Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott–Aldrich syndrome-like platelet defect

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Key Points• Dynein-dependent microtubule sliding drives proplatelet elongation under static and physiological shear stress conditions. • Proplatelet formation is a process that can be divided into repetitive phases: extension, pause, and retraction.Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged b1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia. (Blood. 2015;125(5):860-868)

Section: Org Frommentioning

confidence: 88%

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein

Bender

Thon

Ehrlicher

et al. 2015

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“…Incubation of platelets from controls at 4°C caused disassembly of microtubules, which then reassembled to the marginal band after rewarming to 37°C, as previously reported. [22][23][24] In contrast, cold incubation or rewarming did not grossly affect the microtubules in platelets from the DIAPH1 R1213* cases ( Figure 6A-B), suggesting that the increased microtubule content resulted from increased microtubule stability.…”

mentioning

confidence: 83%

“…Reassembly was allowed by subsequent rewarming at 37°C as previously reported. [22][23][24] Detailed methods and uncropped images of western blots are provided in supplemental Methods and supplemental Figures, respectively, available on the Blood Web site.…”

Section: Microtubule Sedimentation and Cold-induced Disassemblymentioning

confidence: 99%

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss

Stritt

Nurden

Turro

et al. 2016

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134

150

Key Points• A gain-of-function variant in DIAPH1 causes macrothrombocytopenia and hearing loss and extends the spectrum of DIAPH1-related disease.• Our findings of altered megakaryopoiesis and platelet cytoskeletal regulation highlight a critical role for DIAPH1 in platelet formation.Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease andprovides new insight into the autoregulation of DIAPH1 activity.

“…[45][46][47][48] In fact, both murine and human WASP KO MKs in culture display proplatelet formation. [45][46][47][48] Therefore, these data are consistent with our model, suggesting WASP is not essential for (pro)platelet formation.…”

Section: Discussionmentioning

confidence: 99%

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation

Machlus

Stumpo

et al. 2016

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Key Points• Proteomic analyses and polysome profiling of developing MKs identified a striking increase in the levels of a novel protein, MARCKS, during proplatelet formation.• MARCKS deletion, inhibition, or phosphorylation inhibits proplatelet formation associated with activation of the actin-binding protein Arp2/3.Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4-and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP 2 . In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP 2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).