title: SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models.
abstract: Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, 513 Stellar-Chance Laboratories, 422 Curie Blvd., Philadelphia, PA 19104-6100, USA.
authors.forename: Ronghua
authors.initials: R
authors.lastname: Meng
authors.forename: Yuhuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Yao
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.forename: Dawn C
authors.initials: DC
authors.lastname: Harper
authors.forename: Harry F G
authors.initials: HF
authors.lastname: Heijnen
authors.forename: Anand
authors.initials: A
authors.lastname: Sitaram
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Graça
authors.initials: G
authors.lastname: Raposo
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Weiss
authors.forename: Mortimer
authors.initials: M
authors.lastname: Poncz
authors.forename: Michael S
authors.initials: MS
authors.lastname: Marks
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000097:blood; D002454:Cell Differentiation; D003594:Cytoplasmic Granules / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000097:blood; D004195:Disease Models, Animal; D011992:Endosomes / Q000378:metabolism; D022861:Hermanski-Pudlak Syndrome / Q000097:blood* / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins / Q000097:blood; D008297:Male; D008533:Megakaryocytes / Q000378:metabolism* / Q000473:pathology; D008544:Melanocytes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D016253:Microscopy, Immunoelectron; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000235:genetics*; D050505:Mutant Proteins / Q000097:blood / Q000235:genetics; D050765:Qa-SNARE Proteins / Q000097:blood; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D014157:Transcription Factors / Q000097:blood
chemical_list: C513476:Bloc1s6 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; D050765:Qa-SNARE Proteins; D012333:RNA, Messenger; C518133:Slc35d3 protein, mouse; D014157:Transcription Factors; C060759:enhancer-binding protein AP-3; C496153:syntaxin 12, mouse