Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Daimon, Tateo; David, Heinz

doi:10.1007/bf00490898

Cited by 16 publications

(12 citation statements)

References 16 publications

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“…Although DG precursors in MKs were identified long ago, 6,44 difficulties in their visualization 7,45 and a lack of DG-restricted protein markers have hampered their characterization. CD63 was proposed as a DG-restricted cargo in human platelets because it rapidly translocates to the plasma membrane on platelet activation, cofractionates with DGs, and is depleted in platelets from HPS1 patients.…”

Section: Discussionmentioning

confidence: 99%

“…Nonpigmented early-stage melanosomes derive from multivesicular endosomes and mature to pigmented, late-stage melanosomes through subsequent BLOC-1-and/or AP-3-dependent cargo delivery from early endosomes. 42 By analogy, a CD63-containing precursor in MKs, perhaps corresponding to mepacrine-labeled structures 6,7 that are malformed in BLOC-3-deficient HPS1 patients, 20,46 may mature into a functional DG by the BLOC-1-and/or AP-3-dependent delivery of cargoes such as SLC35D3 during a late stage of MK differentiation (supplemental Figure 6B and see next paragraph). This model is consistent with the accumulation of SLC35D3 in early endosomes in MKs and with the requirement for BLOC-1 and AP-3 but not BLOC-3 for SLC35D3 accumulation in platelets, and would explain why platelets from HPS model mice label with mepacrine 30 but lack normal DG contents 43 ; they would contain the precursors, but not selected cargoes required for DG maturation.…”

Section: Discussionmentioning

confidence: 99%

“…MKs in early stages of differentiation generate DG precursors that accumulate mepacrine and nucleotides, 6,7 and the numbers of DG precursors increase as MKs mature and enlarge, 21,50 but the stage of MK differentiation at which DGs mature to completion is not known. The megakaryocytoid cells analyzed herein likely represent intermediate MK-differentiation states, because GATA-1-expressing G1ME cells do not form proplatelets and the FLC-derived MKs were analyzed before proplatelet formation.…”

Section: Discussionmentioning

confidence: 99%

“…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) are dysfunctional. HPS is characterized minimally by ␦-SPD and oculocutaneous albinism due to malformation of platelet DGs and pigment cell melanosomes.…”

Section: Introductionmentioning

confidence: 99%

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SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Meng

Wang

Yao

et al. 2012

Blood

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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) ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Meng

Wang

Yao

et al. 2012

Blood

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“…Electron dense "bullseye" structures in MK thin sections labeled with standard dyes or the uranaffin method (which labels biogenic amines 26 ) have been interpreted as DGs 10,27,28 , but distinguishing these from the more abundant and heterogeneous α-granules is challenging. Refinements in cryopreservation, specifically by high-pressure freezing and freeze substitution, have yielded platelet α-granules more uniform in appearance and without an electron-dense core, and therefore may be a promising avenue to distinguish granule populations in MKs 29,30 , but thus far dense granules have not been unambiguously identified by this technique.…”

Section: Introductionmentioning

confidence: 99%

Platelet dense granules begin to selectively accumulate mepacrine during proplatelet formation

et al. 2017

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Platelet dense granules (DGs) are storage organelles for calcium ions, small organic molecules such as ADP and serotonin, and larger polyphosphates that are secreted upon platelet stimulation to enhance platelet activation, adhesion, and stabilization at sites of vascular damage. DGs are thought to fully mature within megakaryocytes (MKs) prior to platelet formation. Here we challenge this notion by exploiting vital fluorescent dyes to distinguish mildly acidic DGs from highly acidic compartments by microscopy in platelets and MKs. In isolated primary mouse platelets, compartments labeled by mepacrine — a fluorescent weak base that accumulates in DGs — are readily distinguishable from highly acidic compartments, likely lysosomes, that are labeled by the acidic pH indicator, LysoTracker, and from endolysosomes and alpha granules labeled by internalized and partially digested DQ™ BSA. By contrast, in murine fetal liver- and human CD34+ cell-derived MKs and the megakaryocytoid cell lines, MEG-01 and differentiated G1ME2, labeling by mepacrine overlapped nearly completely with labeling by LysoTracker and partially with labeling by DQ™ BSA. Mepacrine labeling in G1ME2-derived MKs was fully sensitive to proton ATPase inhibitors, but was only partially sensitive in platelets. These data indicate that mepacrine in MKs accumulates as a weak base in endolysosomes but is likely pumped into or retained in separate DGs in platelets. Fluorescent puncta that labeled uniquely for mepacrine were first evident in G1ME2-derived proplatelets, suggesting that DGs undergo a maturation step that initiates in the final stages of MK differentiation.

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Megakaryocyte Biology

Jackson¹,

Arnold²,

Pestina³

et al. 1997

Thrombopoiesis and Thrombopoietins

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Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Cited by 16 publications

References 16 publications

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Platelet dense granules begin to selectively accumulate mepacrine during proplatelet formation

Megakaryocyte Biology

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