO cells it also decorated cytoplasmic microtubules and was excluded from the nucleus, suggesting that this may be a common consequence of inhibiting motor function of kinesin-like proteins (Minestrini, 2002 and references therein).

The microtubule arrays that accumulate when overexpressed Pav-KLP cannot enter the nucleus are more resistant to the microtubule-depolymerizing drug colchicine than the microtubule arrays of wild-type egg chambers. Although they retain some specificity for microtubule-associated proteins, shown by their binding of the Orbit but not the Mini spindles protein, it is unlikely that they function normally. Ultimately overexpression of GFP-PavNLS(4-7)* or GFP-PavDEAD leads to a breakdown of the cortical cytoskeleton of the germ line cells. The GFP-tagged Pav-KLP molecules and their associated microtubules appear to colocalize with actin in the cortical cytoplasm, resulting in disruption of cell membranes. This in turn leads to the formation of aggregates enriched in Pav-KLP, tubulin, actin and actin-binding proteins such as Huli tai shao, which is a homolog of adducin that acts as an assembly factor for the spectrin-actin network. It is possibly released from the ring canals as they dissociate and then associates with the actin aggregates. It is not clear whether this effect on the actin cytoskeleton is a direct consequence of the accumulation of microtubules at the cell cortex or whether the ectopic Pav-KLP is sequestering actin regulatory proteins at this site. This might be detrimental when permitted to occur at the cell cortex but may have little effect if restricted to structures such as ring canals, which could occur following overexpression of the stalk domain alone. The breakdown of nurse cell membranes is also caused by mutations in sko, a gene encoding filamin, which cross-links F-actin to nurse cell membranes and ring canals. Like the dominant Pav-KLP mutants, sko mutant egg chambers also have nurse cell nuclei that transgress into the oocyte compartment in addition to abnormal ring canals and actin cables. These findings suggest that accumulated cytoplasmic Pav-KLP leads to a progressively abnormal distribution of actin and its binding proteins and that this is associated with the breakdown of cell borders. This may also facilitate the fusion of egg chambers that can occur at all stages during oogenesis in the dominant mutants (Minestrini, 2002 and references therein).

The function of Pav-KLP will not be fully understood until it is known precisely what molecules Pav-KLP interacts with and the nature of the cargoes it carries. As a kinesin-like protein, it might be expected to interact with microtubules as is demonstrated by these studies. The present findings imply that the protein may also interact with the contractile actin ring or proteins that regulate its function during cytokinesis to become incorporated into the cytokinesis remnant. To date the closest hint of any possible interaction of the protein with any potential regulator of the actin cytoskeleton is given by the observation that recruitment of the C. elegans MKLP-1 homolog ZEN-4 to the spindle midzone requires interaction with a GTPase-activating protein CYK-4. GTPase-activating proteins of this type regulate the activity of Rho family GTPases, which have a function in contractile ring assembly. Knowledge of the interactions that Pav-KLP makes with other molecules to coordinate microtubule and microfilament behavior together with the dynamics of these processes will be essential for understanding its roles in mitosis and interphase (Minestrini, 2002 and references therein).

RacGAP50C directs perinuclear gamma-tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. A novel postmitotic function has been identified for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. The localization of the MT nucleator gamma-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and this change in localization depends on RacGAP50C. RacGAP50C and gamma-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants gamma-tubulin remains dispersed throughout the cytoplasm. Furthermore, the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute gamma-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process (Guerin, 2009).

The reorganization of the actin and MT cytoskeletons during myogenesis has long been observed in cell culture as individual myoblasts fuse into multinucleated myotubes. Although the actin cytoskeleton has been shown to be indispensable for mediating myoblast fusion, little is known about how the MT network is remodeled during muscle development or the developmental significance of this event. This study provides evidence that RacGAP, a known regulator of the MT cytoskeleton during cytokinesis, and Pav, a kinesin-like RacGAP-binding protein, play a novel and important role in MT organization in vivo by localizing γ-tubulin to perinuclear sites in myotubes. Furthermore, the organization of the MT network in multinucleated myotubes is important for muscle attachment site (MAS) selection. Muscles that are mutant for RacGAP or pav have defects in MT polarity and fail to properly extend towards their attachment sites, resulting in defects in somatic muscle patterning (Guerin, 2009).

