InteractiveFly: GeneBrief

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


Gene name - sticky

Synonyms - CG10522, citron

Cytological map position - 69C4

Function - signaling

Keywords - cytokinesis

Symbol - sti

FlyBase ID: FBgn0036295

Genetic map position - 3L

Classification - Serine/Threonine protein kinase family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Pebble (Pbl)-activated RhoA signalling is essential for cytokinesis in Drosophila melanogaster. The Drosophila citron gene, [a. k. a. sticky (sti)], encodes an essential effector kinase of Pbl-RhoA signalling in vivo. Drosophila citron is expressed in proliferating tissues but is downregulated in differentiating tissues. Citron can bind RhoA and localisation of Citron to the contractile ring is dependent on the cytokinesis-specific Pbl-RhoA signalling. Phenotypic analysis of mutants showed that citron is required for cytokinesis in every tissue examined, with mutant cells exhibiting multinucleate and hyperploid phenotypes. Strong genetic interactions were observed between citron and pbl alleles and constructs. Vertebrate studies implicate at least two Rho effector kinases, Citron and Rok, in cytokinesis. By contrast, no evidence was found of a role for the Drosophila ortholog of Rok in cell division. It is concluded that Citron plays an essential, non-redundant role in the Rho signalling pathway during Drosophila cytokinesis (Shandala, 2004).

RNA interference-mediated silencing of sticky/citron in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such Sticky-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes, suggesting that these GTPases antagonistically regulate Sticky functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. Antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis (D'Avino, 2004).

It is unclear at this point which of the two names will be the accepted term for this gene. The priority should go to the 15 year old term 'sticky', applied to a series of mutations described in 1989. In a screen for P element-induced Drosophila mutants, showing abnormal mitotic figures in neuroblasts of third instar larvae (Deak, 1997), two different lines were identified that displayed a polyploid phenotype consistent with a defect in cytokinesis. These mutations were allelic to each other but the P elements were not responsible for the sti phenotypes. The mutations were mapped to a region containing a gene named l(3)7m-62, or sti, that exhibits very similar mitotic defects (Gatti, 1989). Complementation analysis has revealed that these mutants are allelic to sti and thus the alleles were named sti4 and sti5. The mitotic index of sti mutants is very similar to that of wild-type larvae, suggesting a primary defect in cytokinesis. Moreover, the majority of polyploid neuroblasts exhibit normal chromosomes, indicating that sister chromatids segregate normally. Only a small fraction of cells showed hypercondensed chromosomes characteristic of karyokinesis defects. However, these cells were also highly polyploid (8N or more), suggesting that defects in chromosome segregation are a secondary effect due to the presence of supernumerary centrosomes and consequent multipolar spindles (D'Avino, 2004 and references therein).

Cytokinesis, the cellular function regulated by sticky/citron, is the final step in the cell division cycle when two prospective daughter cells are separated by ingression of the plasma membrane between separating chromosomes. Although still poorly understood, the strict spatial and temporal coordination of cytokinesis with the other events of mitosis appears to be mediated by a number of proteins that form a complex regulatory network. A major component of this network is the Rho small GTPase, which serves as a switch in a wide variety of signal transduction pathways that regulate cytoskeletal dynamics in cellular processes such as cell migration, adhesion, morphogenesis, axon guidance and cytokinesis. Guanine nucleotide exchange factors (GEFs) catalyse the formation of the GTP-bound active form of Rho GTPases, which can bind and activate downstream effectors. It has been hypothesised that the activation of Rho GTPases by particular GEFs specifies the downstream Rho effector proteins and, therefore, the pathway that is activated (Shandala, 2004).

Drosophila pebble (pbl) and its mouse and human homologues, named Ect2, encode GEFs that specifically activate RhoA signalling during cytokinesis. Downstream effectors of Rho small GTPases in different cellular contexts are starting to be defined. With respect to cytokinesis, the vertebrate Citron kinase (Citron) is postulated to be an in vivo target of RhoA during cytokinesis. Citron was isolated in a yeast two-hybrid screen as a protein capable of binding preferentially to GTP-bound RhoA (Madaule, 1995). It is a member of a conserved family of serine/threonine kinases described in mouse, rat, human and fly. Consistent with a role in activating myosin II during cytokinesis, Citron can phosphorylate the myosin regulatory light chain (MRLC: spaghetti-squash in Drosophila) in vitro (Yamashiro, 2003). Citron and the GTP-bound form of Rho localise to the cleavage furrow and midbody during cell division in mouse, rat and human cells (Di Cunto, 1998; Eda, 2001). Furthermore, inhibition of Rho in HeLa cells by botulinum C3 exoenzyme abolishes Citron transfer from the cytoplasm to the cleavage furrow (Eda, 2001; Sarkisian, 2002), suggesting strongly that Rho and Citron interact during cytokinesis. Overexpression of truncated Citron in cell culture blocks cytokinesis, though this phenotype is not induced by full-length or kinase dead protein. Embryos homozygous for a mutation in the mouse Citron gene have multinucleate testis and brain cells, indicating a role in cytokinesis (Di Cunto, 2002). However, proliferation is not blocked in most tissues, suggesting that other factors may compensate for the loss of Citron. One such candidate is another Rho effector kinase, Rok. The overall domain structure of Rok resembles that of Citron. Rok has recently been implicated in the control of cytokinesis; inhibition of human Rok delays completion of cytokinesis. Consistent with a level of functional redundancy in their involvement in cytokinesis, both Citron and Rok localise to the cleavage furrow during cytokinesis (Madaule, 2000), they are both capable of binding to the same region of Rho (Fujisawa, 1998; Yamashiro, 2003) and they both phosphorylate the regulatory subunit of myosin II in vitro (Ueda, 2002). The vertebrate studies are consistent with Citron being a Rho effector during cytokinesis, but the tissue-restricted mutant phenotypes observed in vivo suggest that Citron is not an essential Rho effector for cytokinesis. With the possibility that phenotypes are being masked by redundancy with Rok, it seemed desirable to analyse the role of Citron in Drosophila, which often shows less genetic redundancy than in vertebrates and is more readily amenable to genetic analysis. citron is shown to be expressed specifically in proliferating tissues and is downregulated in differentiating tissues. citron plays a non-redundant role in cytokinesis and, unlike rok, exhibits strong genetic interactions with pbl, consistent with a role as a downstream target of the Pebble (Pbl)-activated Rho intracellular signalling pathway during cytokinesis (Shandala, 2004 and references therein).

citron gene is essential for normal cell division in the different tissues tested, the central and peripheral nervous systems, larval brain cells and larval imaginal tissues. Consistent with a role specifically in cell division, citron transcripts were detected in proliferating tissues, and citron expression is downregulated in post-proliferative cells. This contrasts with the ubiquitous expression of Rho and another Rho effector kinase, Rok, both of which play roles in non-proliferating cells during Drosophila development. Analysis of the Drosophila citron mutant phenotype revealed a widespread function in proliferation. In S2 cultured cells treated with citron dsRNA, binucleate cells were observed. Furthermore, the PNS of transheterozygous citron mutant embryos exhibited a loss of neurons and concomitant appearance of multinucleate neurons, similar to the phenotype seen in hypomorphic pbl and other cell cycle control mutants. citron mutant larval brain cells also show a significant number of binucleate cells. These binucleate cells presumably occur due to the failure of a single round of cell division. Some of the brain cells had undergone multiple rounds of mitosis without cell division, as evidenced by multiple microtubule spindles, an increase in the number of chromosome complements and polyploid anaphase figures, indicating assembly of an effective mitotic spindle. However, with further increases in cell ploidy and centrosome numbers, the spindle did not form properly and chromosomes were lost at metaphase, but the cells evidently continued to cycle, eventually producing giant cells filled with chromosomes. This brain phenotype is similar to spaghetti squash (myosin regulatory light chain) and diaphanous mutant phenotypes, but is different from phenotypes of mutants such as makós or aurora that arrest in metaphase. It is concluded, therefore, that Drosophila Citron has an important and conserved function during cytokinesis. In contrast to larval brain cells and embryonic PNS cells, polyploidy and binucleate cells were not observed in larval imaginal tissues. Rather, the discs were dramatically reduced in size and exhibited high levels of apoptosis. The analysis of other tissues shows that citron is not generally required for cell survival, and inhibition of apoptosis in citron mutant discs results in the accumulation of multinucleate cells. Therefore, it was reasoned that imaginal cells differ from their brain counterparts by possessing a checkpoint control mechanism that triggers apoptosis following the failure of cell division. The key cytokinetic mutant diaphanous (dia) exhibits similar hyperploid neuroblasts and loss of larval imaginal tissue (Shandala, 2004).

