TY - JOUR

T1 - Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42

AU - Pleines, Irina

AU - Dütting, Sebastian

AU - Cherpokova, Deya

AU - Eckly, Anita

AU - Meyer, Imke

AU - Morowski, Martina

AU - Krohne, Georg

AU - Schulze, Harald

AU - Gachet, Christian

AU - Debili, Najet

AU - Brakebusch, Cord Herbert

AU - Nieswandt, Bernhard

PY - 2013/10/31

Y1 - 2013/10/31

N2 - Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.

AB - Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.

KW - Animals

KW - Blotting, Western

KW - Cytoskeleton

KW - Hemostasis

KW - Megakaryocyte Progenitor Cells

KW - Megakaryocytes

KW - Mice

KW - Mice, 129 Strain

KW - Mice, Inbred C57BL

KW - Mice, Knockout

KW - Microscopy, Confocal

KW - Microscopy, Electron, Scanning

KW - Microscopy, Electron, Transmission

KW - Microtubules

KW - Pseudopodia

KW - Thrombocytopenia

KW - Thrombosis

KW - Tubulin

KW - cdc42 GTP-Binding Protein

KW - rac1 GTP-Binding Protein

U2 - 10.1182/blood-2013-03-487942

DO - 10.1182/blood-2013-03-487942

M3 - Journal article

C2 - 23861250

VL - 122

SP - 3178

EP - 3187

JO - Blood

JF - Blood

SN - 0006-4971

IS - 18

ER -