RESUMEN
At mitotic entry, reorganization of the actomyosin cortex prompts cells to round-up. Proteins of the ezrin, radixin, and moesin family (ERM) play essential roles in this process by linking actomyosin forces to the plasma membrane. Yet, the cell-cycle signal that activates ERMs at mitotic entry is unknown. By screening a compound library using newly developed biosensors, we discovered that drugs that disassemble microtubules promote ERM activation. We further demonstrated that disassembly of interphase microtubules at mitotic entry directs ERM activation and metaphase cell rounding through GEF-H1, a Rho-GEF inhibited by microtubule binding, RhoA, and its kinase effector SLK. We finally demonstrated that GEF-H1 and Ect2, another Rho-GEF previously identified to control actomyosin forces, act together to drive activation of ERMs and cell rounding in metaphase. In summary, we report microtubule disassembly as a cell-cycle signal that controls a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.
Asunto(s)
Actomiosina , Interfase , Microtúbulos , Factores de Intercambio de Guanina Nucleótido Rho , Actomiosina/metabolismo , Forma de la Célula , Células HEK293 , Humanos , Microtúbulos/metabolismo , Mitosis , Proteínas Proto-Oncogénicas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
The COVID-19 pandemic has highlighted the need to modernize healthcare systems to the reality of the 21st century. The first world-wide Strategic Committee to launch Collaborative Value-Based Healthcare (C-VBHC) anchored on populational Real World Data and structured collaboration, took place in Montreal, via TOWWERS showcase project. The meeting covered a broad range of topics from the perspective of each of the various Real-World healthcare actors, the 5P+: Patient, Prescriber, Producer, Policymaker, Payer, including Data and Research stakeholders. Attended by approximately 20 participants from North America and Europe, the meeting provided a valuable opportunity to unit the 5P+ around common goals and exchanging on solutions. TOWWERS Strategic committee identified key elements required to continue the transformation.
Asunto(s)
COVID-19 , Pandemias , Atención a la Salud , Europa (Continente) , HumanosRESUMEN
Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.
Asunto(s)
Técnicas Biosensibles , Proteínas del Citoesqueleto , Transferencia de Energía , Proteínas de la Membrana , Proteínas de MicrofilamentosRESUMEN
Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Células Epiteliales/fisiología , Mitosis , Morfogénesis , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Animales , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliales/citología , Ensayos Analíticos de Alto Rendimiento , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.
Asunto(s)
Actomiosina/genética , Movimiento Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Oogénesis/genética , Proteínas Serina-Treonina Quinasas/genética , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Algoritmos , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Modelos Genéticos , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARNRESUMEN
The tumor suppressor PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 Here, we make the unexpected discovery that in Drosophila melanogaster PTEN reduces PtdIns(4,5)P2 levels on endosomes, independently of its phosphatase activity. This new PTEN function requires the enzymatic action of dPLCXD, an atypical phospholipase C. Importantly, we discovered that this novel PTEN/dPLCXD pathway can compensate for depletion of dOCRL, a PtdIns(4,5)P2 phosphatase. Mutation of OCRL1, the human orthologue of dOCRL, causes oculocerebrorenal Lowe syndrome, a rare multisystemic genetic disease. Both OCRL1 and dOCRL loss have been shown to promote accumulation of PtdIns(4,5)P2 on endosomes and cytokinesis defects. Here, we show that PTEN or dPLCXD overexpression prevents these defects. In addition, we found that chemical activation of this pathway restores normal cytokinesis in human Lowe syndrome cells and rescues OCRL phenotypes in a zebrafish Lowe syndrome model. Our findings identify a novel PTEN/dPLCXD pathway that controls PtdIns(4,5)P2 levels on endosomes. They also point to a potential new strategy for the treatment of Lowe syndrome.
Asunto(s)
Proteínas de Drosophila/genética , Síndrome Oculocerebrorrenal/genética , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas/genética , Fosfolipasas de Tipo C/genética , Animales , Citocinesis/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Endosomas/genética , Endosomas/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/patología , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de SeñalRESUMEN
Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/genética , Anafase , Animales , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular , Proteínas del Citoesqueleto/genética , Drosophila/genética , Drosophila/metabolismo , Humanos , Interfase , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Metafase , Microtúbulos/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Proteína Fluorescente RojaRESUMEN
The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division.
Asunto(s)
Anafase/fisiología , Forma de la Célula/fisiología , Citocinesis/fisiología , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Regulación hacia Abajo/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de la Membrana/genética , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas Fosfatasas/genéticaRESUMEN
Three Phanerochaete chrysosporium endo-1,4-beta-xylanase genes were cloned and expressed in Aspergillus niger. Two of these genes, xynA and xynC, encode family 10 glycoside hydrolases, while the third, xynB, codes for a family 11 glycoside hydrolase. All three xylanases possess a type I carbohydrate-binding domain connected to the catalytic domain by a linker region. The three xylanases were purified to homogeneity by weak anion or Avicell column chromatography and subsequently characterized. The XynA, XynB and XynC enzymes have molecular masses of 52, 30 and 50 kDa, respectively. Optimal activity was obtained at pH 4.5 and 70 degrees C with the family 10 xylanases and pH 4.5 and 60 degrees C with the family 11 xylanase. The measured Km when using birchwood xylan as the substrate was 3.71 +/- 0.69 mg/ml for XynA and XynC and was 9.96 +/- 1.45 mg/ml for XynB. Substrate specificity studies and the products released during the degradation of birchwood xylan suggest differences in catalytic properties between the two family 10 xylanases and the family 11 xylanase.