Drosophila Citron, like its mammalian counterparts, is localised to the cleavage furrow during cytokinesis. Many of the important regulators of cytokinesis such as the RhoA GTPase and its activators, and structural components such as myosin and actin, are concentrated in this structure. Importantly, Drosophila Citron localisation to the cleavage furrow depends on proper activation of the RhoA cytokinesis signalling pathway. In pbl mutant embryos lacking the Pbl Rho-GEF, which activates RhoA during cytokinesis, accumulation of Citron-GFP into the contractile ring does not occur. In vivo confirmation of the importance of Citron function in the Rho cytokinesis signalling pathway came from strong genetic interactions between cit and pbl mutants using two independent genetic assays, one based on the appearance of multinucleate cells in the PNS and one based on a developing wing phenotype. In both cases, loss of function of one of the genes strongly enhances the phenotype of the other. The citron mutant cytokinesis phenotypes and the enhancement of cit and pbl phenotypes by a reciprocal reduction in their gene activity provides clear genetic evidence for the involvement of Citron in the Rho cytokinesis signalling pathway. The interaction between active Rho and Citron observed in yeast two-hybrid assays and the loss of Citron localisation to the cleavage furrow in pbl mutant embryos (Shandala, 2004) and in response to Rho inhibitors (Eda, 2001) demonstrate that the role of Citron in this pathway is as a downstream effector of Pbl-activated Rho (Shandala, 2004).

The relationship between the two Rho effector kinases, Citron and Rok, is the subject of ongoing discussion (see Li, 2003). A non-redundant essential role was found for Citron in cell division in all Drosophila tissues examined in this study. Drosophila Rok plays an important role in planar cell polarity (PCP) via regulatory phosphorylation of myosin light chain (MLC) and subsequent activation of non-muscle myosin II. No PCP specific phenotype was observed in citron mutants. Moreover, overexpression or loss of one copy of citron has no effect on a PCP-specific dishevelled1 mutant phenotype, arguing against any involvement of Citron in the control of planar polarity by Rho. Conversely, no evidence was found of a role for Rok in cell division in Drosophila. Evidence exists for such a role for human Rok, with depletion of its activity in HeLa cells by chemical inhibitors leading to a substantial delay in the completion of cytokinesis. However, other studies using the same cells and Rok inhibitor saw no effect or early completion of cytokinesis. Higher doses of Rok inhibitor could effectively prevent cytokinesis, but these concentrations also inhibit Citron. In Drosophila, homozygous rok2 eye clones do not differ in size from their homozygous wild-type twin spots. Analysis of genetic interactions also failed to produce any evidence of a role for Rok in Rho cytokinesis signalling (Shandala, 2004).

In summary, genetic analyses have demonstrated an essential role for Drosophila Citron in cell division and provided clear genetic evidence for its function, in vivo, as an effector in Pbl-activated Rho signalling during cytokinesis. This analysis has also established a set of genetic tools that will allow a detailed dissection of the roles of Citron within the context of a developing organism (Shandala, 2004).


REGULATION

Protein Interactions

Citron, like the mammalian Citron, binds active Rho. In a yeast two-hybrid assay, the C-terminal-most 416 amino acids of Drosophila Citron interact specifically with constitutively active RhoA. Surprisingly, this interaction region, which encompasses the CNH (Citron homology domain), is distinct from the Rho interaction domain of mammalian Citron that lies N-terminal to the CNH and C1 (lipid binding) domains, within a coiled-coil region. Thus, although the interaction between Citron and active RhoA is functionally conserved, the interaction domain appears to be different (Shandala, 2004).

Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. This paper shows that, contrary to previous models, the Drosophila CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, the Sti Citron-Nik1 homology domain was found to interact with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion causes an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. It is proposed that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics (Bassai, 2011).

Current models propose that in human cells, RhoA recruits and then activates CIT-K, which in turn phosphorylates MRLC at the CF. Results from this study indicate that, at least in Drosophila, this model is incorrect. Indeed, Sti recruitment to the CF does not require interaction with RhoA, and instead, Sti appears necessary to maintain proper RhoA localization at the CF specifically in late cytokinesis. In addition, Sti depletion causes an increase and not a decrease of mono- and diphosphorylated MRLC at the cleavage site in late cytokinesis. Because MRLC phosphorylation is controlled by RhoA signaling, it is proposed that aberrant RhoA localization after Sti depletion might cause an increase of phosphorylated MRLC at the CF. MRLC phosphorylation has been described to control actin dynamics, and thus, this model could well explain the CR disorganization observed after Sti knockdown. Why does Sti depletion cause an increase of MRLC phosphorylation? The simplest hypotheses are that Sti kinase activity could, directly or indirectly, promote the activity of an MRLC phosphatase or inhibit RhoA and/or an MRLC kinase. Identification of Sti targets will be necessary to fully comprehend how this kinase controls RhoA localization during cytokinesis and to distinguish between the aforementioned hypotheses (Bassai, 2011).


DEVELOPMENTAL BIOLOGY

Patterns of gene expression can provide clues to gene function. In situ hybridisation showed that Drosophila citron transcripts occur in nurse cells during oogenesis and ubiquitously in early blastoderm embryos. This indicates the presence of a significant maternal store of transcript. The maternal transcripts persist only until germ band extension, because transcripts are not seen after this stage in embryos lacking zygotic citron. With the progression of embryogenesis, citron transcripts are restricted to cells in the central and peripheral nervous systems (CNS and PNS respectively). citron expression is gradually lost from the CNS and PNS, correlating with differentiation within these tissues. In third instar larvae, citron transcripts were uniformly distributed in all imaginal discs, with the exception of the posterior, differentiating region of the eye disc. This pattern of expression, which is initially ubiquitous then restricted to the nervous system is very similar to that observed for known cell cycle genes (e.g., three rows), consistent with a role for citron in proliferation. The association of citron expression with proliferating tissues contrasts with the widespread expression of another Rho effector kinase rok, and of RhoA, the putative upstream activator of Citron, consistent with Rok and RhoA having roles beyond proliferation (Shandala, 2004).

To explore the intracellular localization of Citron, transgenic UAS-citron-GFP flies were generated to produce Citron fused at the C terminus to Green Fluorescent Protein (GFP). The Citron-GFP fusion protein expressed in embryos and larval brains is scattered throughout the cytoplasm during interphase. In dividing cells, Citron-GFP is observed to accumulate at the constricting membrane following anaphase; it persists in the midbody between divided daughter cells upon completion of cytokinesis. The cleavage furrow is enriched in many proteins that are required for the progression of cell division, including RhoA, MRLC and myosin II. Citron-GFP localises to this contractile ring; an overlap of the GFP signal is seen with a stain for RacGAP50C, a known cytokinesis regulator that associates with the contractile ring, and by live imaging of contraction. The striking similarity in intracellular localisation of fly, mouse, rat and human homologues of Citron suggests that the function of these proteins during mitosis is conserved. Localisation of mammalian Citron to the cleavage furrow during cytokinesis is blocked by treatment with a Rho GTPase inhibitor. The Rho-GEF, Pbl, targets RhoA during cytokinesis. By examining Citron-GFP localisation in a pbl mutant embryo, the drastic disruption that could occur if Rho signalling was blocked indiscriminately is avoided. In the absence of Pbl, Citron-GFP fusion protein does not accumulate at the cleavage furrow at telophase. These data indicate that Citron accumulates at the contractile ring in response to activation of Rho by the Rho-GEF Pbl (Shandala, 2004).

