F-Actin Interactome Reveals Vimentin as a Key Regulator of Actin Organization and Cell Mechanics in Mitosis.

Most metazoan cells entering mitosis undergo characteristic rounding, which is important for accurate spindle positioning and chromosome separation. Rounding is driven by contractile tension generated by myosin motors in the sub-membranous actin cortex. Recent studies highlight that alongside myosin activity, cortical actin organization is a key regulator of cortex tension. Yet, how mitotic actin organization is controlled remains poorly understood. To address this, we characterized the F-actin interactome in spread interphase and round mitotic cells. Using super-resolution microscopy, we then screened for regulators of cortex architecture and identified the intermediate filament vimentin and the actin-vimentin linker plectin as unexpected candidates. We found that vimentin is recruited to the mitotic cortex in a plectin-dependent manner. We then showed that cortical vimentin controls actin network organization and mechanics in mitosis and is required for successful cell division in confinement. Together, our study highlights crucial interactions between cytoskeletal networks during cell division

F-Actin Interactome Reveals Vimentin as a Key Regulator of Actin Organization and Cell Mechanics in Mitosis.

Most metazoan cells entering mitosis undergo characteristic rounding, which is important for accurate spindle positioning and chromosome separation. Rounding is driven by contractile tension generated by myosin motors in the sub-membranous actin cortex. Recent studies highlight that alongside myosin activity, cortical actin organization is a key regulator of cortex tension. Yet, how mitotic actin organization is controlled remains poorly understood. To address this, we characterized the F-actin interactome in spread interphase and round mitotic cells. Using super-resolution microscopy, we then screened for regulators of cortex architecture and identified the intermediate filament vimentin and the actin-vimentin linker plectin as unexpected candidates. We found that vimentin is recruited to the mitotic cortex in a plectin-dependent manner. We then showed that cortical vimentin controls actin network organization and mechanics in mitosis and is required for successful cell division in confinement. Together, our study highlights crucial interactions between cytoskeletal networks during cell division

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability

Etude des fonctions indépendantes des cyclines/CDKs de p27Kip1 au cours de l'oncogénèse et de la progression mitotique

Le cycle de division cellulaire est gouverné par les complexes cycline-CDK dont l'activité est finement régulée, notamment par des inhibiteurs de CDK (CKI) dont p27Kip1 (p27) fait partie. p27 a également des rôles indépendants de la régulation des complexes cycline-CDK dans le cytoplasme, comme l'inhibition de la GTPase RhoA. Pour étudier les nouvelles fonctions de p27, des souris knock-in dans lesquelles le gène de p27 est remplacé par un allèle (p27CK-) qui ne peut plus lier ou inhiber les complexes cycline-CDK ont été générées. Les animaux p27CK- développent spontanément des tumeurs dans divers organes, suggérant un rôle oncogénique de p27. Ces souris présentent également un phénotype de multinucléation et polyploïdie dans plusieurs tissus. Le but du projet de recherche a été de déterminer dans quelle mesure p27CK- contribue au processus de transformation et peut causer un phénotype de multinucléation et polyploïdie. Une étude de tumorigénèse induite par l'uréthane, qui cause des mutations activant K-Ras montrent que les souris p27CK- développent plus de tumeurs dans le poumon que les animaux p27-/- et p27+/+. Nos résultats suggèrent également que le p27 cytoplasmique peut contribuer à la transformation cellulaire et coopérer avec l'activation de voies oncogéniques spécifiques qui causent sa translocation dans le cytoplasme. Dans un deuxième temps, nous avons caractérisé le phénotype de polyploïdie et de multinucléation des souris p27CK-. Nos résultats suggèrent que p27CK- entraîne la multinucléation en interférant avec l'activation de Citron-K par RhoA ce qui conduit à un défaut de cytocinèse.The cell cycle inhibitor p27Kip1 (p27) also has cyclin-CDK independent functions. Genetic evidence for these functions was provided by p27CK- knock-in mice in which p27 cannot bind or inhibit cyclin-CDK complexes. p27CK- mice have an increased incidence of tumor development in multiple organs compared to wild-type and p27-/- mice, revealing an oncogenic role for p27. In the first part of my PhD thesis, I studied the roles played by p27 during tumorigenesis. The susceptibility to urethane-induced tumorigenesis of mice either entirely lacking p27 or expressing the p27CK- allele was compared. Our results indicate a cooperation between p27CK- and activated Ras. We propose that the p27CK- protein needs to be localized in the cytoplasm in order to function as an oncogene, otherwise it just behaves similarly to a null allele. In the second part of my PhD thesis, a phenotype of multinucleation and polyploidy was identified in p27CK- mice that is not present in p27-/- animals, suggesting a role for p27 in G2/M independent of cyclin-CDK regulation. We identified the Rho effector Citron-Kinase as a p27-interacting protein in vitro and in vivo. Our data suggests that p27 may play a role during cytokinesis via the regulation of Citron-Kinase activity

