Platelet dense granules are members of a family of tissue-specific, lysosomerelated 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 epitopetagged 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 HPSassociated 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.
IntroductionPlatelet functions are largely mediated by soluble factors released from membrane-bound storage organelles, including dense granules (DGs), ␣-granules, and lysosomes. 1 DGs store calcium, ATP, ADP, phosphates, and serotonin. 2 The high calcium concentration makes them electron dense, and 4-8 DGs per platelet can be identified by whole-mount electron microscopy. 3 Contents released from DGs after platelet activation amplify coagulation at sites of vascular injury. 2 Defective DG biogenesis causes ␦-storage pool deficiency (␦-SPD), characterized by reduced or undetectable dense core structures by whole-mount electron microscopy, depleted DG components, and reduced DG content release after stimulation. This cellular defect causes bleeding diathesis with potential severe pathology or lethality. 2,4 Understanding the cellular mechanisms that underlie DG formation in megakaryocytes (MKs) and platelets is crucial to improving ␦-SPD diagnostic tools and therapies.DGs harbor membrane transporters to import their contents from the MK or platelet cytosol, but few such transporters have been characterized. 5 The paucity of known integral membrane proteins that localize specifically to DGs has hampered efforts to define DG intermediates as they form from electron lucent precursors during MK differentiation. 6,7 Our understanding of DG biogenesis derives largely from analyses of platelets in syndromic forms of ␦-SPD, such as Hermansky-Pudlak syndrome (HPS) 8,9 and Chediak-Higashi syndrome, 10 in which DGs and other tissuespecific lysosome-related organelles (LROs) ...