In interphase S2 cells, affinity-purified Sti antibody detects punctate signals in the cytoplasm and in the nucleus. In addition, some cells show strong dot-like Sti staining that is thought to be midbody remnants. A similar staining pattern was observed in metaphase and anaphase, but as cells enter telophase, Sti localizes to the ingressing cleavage furrow and forms a ring around the midzone microtubules to concentrate into the midbody at the end of telophase. In cells treated with sti dsRNA, the signal detected throughout the cell cycle is either absent or extremely weak. Co-staining to detect F-actin reveals Sti localizes underneath the actomyosin ring. This localization becomes particularly evident in cells at the midbody stage. In addition, Sti also appears to colocalize with the septin peanut. These results suggest that Sti might be a component of the contractile ring, but probably does not interact directly with the cell membrane. This is consistent with Sti not having a PH domain like its mammalian counterpart CIT-K (D'Avino, 2004).

Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring

During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms. Using time-lapse microscopy of Drosophila melanogaster S2 cells, this study shows that the transition from the CR to the MR proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of the scaffold protein Anillin. The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR. Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process. These findings highlight the relatedness of the CR and MR and suggest that membrane removal is coordinated with CR disassembly (El Amine, 2013).


EFFECTS OF MUTATION

To test citron function, Citron expression in Drosophila Schneider line 2 (S2) cultured cells was initially inhibited using RNAi. Many citron dsRNA-treated cells (~30%, compared with <2% in controls) become multinucleate, comparable to pbl dsRNA treatment, indicating that mitosis is not accompanied by cell division. This result is in line with the identification of citron by cell culture-based RNAi screening with a multinucleate phenotype (Kiger, 2003; Rogers, 2003). To assess whether Citron is required for cytokinesis in vivo, use was made of the available citKG01697 and citGS9053 alleles, which have P-element transposon insertions in the 5'UTR of the Drosophila citron locus. These alleles are referred to as cit1 and cit2 respectively. To avoid the possibility of homozygosing second site mutations, all analyses used transheterozygotes between cit1, cit2 or Df(3)iro-2, a small deficiency spanning the citron locus. All homozygous and transheterozygous genotypes were lethal, showing that citron is essential for Drosophila development. A considerable number of citron transheterozygotes die during larval development, and the remainder die before eclosion. Transheterozygotes between citron alleles and the deficiency did not show an earlier lethal phase or stronger phenotypes than citron mutant homozygotes or transheterozygotes, indicating that cit1 and cit2 are strong alleles if not genetically null. The insertions were responsible for the lethality observed, since both alleles can be reverted to viability by the expression of P transposase. Furthermore, since the cit2 insertion carries a UAS element, it was possible to show that ubiquitous citron gene expression rescues cit2/Df(3)iro-2 transheterozygotes to viability, confirming that loss of the citron transcript is responsible for the phenotypes described. Depletion of maternal deposition of citron using the FLP/FRT ovoD1 system led to female sterility, indicating that Citron is required during oogenesis (Shandala, 2004).

To characterize the citron mutants, disruption to neural cell development was examined. Maternal citron seems to be depleted by stage 10, the stage at which embryonic sensory organ precursors commence a wave of cell divisions. It was reasoned that loss of zygotic citron might have some effect on these PNS cell divisions and on neural divisions in the larva. In wild-type embryos the PNS is organised in distinct, highly stereotyped clusters within each segment. For instance, the dorsal external sensory (DES) cluster forms a grape-like structure with cells closely linked to one another. The lateral chordotonal organ (CH) cluster typically contains five oval-shaped cells of similar size, extending parallel processes dorsally. Examination of the PNS in cit1/Df(3)iro-2 and cit2/Df(3)iro-2 transheterozygous embryos using the 22C10 monoclonal antibody reveals a modest disruption to nervous system organization with some disorganisation of clusters and defects in axonal projections, suggestive of a role in neuronal morphology and axonogenesis. Similar phenotypes have previously been shown for hypomorphic alleles of pbl, which encodes an upstream activator of the Rho GTPase. Importantly, there is a loss of neurons and concomitant appearance of binucleate neurons in approximately 15% of citron mutant PNS cells, consistent with the failure of cytokinesis (Shandala, 2004).

The presence of maternal citron transcripts and the late embryonic PNS phenotype suggests that maternally-derived Citron permits early proliferation, but is lost later in embryogenesis. After embryogenesis there is little proliferation in the nervous system until the CNS begins to divide rapidly at the end of the second larval instar, by which stage the zygotic mutant phenotype should be evident. citron mutant larval brains contain massively enlarged nuclei: DNA stains show large diffuse aggregates of apparently hyperploid cells. In addition, dissociated mutant larval brain cells from any allelic combination are binucleate at a far higher rate (26/103) than normal (1/100). Chromosome preparations from mutant brains show tetraploid metaphase figures, confirming that citron mutant brain cells can complete S phase and enter mitosis following a previously failed mitosis or cytokinesis. Hyperploid cells were observed undergoing anaphase, suggesting that a failure of chromosome disjunction is not the primary defect. Furthermore, the frequency of diploid anaphases and telophases is similar in all citron allelic combinations (approximately 37% of mitotic cells) and doesn't significantly differ from wild-type control (39%). Consistent with this, immunohistochemical analysis shows that centrosomes separate normally in the polyploid cells, since an assembly of microtubule spindles was observed, apparently emanating from multiple centrosomes. Some mutant brain cells are massively hyperploid, showing that in the absence of Citron, brain cells can undergo multiple mitotic cycles without dividing. However, with increasing cell ploidy, microtubules form abnormal spindles and chromosomes fail to assemble at the metaphase equator. These defects are clearly the later consequences of an earlier cell division failure, so it is concluded that the primary citron larval brain phenotype is a failure of cell division (Shandala, 2004).

Examination of citron mutant larvae reveals a variable but marked reduction in the size of their imaginal discs. In line with these observations, only one or two cell somatic clones of homozygous mutant cells in the wing could be generated. Cell sizes are not obviously different in the mutants, but wing and leg discs stained with anti-phospho Histone H3 (anti-PH3) show a mild reduction in the number of mitotic cells relative to wild-type (down to 60% of wild-type for cit1and 77% for cit2). Since this level of perturbation might not be expected to produce such small discs, the possibility was examined that a stronger cell division phenotype could have been masked by the rapid clearance of mutant cells by apoptosis. The TUNEL assay for apoptotic cells and staining with the cell death marker Acridine Orange, revealed an increased level of apoptosis in mutant imaginal discs. Confirmation that apoptosis was clearing cells with a cytokinesis defect came from expression of the apoptosis inhibitor, p35, in citron mutant imaginal discs; this led to the accumulation of multinucleate cells (Shandala, 2004).

Citron has been proposed to act downstream of Rho in the regulation of cytokinesis. However, little in vivo evidence has been found to support this proposition. To test whether Citron participates in Rho signalling, genetic interactions were examined between citron and a known regulator of the Rho pathway, the Rho-GEF-encoding gene, pebble. The first assay chosen was the ability to modify the moderate citron embryonic PNS phenotype. pbl mutants were chosen rather than Rho mutants because Pbl appears to be a specific Rho activator for cytokinesis, whereas loss of Rho also affects many other processes. Removing one copy of pbl in cit mutants results in a significant reduction in the overall number of cells in the PNS, while most of the remaining cells (52%) appear to be multinucleate. Therefore, a mild reduction in Pbl-mediated Rho activation during cytokinesis results in a significant enhancement of the cit mutant embryonic PNS defects (Shandala, 2004).