Proteomic analysis of the actin cortex in interphase and mitosis.

Funder: Fonds de la recherche du Québec-Santé; Id: http://dx.doi.org/10.13039/501100000156Many animal cell shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network supporting the plasma membrane. Elucidating cortical tension control is thus essential for understanding cell morphogenesis. Increasing evidence shows that alongside myosin activity, actin network organisation and composition are key to cortex tension regulation. However, owing to a poor understanding of how cortex composition changes when tension changes, which cortical components are important remains unclear. In this article, we compared cortices from cells with low and high cortex tensions. We purified cortex-enriched fractions from cells in interphase and mitosis, as mitosis is characterised by high cortical tension. Mass spectrometry analysis identified 922 proteins consistently represented in both interphase and mitotic cortices. Focusing on actin-related proteins narrowed down the list to 238 candidate regulators of the mitotic cortical tension increase. Among these candidates, we found that there is a role for septins in mitotic cell rounding control. Overall, our study provides a comprehensive dataset of candidate cortex regulators, paving the way for systematic investigations of the regulation of cell surface mechanics. This article has an associated First Person interview with the first author of the paper

Proteomic analysis of the actin cortex in interphase and mitosis

Peer reviewed: TrueAcknowledgements: We thank all present and past Paluch lab members for critical discussions, Kevin Chalut, Ruby Peters and Iskra Yanakieva for comments on the manuscript, Guillaume Charras for feedback on the project and manuscript. Proteomic analyses were performed by the Center for Advanced Proteomics Analyses (CAPCA), a Node of the Canadian Genomic Innovation Network that is supported by the Canadian Government through Genome Canada.Publication status: PublishedFunder: Fonds de la recherche du Québec-Santé; doi: http://dx.doi.org/10.13039/501100000156Many animal cell shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network supporting the plasma membrane. Elucidating cortical tension control is thus essential for understanding cell morphogenesis. Increasing evidence shows that alongside myosin activity, actin network organisation and composition are key to cortex tension regulation. However, owing to a poor understanding of how cortex composition changes when tension changes, which cortical components are important remains unclear. In this article, we compared cortices from cells with low and high cortex tensions. We purified cortex-enriched fractions from cells in interphase and mitosis, as mitosis is characterised by high cortical tension. Mass spectrometry analysis identified 922 proteins consistently represented in both interphase and mitotic cortices. Focusing on actin-related proteins narrowed down the list to 238 candidate regulators of the mitotic cortical tension increase. Among these candidates, we found that there is a role for septins in mitotic cell rounding control. Overall, our study provides a comprehensive dataset of candidate cortex regulators, paving the way for systematic investigations of the regulation of cell surface mechanics. This article has an associated First Person interview with the first author of the paper

MiniBAR/KIAA0355 is a dual Rac and Rab effector required for ciliogenesis

Posted July 24, 2023 on bioRxiv.Cilia protrude from the cell surface and play critical roles in in-tracellular signaling, environmental sensing and development. Actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1-and Rab35-binding protein with a truncated BAR domain that we named MiniBAR (aka KIAA0355/GARRE) which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles traffick-ing to the ciliary base and exhibits remarkable fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia resulting from abnormal Rac-GTP/Rho-GTP levels, increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hall-marks of ciliopathies including left-right asymmetry defects. Thus, MiniBAR is a unique dual Rac and Rab effector that con-trols both actin cytoskeleton and membrane trafficking for cili-ogenesis

MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis

International audienceCilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis