Cdc42‐dependent F‐actin dynamics drive structuration of the demarcation membrane system in megakaryocytes

Abstract

EssentialsInformation 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.Summary. 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). F€ orster 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.

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

“…Given that all markers of MK maturity (such as CD41, CD42b, and the TPO receptor) were present, this underdeveloped DMS seems to be the consequence of a reduced capacity to generate these membranes. Consecutive stages of DMS organization do rely on F-actin dynamics to polarize membranes (24) and are also controlled by ROCK (34). It seems thus conceivable that the insufficiently developed membrane reservoir is in part responsible for the reduced platelet count and/or the increased platelet size in these 2B mice through dysregulation of F-actin dynamics and RhoA activity, both found to be involved in proplatelet formation defects in 2B MKs.…”

“…Because 2B MKs failed to develop normal DMS and proplatelet extensions, we investigated the structure of actin, a critical component of the cytoskeleton controlling MK cytoplasmic morphogenesis (8,23,24). Actin distribution and organization were investigated in cultured MKs that were allowed to spread on a fibrinogen matrix.…”

On the Way to in vitro Platelet Production

“…The importance of podosomes in thrombopoiesis is further suggested by the occurrence of thrombocytopenia in primary genetic deficiencies affecting podosome formation (WASP, CDc42, α-actinin, or CD44) (54, 55). Work by Antkowiack et al also indicates that EC contacts triggering podosome formation could participate in the DMS polarization preceding proplatelet extension (56). …”

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis

“…This involves a cross-regulation between Cdc42 and RhoA, which tunes GTPase function in different cell types3031. It was recently demonstrated that Cdc42 is important to polarize the MK DMS towards BM sinusoids32. Moreover, we have previously shown that mice with MK/platelet-specific RhoA-deficiency ( RhoA fl/fl Pf4-cre , further referred to as RhoA −/− ) exhibit pronounced macrothrombocytopenia (Supplementary Fig.…”

“…To decipher the underlying mechanism, we took advantage of lentiviral FRET biosensors derived from the Raichu probe to monitor Cdc42 activity in the polarized DMS32. Wt MKs showed a polarized Cdc42 activity at the DMS/F-actin complex, which was reduced by 68% in Gp1ba-Tg MKs (Fig.…”