A complementary approach monitored whether under- or over-expression of citron could modify a loss-of-Pbl phenotype. Since strong pbl phenotypes arise too early and are too drastic to be of use, an RNAi construct was generated to inhibit Pbl synthesis later in development. Expression of this pblRNAi construct in the posterior half of the wing resulted in a decrease in the size of the corresponding region. Analysis of the affected area revealed that more than 67% of cells produce multiple hairs in contrast to the invariably single-haired cells in wild-type, a phenotype observed when cytokinesis is blocked, for example by inhibition of RacGAP50C. As expected, co-staining of pupal wings with phalloidin and the DNA stain Hoechst 33258 revealed that the pblRNAi-expressing cells were abnormally large and typically multinucleate, resembling the embryonic phenotype of pbl mutants. The intermediate nature of the en-GAL4>UAS-pblRNAi wing size and multiple hair phenotypes allowed detection of enhancement and suppression by prospective interactors. To test the specificity of this assay system, the pblRNAi phenotype was examined in a RhoA/+ background. Significant diminution of the pbl-depleted region of the wing shows that the pblRNAi phenotype is enhanced by removal of one copy of wild-type RhoA, as seen in other genetic assays for pbl function. The multiple hair phenotype was quantified in a defined wing region posterior to vein L5. A significant increase in the proportion of multihaired cells from 67% to 84% upon loss of one copy of RhoA shows that this assay could detect reductions in the dose of cytokinesis effector genes. Removal of one copy of wild-type citron also reduces the size of the posterior half of the wing in en-GAL4>UAS-pblRNAi flies and enhances the multiple hair phenotype. Identical effects were observed in Df(3)iro-2 heterozygous mutants . The genetic interactions between loss of function citron and pbl phenotypes support the role of Citron as a Rho effector in cytokinesis. Ectopic expression of citron in various Drosophila tissues generates no dramatic phenotype in wild-type or pblRNAi backgrounds, suggesting that the activity of Rho is rate limiting for Citron function (Shandala, 2004).

It has been suggested that the human Rho effector kinase Rok plays some role in cytokinesis since inhibition of Rok function by Y-27632 leads to significantly prolonged ingression of the cleavage furrow, although cytokinesis was eventually completed. Drosophila Rok is not required for cytokinesis in wing cells; somatic mutant rok clones do not display any reduction of size. However this test does not address the issues of delayed completion of cytokinesis or redundancy between Rok and Citron. Drosophila rok is uniformly expressed throughout development and so is present in cells expressing citron, making redundant function possible. If the cytokinetic functions of Rok and Citron were redundant, it would be expected that halving the dose of one would enhance the cytokinetic mutant phenotype of the other. Since rok mutants have no described cytokinetic phenotype, tests were run for genetic interactions between rok2, a strong loss-of-function allele, and Rho signalling in cytokinesis, by introducing the rok2 allele into the sensitised pbl RNAi wing assay. In contrast to the enhancement observed by removing one copy of citron, removal of one copy of rok makes no significant difference to the pblRNAi mutant phenotype (67% and 65% respectively). Therefore no evidence was found in support of a key role for Rok in Pbl-Rho signalling during cytokinesis (Shandala, 2004).

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

To evaluate each sticky/citron (sti) allele, complementation and lethal stage analyses of all sti allelic combinations were performed. A deficiency was used (Df(3L)F10) that completely removes the sti genomic region, and five different sti alleles, were used. The sti1 allele was originally described by Gatti (1989); sti2 and sti3 mutations were generated by chemical mutagenesis and sti4 and sti5 were isolated from a P element screen. The data reveal that in most homo- and hetero-allelic combinations, sti mutants die at the onset of metamorphosis (prepupal stage, PP) because they have very small imaginal discs. This indicates that sti is essential for imaginal cell division. Lethal phase analysis shows that all sti alleles behave as hypomorphs with sti3 being the most severe and sti5 the weakest. All other alleles appear to have similar strength (D'Avino, 2004).

Precise excisions of the P elements present in sti4 and sti5 mutants rescue neither the lethality nor the polyploidy observed in these animals, indicating that these elements are not inserted within the sti gene. Using both deficiency and recombination mapping, the sti gene was mapped to the 69D2-3 polytene region in an area uncovered by two deficiencies, Df(3L)F10 and Df(3L)E44. Although the P elements were not inserted in the sti gene, it was surmised that some rearrangements might have occurred in the genomes of these lines during the mutagenesis. Thus, possible RFLPs (restriction fragment length polymorphisms) were sought in the genomic region uncovered by the two deficiencies in sti4 and sti5 mutants. As a starting point, another gene studied in the lab, vihar, was used, which also maps to the same region. In this way, two RFLPs were identified in two adjacent EcoRI fragments that harbor a single transcription unit, identified as CG10522 by the Drosophila genome project. The sequence of four different CG10522 cDNAs revealed a single ORF coding for a 1,854-amino acid protein of a predicted mass of 211 kD. Northern blot analysis indicates that CG10522 encodes a single transcript of ~6.1 kb that is prematurely truncated (and slightly more abundant) in sti5 mutants and very unstable in sti4 animals. To further confirm that CG10522 is indeed sti, its genomic region was sequenced in all sti mutants and two hobo transposable element insertions were found in sti4 and sti5 and three premature termination codons in sti1, sti2, and sti3 at residues 1093, 1126, and 348, respectively. An antibody was raised against the CG10522 protein and its expression was analyzed in sti mutant larval brains by Western blot. This antibody recognizes a single product of ~220 kD, in good agreement with the CG10522 predicted molecular weight. This protein is truncated prematurely in sti1, sti2, and sti5 and undetectable in sti4, exactly as predicted by sequence and Northern blot analysis. Thus, CG10522 is the sti gene (D'Avino, 2004).

sti3 encodes a truncated protein containing only part of the kinase domain, whereas sti1, sti2, and sti5 encode products lacking the C1 and CitroN homology domains and the last portion of the coiled-coil region. Finally, it is noteworthy that although sti5 is a much weaker allele than sti2, the difference in length between the two mutated proteins is only 53 aa. This indicates that the region lying between residues 1125 and 1178 is important for Sti activity (D'Avino, 2004).

To further show that sti is essential for cytokinesis, RNAi was used to inactivate its function(s) in Drosophila Schneider 2 (S2) cells. Most of the Sti protein is depleted after incubation of S2 cells with sti dsRNA for 48 or 72 h. Several bands were detected in S2 cell extracts and all were specifically depleted after RNAi treatment. These bands are believed to correspond to degradation products in S2 cells, since multiple protein or mRNA isoforms were never detected in previous experiments. Moreover, immunolocalization experiments also suggested that Sti protein is likely to be degraded at the end of cell division (D'Avino, 2004).

Flow cytometric analysis (FACS®) revealed that depletion of Sti causes an increase of polyploid (8N or more) together with aneuploid cells. In addition, the number of putative apoptotic cells was higher in Sti RNAi cells, respective to the controls, suggesting that the increase in polyploidy can, at some point, trigger cell death. Immunostaining experiments confirm that polyploidy in Sti-deficient cells results from defects in cytokinesis. The percentage of bi/multinucleate cells increased after incubating for 48 and 72 h with sti dsRNA by ~21 and 42 times, respectively. The nuclei present in binucleate cells following sti RNAi after 48 h were of similar size suggesting that inactivation of sti in S2 cells produces a block in cytokinesis. Very large cells containing up to 10-12 nuclei could be observed in cultures treated for 72 h, indicative of failure of cytokinesis in multiple cycles (D'Avino, 2004).

To confirm directly a failure of cytokinesis after sti RNAi, cells were observed by time-lapse microscopy. In control, GFP dsRNA transfected cells, the cytoplasm began to constrict symmetrically at the spindle equator within 5 ± 1 min of anaphase onset. Furrow ingression was rapid and a midbody formed shortly thereafter, linking the two daughter cells together for the duration of filming. In Sti-depleted cells, chromosome congression, alignment and segregation appeared normal and the timing of furrow initiation was also unaffected. However, in about half of these cells a 'unilateral furrow' was initiated and advanced across the cytoplasm before joining with one or two less robust furrows. This asymmetric furrowing was accompanied by a series of highly dynamic equatorial protrusions that could culminate in the formation of ectopic contractile rings. Although some blebbing also occurred in control cells, this was usually confined to nonequatorial locations and was much less dramatic than observed in Sti-depleted cells. Abnormal blebbing was observed only during cytokinesis in sti RNAi cells. Unfortunately, it was not possible to follow cells for longer than 35 min after anaphase onset and this prevented direct observation of cell abscission in both control and sti RNAi treated cells. Nonetheless, the experiments clearly reveal that sti-deficient cells become multinucleate. Therefore, it is proposed that these cells are unable to complete cytokinesis as a consequence of the altered dynamics of events occurring during furrow ingression (D'Avino, 2004).

To understand these aberrant phenotypes, the localization of several central spindle and contractile ring components were examined after sti RNAi. The morphology of the central spindle and the localization of factors essential for its formation, including the Aurora B kinase, PAV-KLP, and RacGAP50C, appeared completely normal in Sti-deficient cells. In contrast, contractile ring components such as F-actin, Anillin, and the septin Peanut localized normally at cleavage furrow formation, but displayed aberrant accumulation and distribution in late telophase. F-actin appeared very disorganized in sti RNAi cells and less compact than in control cells. Actin also appeared to diffuse inside the equatorial membrane protrusions and sti-deficient cells showed gaps in the actin ring structure that may reflect unilateral furrowing. Anillin, an actin-binding protein that has been postulated to interact with the membrane and the actomyosin ring, also accumulated abnormally in sti RNAi late telophases. In some severe situations, Anillin also marked the formation of ectopic contractile rings. Finally, it should be noted that, although depletion of Sti altered both the morphology and organization of the contractile ring, the furrow was still able to constrict completely to form, at least transiently, a midbody-like structure. This observation indicates that actomyosin filaments can contract normally when Sti is depleted, in contrast with what has been proposed (Madaule, 2000) for its mammalian counterpart, CIT-K (D'Avino, 2004).

Several lines of evidence have indicated that RhoA activates CIT-K in vitro (Madaule, 1995; Di Cunto, 1998; Eda, 2001). To investigate Sti regulation by the family of small Rho GTPases in vivo, a genetic approach was used. To this end, transgenic fly lines were created in which sti functions can be partially silenced by RNAi in specific tissues using the GAL4/UAS system. The expression of sti dsRNA in the developing eye tissue using the ey-GAL4 driver and two copies of a SymUAS-sti reporter led to the formation of multinucleate cells. As a likely consequence, the emerging adults had eyes considerably smaller than wild type and composed of large and disorganized ommatidia. This 'rough' eye phenotype was enhanced by the presence in the genome of a strong sti mutation, sti3, consistent with the effect being caused by inactivation of the sti gene. The sti eye phenotype was also significantly enhanced by null mutations in the single Drosophila RhoA homologue, Rho1. Interestingly, a chromosome mutated in the three Drosophila Rac genes, Rac1, Rac2, and Mtl (triRac), was able to dominantly suppress the sti phenotype almost completely, reverting the eye to wild-type size and appearance. A very similar suppression was obtained with mutations only in the Rac1 and Rac2 genes (unpublished data) (D'Avino, 2004).

Yamashiro (2003) has recently shown that CIT-K is able to phosphorylate the myosin regulatory light chain (MRLC) at both serine-19 and threonine-18 residues in vitro. To investigate if the MRLC encoded by the spaghetti squash (sqh) gene is a Sti target in vivo, a transgenic fly stock carrying a mutated form of the sqh gene, sqhE20E21, was used in which both Ser-21 and Thr-20 (equivalent to vertebrate Ser-19 and Thr-18) were replaced by the phosphomimic residue glutamic acid (E). This mutated protein behaves in vivo as a phosphorylated version of Sqh, inasmuch as it is able to rescue the lethality associated with strong mutations in the Drosophila ROK (rok) gene. A single copy of the sqhE20E21 transgene is able to suppress considerably the sti rough eye phenotype, suggesting the possibility that Sqh can be phosphorylated by Sti in vivo (D'Avino, 2004).

Genetic evidence that Rac genes dominantly suppress the sti RNAi phenotype suggests the possibility that Rac GTPases may play an inhibitory role during cytokinesis. Loss of function studies in Drosophila and other animals have not previously implicated Rac in cytokinesis. These studies, however, could only indicate that Rac GTPases are not essential for this process, but do not rule out the possibility that the activity of these GTPases needs to be down-regulated during cytokinesis. Identification of a Rac repressor that is essential for cytokinesis would provide further evidence for such an inhibitory role. The Drosophila RacGAP50C has been implicated in cytokinesis, but its target GTPase has not been clearly identified yet. Thus, whether RacGAP50C could inhibit Rac activity was investigated in vivo. Expression of RacGAP50C dsRNA in developing imaginal tissues led to the formation of multinucleate cells and its RNAi-mediated silencing during eye development resulted in a significant reduction of the adult eye. These eyes contained large and disorganized ommatidia, very similar to sti RNAi mutants. A chromosome carrying mutations in all three Rac genes, (triRac), dominantly suppressed this phenotype, whereas Rho mutations acted as strong enhancers. These observations are consistent with RacGAP50C inhibiting Rac and promoting Rho activity. Finally, the strongest sti mutation available, sti3, failed to significantly enhance or suppress the RacGAP50C eye phenotype. This result suggests that sti does not function in a pathway between Rho and RacGAP50C (D'Avino, 2004).

It is important to note three major differences between Sti and mammilian CIT-K; (1) CIT-K is required for cytokinesis only in testes and specific neuronal populations in mice (Di Cunto, 2000), whereas Sti is essential for cell division in all Drosophila proliferating tissues examined, including all the imaginal discs and the brain; (2) multiple CIT-K isoforms have been described in mouse and HeLa cells (Di Cunto, 1998; Madaule, 2000), whereas only a single sti transcript is observed in Drosophila larvae and only one protein in brain extracts; (3) Sti does not contain a PH domain like CIT-K, and consistent with this observation Sti protein does not appear to localize to the cell membrane. Altogether these data suggest that Sti and CIT-K originate from the same ancestral protein, but the mammalian product may have lost some of its original functions and acquired new ones during evolution (D'Avino, 2004).

In sti-depleted cells the recruitment of central spindle and contractile ring components is normal during telophase. However, these cells display uncoordinated furrow constriction and abnormal blebbing. These phenotypes are accompanied by a less compact ring structure, unusual F-actin formations and abnormal accumulation of the contractile ring component Anillin. In some situations, sti RNAi cells also exhibit supernumerary contractile rings. These defects are consistent with problems in controlling the organization and/or structure of the contractile ring. The presence of unusual F-actin formations also suggests that Sti-depleted cells are unable to control actin depolymerization and behavior during cytokinesis. Interestingly, very similar phenotypes have been observed in Anillin-depleted cells (Somma, 2002), suggesting that one sti function might be to regulate the interaction between F-actin and Anillin during cytokinesis (D'Avino, 2004).

The identification of Sti targets will be crucial to fully comprehend how this kinase regulates the organization of the contractile ring during cytokinesis. A genetic experiment indicates that one potential Sti target is the MRLC encoded by the sqh gene, since a phosphomimic version of this protein rescues the sti rough eye phenotype. However, MRLC phosphorylation by Sti does not seem to regulate actomyosin ring contractility but rather its organization and/or structure. How can the MRLC phosphorylation status affect contractile ring structure? A recent study has indicated that CIT-K induces diphosphorylation, rather than monophosphorylation, of the MRLC (Yamashiro, 2003). These study also relates that diphosporylated MRLC shows more constrained localization at the cleavage furrow than the monophosphorylated form. These results, together with previous observations that MRLC diphosphorylation can affect filament assembly, led to the proposal that diphosphorylated MRLC may play a role in cross-linking of actin filaments rather that stimulation of motor activity. In conclusion, these data suggest that diphosphorylation of the MRLC by Sti/CIT-K induces a conformational change of the actomyosin filament structure that is essential for proper assembly of the contractile ring (D'Avino, 2004).

