Mechanism of platelet dense granule biogenesis: study of cargo transport and function of Rab32 and Rab38 in a model system

Abstract

Dense granules are important in platelet aggregation to form a hemostatic plug as evidenced by the increased bleeding time in mice and humans with dense granule deficiency. Dense granules also are targeted by antiplatelet agents because of their role in thrombus formation. Therefore, the molecular understanding of the dense granule and its biogenesis is of vital importance. In this work, we establish a human megakaryocytic cell line (MEG-01) as a model system for the study of dense granule biogenesis using a variety of cell biology and biochemical approaches. Using this model system, we determine the late endocytic origin of these organelles by colocalization of the internalized fluid phase marker dextran with both mepacrine and transmembrane dense granule proteins. By mistargeting of mutant dense granule proteins, we demonstrate that sorting signals recognized by adaptor protein-3 are necessary for normal transport to dense granules.

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

“…Thus a small proportion of Myosin Vc is associated with melanosomes at steady state or the association is transient and does not withstand the lengthy fractionation procedure. Myosin Vc largely co-fractionates with cytosolic proteins, vesicles, and light organelles that are obtained in the first fractions (64). Notice that in addition to a cohort of Rab32 and Rab38 (and other Rabs) that co-fractionate with melanosomes (fractions 5-10), a proportion of these Rabs is obtained in lighter fractions, consistent with their known presence in small transport vesicles and early endosomal domains (24,25,64).…”

“…Electron Microscopy-High pressure freezing was performed as previously described (63,64). Briefly, MNT-1 cells treated with control or Myosin Vc siRNA were subjected to high pressure freezing in 15% dextran (9 -11 kDa) in culture media, freeze-substitution in 0.25% glutaraldehyde, 0.1% uranyl acetate, and embedding in Lowicryl HM20 resin and processed for thin-section transmission electron microscopy.…”

“…Myosin Vc largely co-fractionates with cytosolic proteins, vesicles, and light organelles that are obtained in the first fractions (64). Notice that in addition to a cohort of Rab32 and Rab38 (and other Rabs) that co-fractionate with melanosomes (fractions 5-10), a proportion of these Rabs is obtained in lighter fractions, consistent with their known presence in small transport vesicles and early endosomal domains (24,25,64). Consistently, live cell confocal fluorescence microscopy of MNT-1 cells expressing full-length Myosin Vc-GFP and tyrosinase-Cherry showed a limited number of structures where both proteins colocalized over time (Fig.…”

Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice

“…9 The a-granules are formed early during megakaryopoiesis by the fusion of small vesicles from the transGolgi network and endocytic vesicles into multivesicular bodies (MVBs) 10 then transported through the cytoplasmic extensions of the proplatelet shafts of the mature megakaryocytes (MKs), the bonemarrow residing platelet precursors, where they become trapped in the terminal buds (ie, the nascent platelets). 11,12 Gray platelet syndrome (GPS), is a rare autosomal recessive bleeding disorder first reported in 1970 13 caused by mutations in NBEAL2. [14][15][16] Platelets from GPS patients are large and have a gray appearance caused by an almost complete absence of a-granules.…”

α-granules: a story in the making

“…If so, the identity of its component proteins will provide some hints as to which SNARE and Rab isoforms function in ␣-granule formation. In addition to affinitybased approaches, the development of megakaryocytic vesicle trafficking models to study platelet granule formation 9 will enable identification of additional components involved the generation of ␣-granules. Perhaps with the first of the main actors identified, a more complete story of ␣-granules formation is now attainable.…”