Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein

Abstract

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)

Megakaryocytic Maturation in Response to Shear Flow Is Mediated by the Activator Protein 1 (AP-1) Transcription Factor via Mitogen-activated Protein Kinase (MAPK) Mechanotransduction

“…Previous studies on the impact of shear stress on MK differentiation focus on cytoskeletal and morphological changes (7,31,32), but no signaling pathways have been investigated. MAPK pathways are active in MKs, but they have not yet been linked to mechanotransduction (33).…”

Label free monitoring of megakaryocytic development and proplatelet formation in vitro

“…This intense biosynthetic need in early stages could be the driving force for the detected optical changes in the early MK stages. As MKs further mature, the DMS, which functions as a membrane reservoir, driven by energy consuming cytoskeletal dynamics [26] is utilized to create cytoplasmic expansions that later fragment and lead to the formation of platelets. Cytoskeletal reorganization for proplatelet extension [26] and cargo transportation through the cytoplasmic extensions create a rising demand for ATP availability.…”

“…As MKs further mature, the DMS, which functions as a membrane reservoir, driven by energy consuming cytoskeletal dynamics [26] is utilized to create cytoplasmic expansions that later fragment and lead to the formation of platelets. Cytoskeletal reorganization for proplatelet extension [26] and cargo transportation through the cytoplasmic extensions create a rising demand for ATP availability. The latter in combination with a switch to metabolic pathways found in resting platelets, like de novo fatty acid synthesis and oxidation, could potentially explain the increasing RR and BF trends and the compartmentalized metabolic differences detected during the terminal differentiation stages.…”

Proplatelets slip slidin’ away

“…They also show that physiological shear forces generated in a microfluidic platelet bioreactor accelerate proplatelet extension by reducing the pause phase. 1,2 Better understanding the molecular basis of platelet biogenesis will yield improved strategies for treating thrombocytopenia and thrombocythemia, and optimize conditions for culturing platelets in vitro for experimental and therapeutic purposes. Previous findings by Hartwig and colleagues led to the hypothesis that dynein-driven microtubule sliding underlies proplatelet elongation.…”

“…3 To address this hypothesis, Bender et al used fluorescence loss after photoconversion (FLAC) time lapse and fluorescence recovery after photobleaching (FRAP) to directly visualize and quantify the rates of proplatelet elongation in fetal liver-derived mouse megakaryocytes expressing b1-tubulin tagged with the photoconvertible fluorophore Dendra2 (b1-tubulin-Dendra2). 1 Experiments were performed in the presence and absence of structurally distinct inhibitors of dynein, under static and physiological shear stress. Bender et al establish that proplatelet elongation is not a continuous process once initiated, but rather undergoes repetitive phases of extension, pause, and retraction back to the megakaryocyte cell body, the rate of which is considerably increased when megakaryocytes are exposed to shear.…”