In vitro studies have indicated that RhoA promotes CIT-K enzymatic activity (Di Cunto, 1998). Consistently, in vivo genetic data show that mutations in Rho1, the Drosophila RhoA homologue, dominantly enhance the sti RNAi phenotype. Intriguingly, these experiments also show that Rac mutations dominantly suppress the sti and RacGAP50C RNAi phenotypes, suggesting that Rac GTPases need to be down-regulated during cytokinesis. Previous studies using Rac loss of function mutants probably failed to reveal such an inhibitory role because the absence of negative regulators would not impair cytokinesis. Other studies have also suggested that Rac inhibition might be important for cytokinesis. (1) Fluorescence resonance energy transfer analysis has demonstrated that Rac activity is strongly reduced at the cleavage furrow during cytokinesis (Yoshizaki, 2003). (2) Inducible expression of a constitutive active form of Rac in PAE cells induces the formation of multinucleate cells. Cross-talk among members of different GTPase subfamilies is not unusual and antagonism between Rho and Rac has been described in other cellular processes. Thus, a similar antagonism may also regulate the dynamics of the contractile ring. Why would Rac GTPases need to be repressed during cytokinesis? Several data indicate that the success of cytokinesis depends not only on the contraction of the actomyosin-based machinery (activated by Rho), but also on reduced stiffness at the cortex. Thus, one possible explanation for the current results is that Rac inhibition may diminish cortical stiffness and help furrow ingression (D'Avino, 2004).

Genetic interaction experiments indicate that genes function in the same biological process, but not necessarily in the same pathway. However, because in the current experiments the suppression of sti and RacGAP50C RNAi phenotypes by Rac mutations is dominant, it is conceivable that they act in the same, rather than in a parallel, pathway. In this scenario, RacGAP50C might be expected to inhibit Rac through a direct protein-protein interaction mechanism. In contrast, two opposite explanations exist for the relationship between sti and Rac: either Rac represses Sti or Sti inhibits Rac. Under the second hypothesis, however, Rac inhibition by Sti should be indirect, since GTPase activity is generally regulated by cofactors (i.e., GEFs and GAPs) and not by phosphorylation. Because the experiments indicate that RacGAP50C represses Rac activity, the second hypothesis implicates a linear pathway in which Rho activates Sti, which in turn activates RacGAP50C, which ultimately inhibits Rac GTPases. This is in contrast with findings that sti does not enhance the RacGAP50C RNAi phenotype whereas Rho1 does, suggesting that these factors do not function in a simple linear pathway. For these reasons the alternative model is favored. In this model, RacGAP50C plays a key role by inhibiting Rac and promoting Rho activity, probably through its interaction with the PBL/ECT2 GEF. The two GTPases then antagonistically regulate Sti activity. This regulation could be direct, since both GTPases bind CIT-K in vitro (Madaule, 1995), but the current genetic data do not exclude the possibility that Rac regulates Sti through one or more intermediates. The hypothesis that Rac inhibits the function of proteins that are activated by Sti cannot be excluded. Further functional and structural analysis of Sti will be required to understand the molecular mechanisms that control the activity of this kinase. One implication of the current model is that even slight variations in the equilibrium of the factors could easily alter the dynamics of contractile ring components during cytokinesis. For example, RacGAP50C activation could promote RhoA activity and consequently actomyosin filament assembly and contraction. Conversely, RacGAP50C inhibition would both down-regulate Rho and activate Rac to repress Sti thereby promoting filament disassembly (D'Avino, 2004).

Drosophila citron kinase is required for the final steps of cytokinesis

The mechanisms underlying completion of cytokinesis are still poorly understood. This study shows that the Drosophila orthologue of mammalian Citron kinases is essential for the final events of the cytokinetic process. Flies bearing mutations in the Drosophila citron kinase (dck) gene are defective in both neuroblast and spermatocyte cytokinesis. In both cell types, early cytokinetic events such as central spindle assembly and contractile ring formation were completely normal. Moreover, cytokinetic rings constricted normally, leading to complete furrow ingression. However late telophases of both cell types displayed persistent midbodies associated with disorganized F actin and anillin structures. Similar defects were observed in dck RNA interference (RNAi) telophases, which, in addition to abnormal F actin and anillin rings, also displayed aberrant membrane protrusions at the cleavage site. Together, these results indicate that mutations in the dck gene result in morphologically abnormal intercellular bridges and in delayed resolution of these structures, suggesting that the wild-type function of dck is required for abscission, he terminal step in cytokinesis that severs a cell in two. The phenotype of Dck-depleted cells is different from those observed in most Drosophila cytokinesis mutants but extraordinarily similar to that caused by anillin RNAi, suggesting that Dck and anillin are in the same pathway for completion of cytokinesis (Naim, 2004).

Sequence analysis has shown that the polypeptide encoded by the dck gene is closely related to mammalian Citron kinases. The Dck protein has an overall domain organization that is very similar to that of mammalian CKs and carries a domain with a high sequence similarity with the unique Rho-binding domain of these CKs. Consistent with this high degree of sequence homology, mammalian CKs and Drosophila Dck are both required for cytokinesis, suggesting that Dck is the fly orthologue of mammalian CKs (Naim, 2004).

This study analyzed the consequences of Dck depletion in larval NBs, S2 tissue culture cells, and spermatocytes. Spindle formation and chromosome segregation seemed to be completely normal in each cell type. In addition, the three cell types displayed regular central spindles and actomyosin rings, which constricted normally, leading to complete furrow ingression. However, each cell type showed a number of defects in late telophase figures and failed to complete cytokinesis (Naim, 2004).

The results indicate that in larval NBs of dck mutants, there is an abnormal persistence and an altered morphology of the late midbody (intercellular bridge), suggesting that the wild-type function of dck is required for abscission. Late telophases of mutant NBs also exhibited equatorial anillin signals that were more extended than those seen in their wild-type counterparts. These extended anillin signals were never observed in early and mid-telophases of dck mutants, suggesting that anillin diffusion along the midbody is due to the disorganization of a previously well formed anillin ring (Naim, 2004).

Dck-depleted S2 cells showed cytokinesis phenotypes comparable with those observed in NBs. In dck RNAi cells, late telophases displayed disorganized and extended F actin and anillin rings and frequent membrane protrusions at the cleavage site. The defects in the actin and anillin rings were not observed in early and mid-telophases, suggesting that Dck depletion affects the stability but not the assembly of these cytokinetic structures. In addition, the frequency of telophases in dck RNAi cells was higher than in controls, suggesting that completion of cytokinesis is delayed in the absence of the dck function (Naim, 2004).

The phenotype of dck mutant spermatocytes was partially different from that observed in both NBs and S2 cells, but the cytokinesis defects were restricted to the late stages of the process as in mitotic cells. Late spermatocyte telophases often exhibited extended and disorganized actin rings similar to those seen in NB and S2 cell telophases. However, spermatocyte late telophases never showed extended equatorial anillin signals. Instead, these cells displayed overconstricted anillin rings, which resulted in ring canals smaller than their wild-type counterparts. The phenotypic difference in the anillin ring morphology between somatic and meiotic cells may reflect the peculiar cytokinetic process in spermatocytes. In Drosophila gonial cells and spermatocytes cytokinesis is incomplete, and arrested contractile rings develop into ring canals that are highly enriched in anillin. It is thus likely that in spermatocyte telophases of dck mutants anillin is recruited for the ring canal assembly pathway and does not accompany actin in its diffusion along the midbody (Naim, 2004).