The current model for MT organization in differentiated myotubes has come primarily from cell culture studies, which describe MTs that run parallel to the long axis of the cell and do not appear to be directly associated with any one organizing center. Further studies have demonstrated that proteins involved in MT organization, such as γ-tubulin, are redistributed from the centrosome of individual myoblasts to discrete cytoplasmic puncta as well as along the nuclear membrane in multinucleated myotubes and that these sites are associated with MT growth (Bugnard, 2005; Musa, 2003). The diffuse cytoplasmic distribution of γ-tubulin that is observed in Drosophila myoblasts differs from that in cultured vertebrate myoblasts, in which γ-tubulin is associated with centrosomes. Nonetheless, in both cases, the MT cytoskeleton must be reorganized from either a centrosomal or broadly distributed array in individual myoblasts, to a parallel array in multinucleated myotubes with the plus ends directed outwards. This study shows that RacGAP plays a crucial role in this reorganization. In the absence of RacGAP, MTs are not uniform in their polarity and γ-tubulin remains dispersed throughout the cytoplasm rather than accumulating at the nuclear periphery of multinucleated myotubes. Furthermore, in pav mutants, mislocalization of RacGAP is sufficient to redistribute γ-tubulin to the ends of myotubes (Guerin, 2009).

To date, the perinuclear localization of γ-tubulin in myotubes has only been weakly detected in vitro (Bugnard, 2005). This study shows that the association of γ-tubulin with the nucleus also occurs in vivo and is dependent at least in part on RacGAP. What is the function of γ-tubulin localization to the nuclear periphery in myotubes? One likely possibility is to anchor MT minus ends. Because the nuclei in multinucleated myotubes cluster in the interior of the myotube, this would allow for the polarization of the MT network, which is aligned along the axis of cell migration, with the plus ends at the leading edge. What is the purpose of MT polymerization at the ends of myotubes? Although, conventionally, the driving force for cell motility has been thought to be provided mainly by the reorganization of the actin cytoskeleton, there is increasing evidence that MTs are indispensable for cell migration. It has been hypothesized that MTs form longitudinal arrays in bipolar myotubes in order to facilitate elongation by 'active crawling' of the two ends of the myotube during MAS selection (Musa, 2003). The data point to a MT-based mechanism for myotube extension and MAS selection. In the absence of RacGAP or Pav, the MT network shows non-uniform polarity and many muscle fibers are abnormally shaped and display guidance errors. The effect of RacGAP and pav mutations on muscle morphology is consistent with previous findings in which both RacGAP and Pav have been implicated in regulating axonal outgrowth and maintaining dendritic morphology. RacGAP was identified in a genetic screen by the increased dendritic branching phenotype observed in tum mutants. RacGAP and Pav have also been shown to play a role in regulating the morphogenesis of postmitotic mushroom body neurons in the Drosophila brain. In addition, disruption of the mammalian form of Pav, KIF23 (CHO1; MKLP1), in postmitotic cultured neurons resulted in the rearrangement of MT polarity and in the disruption of dendrite morphology (Guerin, 2009).

There is increasing evidence that morphological processes require regulated coordination of the cytoskeleton by linking actin and MTs. For example, in Drosophila the Rho activator RhoGEF2 is implicated both in Myosin II localization and MT organization via the localization of the plus-end protein Eb1. Likewise, RacGAP provides a connection between the actomyosin ring and the peripheral central-spindle MTs during cytokinesis via its interaction with the actin-binding protein Anillin. In addition, proper formation of the cleavage furrow is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed MT protein Pav. The current data show that similar to its function during cytokinesis, the function of RacGAP in postmitotic myotubes depends on its association with the MT-binding protein Pav. However, the role of RacGAP in regulating γ-tubulin distribution appears to be independent of its interaction with Anillin and the actin cytoskeleton. scraps mutants do not show defects in muscle patterning. Furthermore, the organization of the actin cytoskeleton and two known actin-dependent processes, myoblast fusion and muscle attachment, are not significantly affected in RacGAP mutants. These findings demonstrate a newly described function for RacGAP that is restricted to the modulation of MTs, but not the actin cytoskeleton, in postmitotic cells (Guerin, 2009).

