title: Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.
abstract: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis.
  
  
  
  Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: A
authors.initials: A
authors.lastname: Antkowiak
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: J
authors.initials: J
authors.lastname: Viaud
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: S
authors.initials: S
authors.lastname: Severin
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: M
authors.initials: M
authors.lastname: Zanoun
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: L
authors.initials: L
authors.lastname: Ceccato
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: G
authors.initials: G
authors.lastname: Chicanne
authors.affiliation: INSERM, UMR_S949, Université de Strasbourg, Etablissement Français du Sang-Alsace, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Strassel
authors.affiliation: INSERM, UMR_S949, Université de Strasbourg, Etablissement Français du Sang-Alsace, Toulouse, France.
authors.forename: A
authors.initials: A
authors.lastname: Eckly
authors.affiliation: INSERM, UMR_S949, Université de Strasbourg, Etablissement Français du Sang-Alsace, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Leon
authors.affiliation: INSERM, UMR_S949, Université de Strasbourg, Etablissement Français du Sang-Alsace, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Gachet
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.|Laboratoire d'Hématologie, CHU de Toulouse, Toulouse, France.
authors.forename: B
authors.initials: B
authors.lastname: Payrastre
authors.affiliation: INSERM, UMR1048, Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.
authors.forename: F
authors.initials: F
authors.lastname: Gaits-Iacovoni
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D021621:Imaging, Three-Dimensional; D016086:Lentivirus; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D015398:Signal Transduction; D034061:Thrombopoiesis; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein
keywords: Rho GTPases; actin; cytoskeleton; megakaryocytes; platelets