It is clear that all cytokinesis defects caused by depletion of the Dck protein are restricted to late telophases. In addition, most of these defects seem to be due to a progressive disorganization of structures that formed and behaved normally in earlier stages of cytokinesis. These observations raise the question of whether these defects are the cause of cytokinesis disruption or merely the consequence of delayed abscission. For example, one can imagine that a late deformation of the F actin ring can interfere with the abscission process, preventing the final separation of the daughter cells. Alternatively, it is conceivable that the failure of mutant cells to complete cytokinesis can lead to an abnormal persistence of the intercellular bridge and to a progressive disorganization of the contractile apparatus. The current results do not allow discrimination between these alternatives. It is also possible that some of the observed phenotypes are indeed responsible for failure in cytokinesis, whereas others are the consequence of the abnormal persistence of the intercellular bridge. dck mutant spermatocytes displayed a cytokinesis defect that was never observed in other mutants that disrupt meiotic cytokinesis of Drosophila males. The phenotypic analysis of mutants in 22 cytokinesis genes revealed that they can be subdivided into four different classes: genes required for central spindle assembly, anillin localization, and F actin ring formation; genes required for both central spindle and F actin ring formation but not for anillin localization; genes required for F actin ring constriction; and genes required for actin ring disassembly. The phenotype of dck mutants is similar to that elicited by mutations in the twinstar (tsr) and bird nest soup (bns) genes, which are required for actin ring disassembly. However, in tsr and bns mutant spermatocytes the actin ring not only fails to disassemble but also overgrows, forming a large and persistent actin aggregate. In dck spermatocytes, F actin rings do not seem to overgrow, but only exhibit a late disorganization, consisting in a diffusion of actin along the midbody. Thus, dck mutants identify a fifth class of genes involved in spermatocyte cytokinesis: those required for the very late events of the process (Naim, 2004).

The phenotype of Dck-depleted cells is also different from most phenotypes observed after RNAi for cytokinesis genes. For example, cells depleted of either the Pavarotti kinesin-like protein, the Rho1 GTPase, the Rho GTPase activating protein RacGap50, the Rho GEF encoded by pbl, the RMLC encoded by sqh, Syntaxin1, or the Drosophila homologue of PRC1 encoded by feo all exhibit defective central spindles and contractile rings. Cells depleted of the cofilin encoded by the tsr gene exhibit normal central spindles and contractile rings in early and late telophases, but these rings overgrow, resulting in large masses of F actin that remain associated with the cleavage site in late telophases. However, the phenotype of Dck-depleted cells is extraordinarily similar to that caused by anillin RNAi. In anillin-depleted cells, late telophases exhibit disorganized actin rings and frequent membrane protrusions at the cleavage furrow, as well as central spindles lacking the dark band at their midzones. Because anillin contains an actin-binding domain and a PH domain, these observations have led to the hypothesis that anillin interacts with both the plasma membrane and the actin-based contractile ring, regulating membrane-ring interactions during late stages of cytokinesis. The finding that anillin- and Dck-depleted cells display comparable phenotypes suggests these two proteins are in the same pathway for completion of S2 cell cytokinesis (Naim, 2004).

An elucidation of the role of Drosophila Citron kinase requires identification of its substrates. Studies on mammalian cells have shown that citron kinase phosphorylates the regulatory myosin light chain at both threonine-18 and serine-19 residues in vitro. The Drosophila RMLC is phosphorylated at the threonine-20 and serine-21 residues, which are equivalent to threonine-18 and serine-19 of the mammalian protein, respectively. Mutant sqh genes in which both threonine-20 and serine-21 have been replaced by alanines, behave like severe sqh mutants and disrupt cytokinesis in female germ cells. However, the analysis of spermatocytes and S2 cells has shown that the phenotypes elicited by sqh ablation are very different from those seen in Dck-depleted cells: the defects observed in sqh mutants are in early steps of cytokinesis such as central spindle and contractile ring formation, whereas those associated with dck mutations pertain to the last steps of the process. The simplest interpretation of these results is that Drosophila citron kinase does not phosphorylate the Sqh protein. However, it is also possible that Sqh has a dual function during Drosophila cytokinesis: a Dck-independent function in the early stages of the process and a late function that requires phosphorylation by Dck (Naim, 2004).

Regardless of whether Sqh is a Dck substrate, the Dck targets that need to be phosphorylated in order to ensure completion of cytokinesis are likely to be very few. The finding that a cytokinesis defect comparable with that observed in dck mutant spermatocytes was not observed in any of the 22 cytokinesis mutants that have been characterized to date, strongly suggests that very few proteins are required for the final stages of cytokinesis. At the moment, the best candidate substrate for Dck is anillin, which possesses many serine and threonine residues that can be phosphorylated. Anillin and Dck colocalize at the spermatocyte cleavage furrow throughout ana-telophase, and the anillin depletion phenotype in S2 cells parallels the one caused by Dck depletion. Unfortunately, the NB and spermatocyte phenotypes observed in dck mutants could not be compared with those elicited by mutations in anillin-coding gene. The early lethality associated with scraps (scra), the only extant mutant in the anillin coding gene, has thus far precluded phenotypic analysis of mutant NBs and spermatocytes (Naim, 2004).


EVOLUTIONARY HOMOLOGS

Characterization of Citron

Using the yeast two hybrid system and overlay assays, a putative rho/rac effector, citron, has been identified that interacts with the GTP-bound forms of rho and rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C6H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rho/rac binding site. Others putative rho effectors characterized by a common rho binding motif have been identified. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners (Madaule, 1995).

A novel serine/threonine kinase belonging to the myotonic dystrophy kinase family has been identified. The kinase can be produced in at least two different isoforms: (1) an approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; (2) an approximately 54-kDa protein [CRIK-short kinase (SK)], that consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of constitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types (Di Cunto, 1988).

Mutation of mammalian Citron

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in the regulation of cytokinesis. To further investigate in vivo its biologic functions, Citron-K gene has been inactivated in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. These results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS (Di Cunto, 2000).

Cytokinesis is an essential step in neurogenesis, yet the mechanisms that control cytokinesis in the developing CNS are not well understood. The flathead (fh) mutation in rat results in cytokinesis failure in neural progenitors followed by apoptosis and a dramatic reduction in CNS growth. Evidence is presented that the fh mutation is caused by a single base deletion in exon 1 of the gene encoding Citron-Kinase (Citron-K). This base deletion causes a premature stop codon at the 27th codon in the N-terminal kinase domain of Citron-K, and Western blot and immunocytochemical analysis show that the Citron-K protein is absent in proliferative zones in fh/fh mutant embryos. Citron-K protein is normally expressed along the ventricular zone (VZ) surface and localizes to cleavage furrows of both symmetrically and asymmetrically dividing progenitors. In addition, Citron-K colocalizes with RhoA at cleavage furrows in wild-type (wt) embryos, whereas RhoA expression is reduced at the VZ surface and is absent from many cytokinesis furrows in homozygous fh/fh mutants. These results, together with evidence from a recently described induced mutation in mice, indicate that the flathead mutation is in the Citron-K gene and that Citron-K may act with RhoA to ensure the progression of cytokinesis in neuronal progenitors (Sarkisian, 2002).

Citron Kinase (Citron-K) is a cell cycle-dependent protein regulating the G(2)/M transition in hepatocytes. Synchronization studies demonstrate that expression of the Citron-K protein starts at the late S and/or the early G(2) phase after that of cyclin B1. Expression of Citron-K is developmentally regulated. Levels of Citron-K mRNA and protein are highest in embryonic liver and gradually decrease after birth. Citron-K exists in interphase nuclei and begins to disperse into the cytoplasm at prophase. It concentrates at the cleavage furrow and midbody during anaphase, telophase, and cytokinesis, implicating a role in the control of cytokinesis. However, studies with knockouts show that Citron-K is not essential for cytokinesis in hepatocytes. Instead, loss of Citron-K causes a significant increase of G(2) tetraploid nuclei in one-week-old rat and mouse liver. In addition, Citron-K deficiency triggers apoptosis in a small subset of embryonic liver cells. In summary, these data demonstrate that Citron-K has a distinct cell cycle-dependent expression pattern and cellular localization as a downstream target of Rho-GTPase and functions in the control of G(2)/M transition in the hepatocyte cell cycle (Liu, 2003).