What is the developmental significance of the actin-independent function of RacGAP in myotube extension? The answer might lie in the complex process of myogenesis itself. Building a mature muscle fiber requires the coordination of many morphological processes, including myoblast fusion, myotube extension and muscle attachment. The uncoupling of actin- and MT-based cytoskeletal processes might allow for actin-based myoblast fusion and MT-based myotube elongation to occur simultaneously. This idea is supported by previous findings showing that myoblasts continue to fuse as the myotube elongates to find its attachment sites. In addition, fusion-defective mutant FCs have been observed to extend and attempt to migrate to their targets, demonstrating that the migration machinery is not perturbed in mutants in which fusion is disrupted (Guerin, 2009).

It is not yet clear what serves as the trigger for MT reorganization upon myoblast fusion or how RacGAP is recruited for this process. It also remains to be determined whether RacGAP promotes the nucleation of new MTs at the nuclear periphery, or reorganizes existing MTs from fusing myoblasts. Changes in MT architecture could be regulated through a direct physical interaction between RacGAP and γ-tubulin, or indirectly through a complex with downstream targets of the GAP domain of RacGAP (Guerin, 2009).

This study examined the role of Pavarotti (Pav-KLP), a kinesin-6, in the coordination of spindle and cortical dynamics during mitosis in Drosophila embryos. In vitro, Pav-KLP behaves as a dimer. In vivo, it localizes to mitotic spindles and furrows. Inhibition of Pav-KLP causes defects in both spindle dynamics and furrow ingression, as well as causing changes in the distribution of actin and vesicles. Thus, Pav-KLP stabilizes the spindle by crosslinking interpolar microtubule bundles and contributes to actin furrow formation possibly by transporting membrane vesicles, actin and/or actin regulatory molecules along astral microtubules. Modeling suggests that furrow ingression during cellularization depends on: (1) a Pav-KLP-dependent force driving an initial slow stage of ingression; and (2) the subsequent Pav-KLP-driven transport of actin- and membrane-containing vesicles to the furrow during a fast stage of ingression. It is hypothesized that Pav-KLP is a multifunctional mitotic motor that contributes both to bundling of interpolar microtubules, thus stabilizing the spindle, and to a biphasic mechanism of furrow ingression by pulling down the furrow and transporting vesicles that deliver new material to the descending furrow (Sommi, 2010).

Previous work has shown that the Drosophila kinesin-6, Pav-KLP, plays important roles in coordinating spindle and cortical dynamics by promoting spindle-midzone organization, contractile-ring formation and cleavage-furrow growth. Although the role of Pav-KLP in conventional cytokinesis has been well studied, little is known about Pav-KLP function in early Drosophila embryos, in which mitosis and cytokinesis are adapted for rapid cycles. The aim of this study was to investigate the role of Pav-KLP in the syncytium, in which extremely dynamic interdependent mitotic spindles and furrows reorganize every 10-15 minutes. Pav-KLP, a MT-bundling dimer, stabilizes the mitotic spindle by bundling interpolar MTs (ipMTs) and, at the same time, promotes the growth of mitotic and cellularization furrows (Sommi, 2010).

