ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing

Abstract

The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/ n-cofilin-mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton. (Blood. 2010;116(10):1767-1775) IntroductionThe formation of anucleated blood platelets from their bone marrow resident precursors, the megakaryocytes (MKs), is a unique process in mammalian physiology. 1 Terminally differentiated MKs, the largest cells evolving from hematopoietic stem cells, are polyploid and contain a highly organized cytoplasm with a membranous network, the demarcation membrane system (DMS), which converts into cytoplasmic protrusions during proplatelet formation. 1,2 According to the current model, the tips of these proplatelet protrusions extend into the lumen of a vessel in bone marrow sinusoids and are shed by shear forces generated by the blood flow. Recently, the first direct visualization of this process in situ in the bone marrow of mice using 2-photon microscopy revealed that the released fragments are larger in size than normal platelets. In addition, beaded proplatelets and barbell-shaped platelet pairs were observed in the blood, leading to the hypothesis that final shaping and sizing of platelets may occur in the circulation. 3 In line with this, very recent observations indicate that platelet progeny formation occurs in whole blood, suggesting that thrombopoiesis continues in the bloodstream. 4 Proplatelet formation relies on coordinated cytoskeletal rearrangements with an essential involvement of tubulin-dependent processes. 5 The extension of cytoplasmic protrusions from MKs is mechanically driven by tubulin sliding and assembly. In contrast, the role of actin, the other major cytoskeletal component in platelets, has remained elusive in platelet biogenesis from MKs. In mature platelets, the importance of actin filament dynamics, mainly the uncapping and elongation of actin filaments, for platelet activation and shape changes, has been well documented. 6 A mouse model for the filamentous actin (F-actin) severing protein gelsolin provided in vivo evidence for a physiologic role of actin dynamics in platelet activation. 7 However, surprisingly, gelsolin-d...

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

“…Consequently, actin filaments are stabilized and accumulate in areas enriched in phosphorylated cofilin (Figures 5 and 7). This extends previous findings showing that hyperphosphorylation of cofilin induces a platelet-formation defect (8,35). In addition, mutations in the cofilin partner Aip1/Wdr1 lead to macrothrombocytopenia (37).…”

“…Changes in actin polymerization are key to many processes involving the formation of proplatelets (4-6, 8, 35). In particular, the formation of extensions and proplatelet shaft bifurcations are clearly actin dependent, and it is not surprising that actin plays a fundamental role in the terminal step of proplatelet production (8). Recently, it has been described that proplatelet formation was impaired and MKs displayed a reduced number of proplatelet tips of altered diameter, while actin polymerization was increased in vitro (36).…”

“…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.…”

“…These unusually thick peripheral zones enriched in disorganized F-actin displayed an increased ratio of F-actin to globular actin (G-actin), indicating accumulation of actin polymerization ( Figure 4C and Supplemental Figure 7). Given the disorganization of the actin network, we considered the hypothesis that the activity of the actin turnover-regulating protein cofilin (8) or levels of the actin-binding scaffold protein IQGAP1 (25) could be modulated in 2B MKs. However, levels of IQGAP1 were similar in 2B and WT MKs ( Figure 5A).…”

“…Actin cytoskeleton and microtubules are involved in proplatelet extension and in the final abscission and cleavage leading to platelet formation in the blood stream of marrow sinusoids (5-7). In vivo, actin filament turnover is a critical step in the terminal phase of platelet formation, as well as in the maturation and sizing of platelets to produce the homogeneous mature platelet population (6,8).…”