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).
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).
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).
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).
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).
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date revised: 15 July 2008
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