Biogenesis of the demarcation membrane system (DMS) in megakaryocytes

Abstract

Key Points• Using state-of-the-art three-dimensional electron microscopy approaches, we show that the onset of the DMS formation is at the megakaryocyte plasma membrane.• A pre-DMS structure is formed in the perinuclear region, through a PM invagination process that resembles cleavage furrow formation.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. (Blood. 2014;123(6):921-930) IntroductionThe maturation of megakaryocytes (MKs) includes the development of a unique and extensive membrane system known as the demarcation membrane system (DMS), which divides the cytoplasm into small platelet territories. One MK is thought to produce an average of 4000 platelets. Although it has been known for many years that the DMS ultimately forms the cell membrane of the future platelets, 1,2 the exact mechanism of the formation of this unique membrane system remains unclear. Early electron microscopy (EM) examination of late stage mature MKs led to the proposal that the DMS demarcates already preformed platelets. 3 Observations by De Bruyn of long MK extensions protruding into the sinusoidal lumen modified this idea and suggested that the DMS divides the MK cytoplasm into intertwined and compacted cylindrical regions. 4 Such a model had already been proposed earlier by Thiery and Bessis, who observed that MKs from bone marrow (BM) explants extended elongated projections they called proplatelets. 5 Studies using cultured MKs led to a more refined model, where the DMS appears to function as a membrane reservoir for the extension of proplatelets, which would then fragment into platelets along their length 6 or at their tips. [7][8][9] More recently, using intravital microscopy...

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

“…In midstage MKs, PACSIN2 was confined mostly to a single locus, which showed contact with colabeled CD41 ( Figure 6A; supplemental Videos 3 and 4), consistent with plasma membrane invagination and reminiscent of the recently characterized initiating DMS. 3 In contrast, in mature MKs, PACSIN2 was observed to be spread throughout the cell in foci similar in size to those observed in platelets ( Figure 6B; supplemental Video 5).…”

“…During MK maturation, the demarcation membrane system (DMS) forms as a surface-connected membrane extension that invaginates into the cell body and elaborates to provide membranes for future platelets. [1][2][3] In platelets, the open canalicular system (OCS) forms multiple interconnections between cell surfaces and serves as a membrane reservoir during platelet spreading following activation. 4 The MK DMS and platelet OCS share structural similarities, but little is known about the proteins and mechanisms responsible for their formation.…”

Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

“…Control Dnm2 fl/fl MKs formed the DMS (Figure 2A), the highly organized intracellular membrane structure and membrane reservoir for future platelets that is associated with late-stage mature MKs. [3][4][5] Although present, the DMS was dramatically altered in Dnm2 fl/fl Pf4-Cre MKs ( Figure 2B-C), appearing at times as a compact, narrow twisting membrane system of clathrin-coated vesicles ( Figure 2B, arrowheads), resembling the "clathrin-coated profiles" of Dnm1-null inhibitory synapses. 12,13 Emperipolesis, or the presence of intact nucleated cells within the cytoplasm, was also a common feature of bone marrow Dnm2…”

“…1,2 This process requires extensive intracellular membrane rearrangements in MKs, including the formation of the demarcation membrane system (DMS), a surface-connected membrane extension that invaginates into the cell body and further develops to provide membranes necessary for platelet formation. [3][4][5] The mechanisms responsible for these membrane rearrangements remain poorly understood.…”

Hyaluronan Depolymerization by Megakaryocyte Hyaluronidase-2 Is Required for Thrombopoiesis

“…Indeed, recent evidence indicates that DMS biogenesis is initiated at the plasma membrane in glycoprotein Ibb þ pre-DMS clusters and the newly synthesized membrane becomes invaginated, thereby initiating formation of the DMS. 5 Expansion of the DMS requires vesicular membrane delivery from numerous Golgi complexes within the MK, and it seems plausible that the HA biosynthetic enzymes and Hyal-2 are contained within Golgi vesicles and are targeted to the DMS. An additional noteworthy finding of our study is that although the addition of exogenous hyaluronidase led to a recovery in the number of proplatelets released from MKs, the treatment did not rescue the abnormal morphologic features seen in Hyal-2e deficient MKs, suggesting that Hyal-2 interactions with other proteins may contribute as well ( Figure 4B and Figure 5, C and D).…”