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Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.

ABSTRACT: During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans. However, the importance of the CYK-4 GAP activity and whether it regulates RhoA remain unclear. We found that two separation-of-function mutations in the GAP domain of CYK-4 lead to cytokinesis defects that mimic centralspindlin loss of function. These defects could be rescued by depletion of the GTPase Rac or its effectors, but not by depletion of RhoA. Thus, inactivation of Rac by centralspindlin functions in parallel with RhoA activation to drive contractile ring constriction during cytokinesis.

SUBMITTER: Canman JC

PROVIDER: S-EPMC2736296 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.

Canman Julie C JC Lewellyn Lindsay L Laband Kimberley K Smerdon Stephen J SJ Desai Arshad A Bowerman Bruce B Oegema Karen K

Science (New York, N.Y.) 20081201 5907

During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans. However, the importance of the CYK-4 GAP ...[more]

PMID: 19056985

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Project description:Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.

Project description:BackgroundWith the large-scale production and application of amorphous silica nanoparticles (aSiNPs), its adverse health effects are more worthy of our attention. Our previous research has demonstrated for the first time that aSiNPs induced cytokinesis failure, which resulted in abnormally high incidences of multinucleation in vitro, but the underlying mechanisms remain unclear. Therefore, the purpose of this study was firstly to explore whether aSiNPs induced multinucleation in vivo, and secondly to investigate the underlying mechanism of how aSiNPs caused abnormal cytokinesis and multinucleation.MethodsMale ICR mice with intratracheal instillation of aSiNPs were used as an experimental model in vivo. Human hepatic cell line (L-02) was introduced for further mechanism study in vitro.ResultsIn vivo, histopathological results showed that the rate of multinucleation was significantly increased in the liver and lung tissue after aSiNPs treatment. In vitro, immunofluorescence results manifested that aSiNPs directly caused microfilaments aggregation. Following mechanism studies indicated that aSiNPs increased ROS levels. The accumulation of ROS further inhibited the PI3k 110β/Aurora B pathway, leading to a decrease in the expression of centralspindlin subunits MKLP1 and CYK4 as well as downstream cytokines regulation related proteins Ect2, Cep55, CHMP2A and RhoA. Meanwhile, the particles caused abnormal co-localization of the key mitotic regulatory kinase Aurora B and the centralspindlin complex by inhibiting the PI3k 110β/Aurora B pathway. PI3K activator IGF increased the phosphorylation level of Aurora B and improved the relative ratio of the centralspindlin cluster. And ROS inhibitors NAC reduced the ratio of multinucleation, alleviated the PI3k 110β/Aurora B pathway inhibition, and then increased the expression of MKLP1, CYK4 and cytokinesis-related proteins, whilst NAC restored the clustering of the centralspindlin.ConclusionThis study demonstrated that aSiNPs led to multinucleation formation both in vivo and in vitro. ASiNPs exposure caused microfilaments aggregation and inhibited the PI3k 110β/Aurora B pathway through excessive ROS, which then hindered the centralspindlin cluster as well as restrained the expression of centralspindlin subunits and cytokinesis-related proteins, which ultimately resulted in cytokinesis failure and the formation of multinucleation.

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Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.

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Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.

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