Luisa Capalbo

Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. We employed affinity-purification methodology coupled with mass spectrometry to identify Anillin-interacting molecules in Drosophila cells. We isolated several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex. Using drug and RNA interference (RNAi) treatments we established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins were not recruited to the cleavage site in cells in which Anillin was knocked down by RNAi, but localized to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, we demonstrate that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow.

The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

Summary Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPCs catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues—CHMP4C—in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer.

Regulators of mitosis have been successfully targeted to enhance response to taxane chemotherapy. Here, we show that the salt inducible kinase 2 (SIK2) localizes at the centrosome, plays a key role in the initiation of mitosis, and regulates the localization of the centrosome linker protein, C-Nap1, through S2392 phosphorylation. Interference with the known SIK2 inhibitor PKA induced SIK2-dependent centrosome splitting in interphase while SIK2 depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. We believe these data identify SIK2 as a plausible target for therapy in ovarian cancers.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

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

Regulated RhoA localization at the cleavage site is mediated by the Sticky/Citron kinase and ensures RhoA’s proper activation and appropriate contractile ring dynamics during cytokinesis.

Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis

The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function.

Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1

Claspin mediates the activation of checkpoint kinase 1 (Chk1) by ATM- and Rad3-related kinase (ATR) in response to genomic stress. This process depends upon phosphorylation of Claspin on two critical residues. These phosphorylations allow docking of Claspin with Chk1. In this study, we identified CK1γ1 as a kinase that carries out these key phosphorylations of Claspin.

Regulation of midbody formation and function by mitotic kinases.

Cytokinesis is the final phase of cell division and safeguards the correct distribution of genomic and cytoplasmic materials between the two nascent daughter cells. The final separation, or abscission, of the daughter cells depends on the proper assembly of an organelle at the intercellular bridge, the midbody, which acts as a platform for the recruitment and organisation of various proteins involved in both the control and execution of the abscission process. Recent studies have led to the identification of the mechanisms, signalling pathways and molecules that control the two tightly linked processes of midbody formation and abscission. Here we review our current knowledge of the role that mitotic kinases play in these processes and offer our perspectives on the potential future challenges that await researchers in the field.

New Auroras on the Roles of the Chromosomal Passenger Complex in Cytokinesis: Implications for Cancer Therapies

The chromosomal passenger complex (CPC), composed of a kinase component, Aurora B, the scaffolding subunit inner centromeric protein, Borealin, and Survivin, is a key regulator of cell division. It controls multiple events, from chromosome condensation in prophase to the final separation or abscission of the two daughter cells. The essential functions of the CPC during metaphase, however, have always hindered an accurate study of its role during cytokinesis. The recent development of small molecule inhibitors against Aurora B and the use of elegant technologies such as chemical genetics have offered new approaches to study the functions of the CPC at the end of cell division. Here, we review the recent findings about the roles of the CPC in controlling the assembly of the cleavage furrow, central spindle, and midbody. We will also discuss the crucial function of this complex in controlling abscission timing in order to prevent abscission when lagging chromatin is present at the cleavage site, thereby avoiding the formation of genetically abnormal daughter cells. Finally, we offer our perspective on how to exploit the potential therapeutic applications of inhibiting CPC activity during cytokinesis in cancer cells.

Coordinated regulation of the ESCRT-III component CHMP4C by the chromosomal passenger complex and centralspindlin during cytokinesis

The chromosomal passenger complex (CPC)—composed of Aurora B kinase, Borealin, Survivin and INCENP—surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes in vitro. Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4Cs membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a ‘relay’ mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.