Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration
Isabelle C. Becker,
Adrian R. Wilkie,
Bret A. Unger,
Anthony R. Sciaudone,
Farheen Fatima,
I-Ting Tsai,
Ke Xu,
Kellie R. Machlus,
Joseph E. Italiano
Affiliations
Isabelle C. Becker
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
Adrian R. Wilkie
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
Bret A. Unger
Department of Chemistry, University of California, Berkeley, Berkeley, CA, 94720
Anthony R. Sciaudone
Brigham and Women’s Hospital, 4 Blackfan Circle, Boston, MA, 02115
Farheen Fatima
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
I-Ting Tsai
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
Ke Xu
Department of Chemistry, University of California, Berkeley, Berkeley, CA, 94720
Kellie R. Machlus
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
Joseph E. Italiano
Vascular Biology Program, Boston Children’s Hospital, 1 Blackfan Circle, Boston, MA, 02115; Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115
Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.
