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).

GENE STRUCTURE

Exons - 7

PROTEIN STRUCTURE

Amino Acids - 1854

Structural Domains

The Drosophila genome encodes a set of Serine/Threonine kinases related to Citron that show a similar domain structure: an N-terminal PKC-type kinase domain, a coiled-coil region, a C1 (lipid binding) domain, PH (pleckstrin homology) domain and finally a CNH (Citron homology) domain. CG10522 and Genghis khan (gek) are the only Drosophila genes encoding this domain order. By sequence arrangement and function, Gek is more closely related to the Cdc42 effector, MRCK. CG10522 has no PH domain, although the flanking sequences are conserved. The PH domain is present in Gek and the vertebrate homologues, suggesting that the common ancestor of these kinases had a PH domain that was lost in CG10522. The related Rho effector kinase, Rok, lacks a CNH domain and has a different domain order. The domain organisation, size, and levels of sequence identity all point to CG10522 encoding the fly homologue of Citron, so CG10522 is referred to as citron (Shandala, 2004).

Database searches revealed that sticky encodes a serine/threonine kinase closely related to the mammalian CIT-K. CIT-K and Sticky share several common functional and structural domains. The highest degree of identity lies in the kinase domain (44%), followed by the Citron homology domain (32%), a relatively novel motif of unclear function found in Citron and NIK1-like kinases and yeast ROM1 and ROM2. Finally, both proteins contain a long coiled-coil region and a cysteine rich (C1) domain, but, interestingly, STI lacks a pleckstrin homology (PH) domain, which is thought to target proteins to the membrane or cell cortex (D'Avino, 2004).

date revised: 20 December 2004

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