It was observed that Pav-KLP is localized on the spindle and at the cortex, and it is suggested that Pav-KLP performs distinct roles there. On the spindle, Pav-KLP colocalizes with MTs and is involved in spindle organization: its inhibition generates defects in pole-pole separation. Various degrees of inhibition give rise to phenotypes of varying severity: short spindles, defective chromosome segregation, disorganized or absent telophase midbodies, spindle collapse and fused daughter nuclei. In addition, it was noticed that nuclei originating from spindles with defective midbodies always gave rise to spindles with a very strong defective phenotype in the next mitotic cycle. The spindle collapse after Pav-KLP inhibition occurred at a later stage of mitosis than that observed after anti-KLP61F injection, which causes collapse at the onset of the prometaphase-metaphase spindle elongation, suggesting that Pav-KLP plays an important role in maintaining pole-pole spacing. The observation that Pav-KLP inhibition prevents spindles from elongating during anaphase could reflect its role in stabilizing the ipMTs so that the necessary forces can be generated. These results are consistent with the hypothesis that Pav-KLP is involved in stabilizing the spindle structure, most probably by bundling ipMTs and providing a framework that allows other motor proteins to promote spindle elongation (Sommi, 2010).

Defects in spindle morphogenesis after Pav-KLP inhibition were also consistently accompanied by the formation of interconnected adjacent spindles. Such connections have been observed after inhibition of KLP61F or KLP3A in an ncd-null mutant background; in this case, the spindle lacks two motors and is so disorganized that MTs splay outwards from the spindle and sometimes reach adjacent spindles. The consistent formation of interconnections after Pav-KLP inhibition supports the hypothesis that Pav-KLP is also involved in furrow assembly. Indeed, the data show that Pav-KLP not only colocalizes with actin at the cortex but is also responsible for actin remodeling: when Pav-KLP function was blocked, diminished furrow growth and loss of actin recruitment along the furrows were observed both in the syncytium and during cellularization (Sommi, 2010).

It is suggested that the role of Pav-KLP in spindle morphogenesis and membrane reorganization are distinct and can be explained by considering two Pav-KLP populations defined by the presence of the spindle envelope. Inside the spindle envelope, Pav-KLP could function in spindle morphogenesis, whereas outside the spindle envelope it could function in membrane reorganization (Sommi, 2010).

It was observed that Pav-KLP is also involved in membrane and actin remodeling during cellularization, a process that is similar to conventional cytokinesis. During cellularization, loss of Pav-KLP function impedes both actin recruitment and membrane insertion into the furrow, thereby affecting the overall furrow length and organization. Modeling suggests that, during cellularization, Pav-KLP functions both as a force generator and a transporter by unfolding and pulling down membrane in the first, slow stage of furrow growth and transporting membrane vesicles to the furrow in the next, fast, stage of growth (Sommi, 2010).

Pav-KLP might play a role in membrane and actin remodeling either via direct transport or via transport of factor(s) involved in actin regulation. The interdependence between actin remodeling and membrane recruitment has been previously observed in nuf, dal and discontinuous actin hexagon (dah) mutants, and also in mutants containing the dominant-negative form of Rab11, in which loss of actin localization and furrow-growth defects were observed. It was observed that, in embryos expressing GFP-Nuf, loss of Pav-KLP function affected Nuf distribution. It has been shown that the centrosomal localization of Nuf relies on minus-end-directed dynein-mediated transport of Nuf along astral MTs from the periphery. It is suggested that Pav-KLP, as a plus-end motor, could be responsible for proper Nuf localization and actin recruitment to the cortex. Actin can be delivered to the furrows simply by being attached to the membrane vesicles, as is the case for Dah. it was not possible to determine whether Pav-KLP is responsible for the direct transport of actin to the cortex or whether it promotes the delivery of signaling molecules. Polo kinase has been proposed as a signaling molecule that is required for centrosome function and cytokinesis, and that is transported by Pav-KLP. Using GFP-Polo-expressing embryos, Polo localization to the cortex was observed, in addition to at centrosomes and the spindle midzone, similar to Pav-KLP localization. These observations hint at a role for Pav-KLP in vesicle transport but more work is needed to elucidate the involvement of Pav-KLP in transport pathways in the early Drosophila embryo (Sommi, 2010).