Small GTPases of the rho family regulate the extensive rearrangements of the cytoskeleton that characterize neuronal differentiation. Citron kinase is a target molecule for activated rhoA, previously implicated in control of cytokinesis. In addition, Citron kinase plays an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, it has been shown that citron kinase is involved in the morphologic differentiation of N1E-115 neuroblastoma cells induced by serum starvation. More importantly, quantitative analysis of citron kinase knockout cerebral cortex shows that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons (Di Cunto, 2003).

Citron kinase function during cytokinesis

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. A splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. Overexpression of citron mutants results in the production of multinucleate cells and a kinase-active mutant causes abnormal contraction during cytokinesis. It is proposed that citron kinase regulates cytokinesis at a step after Rho in the contractile process (Madaule, 1998).

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, Citron-K localization in cell cycle was monitored. Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase. Rho colocalizes with Citron-K in the cortex of anaphase to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody. Inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevents its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, active Val14RhoA was expressed in interphase cells expressing GFP-Citron-K. Val14RhoA expression transfers Citron-K to the ventral cortex of interphase cells, where it forms band-like structures in a complex with Rho. This structure localizes at the same plane as actin stress fibers, and they exclude one another. Disruption of F-actin abolishes the band and disperses the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in the cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells (Eda, 2001).

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis reveals that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. These data indicate that Citron kinase is specifically required for cytokinesis of the male germ line (Di Cunto, 2002).

Successful cell division in neural progenitors in the neocortical ventricular zone (VZ), as in all dividing cells, depends critically upon coordinating chromosome segregation during mitosis with cytokinesis. This coordination further suggests that common molecular regulators may link events in mitosis with those in cytokinesis. Recent genetic evidence indicates that cytokinesis in CNS neuronal progenitors, but not in most other cell types of the body, requires the function of citron kinase. In neocortex, citron kinase is most critical for neurogenic cytokinesis. In citron kinase null mutants, a large proportion of neuronal cells within neocortex are binucleate; however, very few glial cells are binucleate. In addition, confocal time-lapse imaging of mitoses at the VZ surface shows that citron kinase is also necessary for phases of the cell cycle just prior to cytokinesis. Deficits in mitosis seen in mutants indicate aberrant mitotic spindle function, and like deficits in cytokinesis, these occur in some but not all cells at the VZ surface. Citron kinase is therefore an essential multifunctional regulator of cell divisions in the VZ, and may serve to coordinate chromosome segregation with cytokinesis in neuronal precursors (LoTurco, 2003).

Citron kinase induces di-phosphorylation of regulatory light chain of myosin II

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. Citron kinase has been shown to phosphorylate regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase does not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. The expression of the kinase domain of citron kinase results in an increase in MLC di-phosphorylation. Furthermore, the kinase domain is able to increase di-phosphorylation and restore stress fiber assembly even when ROCK is inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increases di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis (Yamashiro, 2003).

Citron and Golgi organization

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. The brain-specific Rho-binding protein Citron-N is shown to be enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, these results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics (Camera, 2003).


REFERENCES

Search PubMed for articles about Drosophila Sticky

Bassi, Z. I., et al. (2011). Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis. J. Cell Biol. 195(4): 595-603. PubMed Citation: 22084308

Camera, P., et al. (2003). Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation. Nat. Cell Biol. 5(12): 1071-8. 14595335

D'Avino, P. P., Savoian, M. S. and Glover, D. M. (2004). Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac. J. Cell Biol. 166(1):61-71. 15240570

Deak, P., et al. (1997). P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F. Genetics. 147: 1697-1722. 9409831

Di Cunto, F., Calautti, E., Hsiao, J., Ong, L., Topley, G., Turco, E. and Dotto, G. P. (1998). Citron Rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron. J. Biol. Chem. 273: 29706-29711. 9792683

Di Cunto, F., et al. (2000). Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron 28(1): 115-27. 11086988

Di Cunto, F. D., et al. (2002). Essential role of citron kinase in cytokinesis of spermatogenic precursors. J. Cell Sci. 115(Pt 24): 4819-26. 12432070

Di Cunto, F., et al. (2003). Role of citron kinase in dendritic morphogenesis of cortical neurons. Brain Res Bull. 60(4): 319-27. 12781320

Eda, M., Yonemura, S., Kato, T., Watanabe, N., Ishizaki, T., Madaule, P. and Narumiya, S. (2001). Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells. J. Cell Sci. 114(Pt 18): 3273-84. 11591816

El Amine, N., Kechad, A., Jananji, S. and Hickson, G. R. (2013). Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring. J Cell Biol 203: 487-504. PubMed ID: 24217622 Fujisawa, K., Madaule, P., Ishizaki, T., Watanabe, G., Bito, H., Saito, Y., Hall, A. and Narumiya, S. (1998). Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules. J. Biol. Chem. 273: 18943-18949. PubMed Citation: 9668072

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Kiger, A., Baum, B., Jones, S., Jones, M., Coulson, A., Echeverri, C. and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2: 27. 14527345

Li, X. F. and Minden, A. (2003). Targeted disruption of the gene for the PAK5 kinase in mice. Mol. Cell Biol. 23: 7134-7142. 14517284

Liu, H., Di Cunto, F., Imarisio, S. and Reid, L. M. (2003). Citron kinase is a cell cycle-dependent, nuclear protein required for G2/M transition of hepatocytes. J. Biol. Chem. 278(4): 2541-8. 12411428

LoTurco, J. J., Sarkisian, M. R., Cosker, L. and Bai, J. (2003). Citron kinase is a regulator of mitosis and neurogenic cytokinesis in the neocortical ventricular zone. Cereb. Cortex 13(6): 588-91. 12764032

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Madaule, P., Eda, M., Watanabe, N., Fujisawa, K., Matsuoka, T., Bito, H., Ishizaki, T. and Narumiya, S. (1998). Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 394: 491-494. 9697773

Madaule, P., Furuyashiki, T., Eda, M., Bito, H., Ishizaki, T. and Narumiya, S. (2000). Citron, a Rho target that affects contractility during cytokinesis. Microsc. Res. Tech. 49: 123-126. 10816250

Naim, V., et al. (2004). Drosophila citron kinase is required for the final steps of cytokinesis. Mol. Biol. Cell 15(11): 5053-63. PubMed citation: 15371536

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Sarkisian, M. R., Li, W., Di Cunto, F., D'Mello, S. R. and LoTurco, J. J. (2002). Full Text Citron-kinase, a protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant rat. J. Neurosci. 22(8): RC217. 11932363

Shandala, T., et al. (2004). Citron Kinase is an essential effector of the Pbl-activated Rho signalling pathway in Drosophila melanogaster. Development 131: 5053-5063. 15459099

Somma, M. P., Fasulo, B., Cenci, G., Cundari, E. and Gatti, M. (2002). Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell. 13(7): 2448-60. 12134082

Ueda, K., Murata-Hori, M., Tatsuka, M. and Hosoya, H. (2002). Rho-kinase contributes to diphosphorylation of myosin II regulatory light chain in nonmuscle cells. Oncogene 21: 5852-5860. 12185584

Yamashiro, S., et al. (2003). Citron kinase, a Rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin II. Mol. Biol. Cell 14(5): 1745-56. 12802051

Yoshizaki, H., et al. (2003). Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162(2): 223-32. 12860967


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date revised: 10 February 2014

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