in-vivo-two-photon-imaging-megakaryocytes-proplatelets-mouse-skull

<em>In Vivo</em> Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network-derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis: (1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer. 

Biogenesis of the demarcation membrane system (DMS) in megakaryocytes.

Macrothrombocytopenia in MYH9-related disease (MYH9-RD) results from defects in nonmuscular myosin-IIA function. Thrombopoietin receptor agonists (eltrombopag; romiplostim) seem to improve hemostasis, but little is known about their biologic effects in MYH9-RD. We administered romiplostim to Myh9(-/-) mice (100 μg/kg, every 3 days, during 1 month). MKs increased to similar numbers in Myh9(-/-) and wild-type (WT) mice (with an increase in immature MKs), but Myh9(-/-) platelet count response was much less (2.5-fold vs 8-fold increase). A strong increase in MK nuclei emboli in the lung, in WT and Myh9(-/-) mice, indicates increased transmigration of MKs from the BM. Prolonged (but not acute) treatment with romiplostim decreased expression of GPIb-IX-V complex and GPVI, but not of GPIIbIIIa, and bleeding time increased in WT mice. Microcirculation was not altered by the increased number of large platelets in any of the assessed organs, but in Myh9(-/-) mice a much stronger increase in BM reticulin fibers was present after 4 weeks of romiplostim treatment vs WT mice. These data further encourage short-term use of thrombopoietic agents in patients with MYH9-RDs; however, myelofibrosis has to be considered as a potential severe adverse effect during longer treatment. Reduction of GPIbIX/GPVI expression by romiplostim requires further studies.

Romiplostim administration shows reduced megakaryocyte response-capacity and increased myelofibrosis in a mouse model of MYH9-RD.

Megakaryocyte (MK) differentiation occurs within the bone marrow (BM), a complex 3-dimensional (3D) environment of low stiffness exerting local external constraints. To evaluate the influence of the 3D mechanical constraints that MKs may encounter in vivo, we differentiated mouse BM progenitors in methylcellulose (MC) hydrogels tuned to mimic BM stiffness. We found that MKs grown in a medium of 30- to 60-Pa stiffness more closely resembled those in the BM in terms of demarcation membrane system (DMS) morphological aspect and exhibited higher ploidy levels, as compared with MKs in liquid culture. Following resuspension in a liquid medium, MC-grown MKs displayed twice as much proplatelet formation as cells grown in liquid culture. Thus, the MC gel, by mimicking external constraints, appeared to positively influence MK differentiation. To determine whether MKs adapt to extracellular stiffness through mechanotransduction involving actomyosin-based modulation of the intracellular tension, myosin-deficient (Myh9) progenitors were grown in MC gels. Absence of myosin resulted in abnormal cell deformation and strongly decreased proplatelet formation, similarly to features observed for Myh9 MKs differentiated in situ but not in vitro. Moreover, megakaryoblastic leukemia 1 (MKL1), a well-known actor in mechanotransduction, was found to be preferentially relocated within the nucleus of MC-differentiated MKs, whereas its inhibition prevented MC-mediated increased proplatelet formation. Altogether, these data show that a 3D medium mimicking BM stiffness contributes, through the myosin IIA and MKL1 pathways, to a more favorable in vitro environment for MK differentiation, which ultimately translates into increased proplatelet production.

Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.

The differentiation and maturation of megakaryocytes (MKs) occurs in a 3D environment where the cells must constantly adapt to the external physical and mechanical constraints during their development and migration to sinusoid vessels. In this chapter, we present a method for culture of mouse MKs from bone marrow hematopoietic progenitor cells in a methylcellulose 3D medium with a stiffness mimicking that of bone marrow. In addition, we describe how the MKs can be recovered to allow for analysis of their differentiation and maturation state by transmission electron microscopy, immunofluorescence or flow cytometry techniques and to evaluate their ability to form proplatelets. This approach allows (1) generation of MKs with a morphology that more closely resembles the MKs that differentiate in vivo, (2) recovery of megakaryocyte phenotypes sometimes observed in vivo but not found in classical liquid cultures, and (3) study of mechanotransduction pathways induced by the stiffness of the medium.

Three-Dimensional Culture in a Methylcellulose-Based Hydrogel to Study the Impact of Stiffness on Megakaryocyte Differentiation.

Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models.

Article	Year
Biogenesis of the demarcation membrane system (DMS) in megakaryocytes. Blood\| PubMed ID: 24152908	2014
Romiplostim administration shows reduced megakaryocyte response-capacity and increased myelofibrosis in a mouse model of MYH9-RD. Blood\| PubMed ID: 22234693	2012
Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation. Blood\| PubMed ID: 27503502	2016
Three-Dimensional Culture in a Methylcellulose-Based Hydrogel to Study the Impact of Stiffness on Megakaryocyte Differentiation. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30171577	2018
Cytoskeletal-based mechanisms differently regulate and proplatelet formation. Haematologica\| PubMed ID: 32327502	2021

Fabien Pertuy

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