Altogether, this experimental work identified Pav-KLP as a stabilizing and transporting agent involved in both spindle and cortical dynamics. Furthermore, the quantitative modeling suggested a possible mechanical role for this motor as a force generator pulling down the membrane (Sommi, 2010).

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. Two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. This nuclear localization is essential for their role in Wnt regulation. Thus, two modulators of the Wnt pathway have been identified that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis (Jones, 2010).

This study presents evidence that Tum and its binding partner, Pav, are required for negative regulation of Tcf-mediated transcription. In retrospect, it seems likely that their pleiotropic effects on cytokinesis have obscured their role in regulation of the Wnt pathway until now. For example, neither of these genes was identified in genomics-based screens for Wg pathway components in cultured cells. Reduction of Tum or Pav function cannot be easily assessed in cell culture because tum- or pav-deficient cells cease to divide. The fly embryo, which uses maternally loaded Tum and Pav until midway through development, provided a fortuitous and genetically tractable model that allowed the detection of their involvement in Wnt modulation. This raises the question of how many pathway components have been overlooked because they have other essential cellular functions (Jones, 2010).

The roles of Tum and Pav in cytokinesis are genetically separable from their roles in Wnt signaling. The GAP activity of Tum is required for cytokinesis: TumδEIE, mutated in the gap domain, was unable to support normal cell division. However, TumδEIE had a significant capacity to repress TOPflash activity in cultured cells and to rescue pattern defects in tum mutant fly embryos. Similarly, the Pav-binding domain of Tum is essential for cytokinesis, but TumδPav retained a modest ability to repress Wg/Wnt signaling in cultured cells, indicating that some of the Tum regulatory function is independent of Pav. Conversely, nuclear localization of Pav is irrelevant to its role in cytokinesis, whereas it was found that nuclear localization of both Tum and Pav was required for rescuing pattern defects in embryos and for repressing TOPflash activity in cultured cells. Thus the Tum-Pav interaction is important for both cytokinesis and full Wnt regulation, but the cellular activity of the complex differs both spatially and enzymatically in the two processes (Jones, 2010).

Most molecules that negatively regulate the Wnt pathway act through the ubiquitylation-based pathway that controls βcat stability. However, Tum and Pav were able to repress TOPflash induced by the constitutively active δGSK-βcat and to diminish pattern disruption caused by the analogous mutant form of Arm in fly embryos. This observation places Tum and Pav activity downstream of the well-characterized control exerted by the destruction complex. Tum and Pav did not prevent βcat from entering the nucleus, but did prevent a βcat-Lef fusion protein from successfully activating gene expression. Therefore a model is favored where Tum and Pav have a constitutive role that impedes binding of the βcat-Tcf complex to DNA or that dampens transcriptional activation by the DNA-bound complex. Curiously, the Tum homolog in humans MgcRacGAP has been connected with transcriptional regulation via HIF-1α, with which it interacts physically (Lyberopoulou, 2007). MgcRacGAP has also been associated with nuclear translocation and activation of STAT transcription factors (Kawashima, 2009). Tum itself was shown to regulate activity of the EGF pathway in the Drosophila wing. These observations suggest that Tum/MgcRacGAP has a broader role within the nucleus, interacting with transcription factors in other cellular pathways (Jones, 2010).

Since Drosophila Tum and Pav are able to repress the Wnt response in human cells, this raises the possibility that MgcRacGAP and MKLP-1, their vertebrate counterparts, might also regulate Wnt signaling. It is tempting to speculate that a direct connection between Wnt regulation and cell division is relevant to oncogenesis, in much the same way that Wnt signaling and cell adhesion are integrally connected through the shared component βcat. Recent work has demonstrated that cell-division defects leading to tetraploidy might ultimately lead to cancer. Multinucleate cells are more likely to experience genetic instability, increasing the risk of transformation to malignancy. Thus, any genetic or epigenetic change that reduces the function of Tum/MgcRacGAP or Pav/MLKP-1 could have a double impact on the cell, compromising the cytokinesis machinery and elevating activity of the Wnt pathway (Jones, 2010).


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pavarotti: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 March 2015

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