Large dense-core vesicles (LDCVs) are characterized as a class of lysosome-related organelles (LROs), which undergo regulated release and play important roles in development, metabolism and homeostasis. The Muted protein is a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), which functions in the biogenesis of lysosomes and LROs. CD63 is a membrane component of lysosomes and LROs. Whether and how CD63 is sorted into LDCVs is largely unknown. In this study, we aim to identify the localization of CD63 in chromaffin cells by colocalization, living cell imaging and cell fractionation. We found that a proportion of CD63-YFP colocalized with NPY-dsRed labeled LDCVs. By sucrose density gradient fractionation, a proportion of CD63 was found to be highly enriched in LDCVs fractions. The Muted mutant mouse is a model of Hermansky-Pudlak syndrome (HPS). We also found that the level of CD63 was significantly decreased in Muted-deficient adrenal glands, suggesting that the Muted protein is important for the steady-state level of CD63. Our results suggest that CD63 is a membrane component of LDCVs and the stability of CD63 is dependent on the Muted protein, which provides a clue to the pathogenesis of LRO defects in HPS.
[1] Kim T, Gondré-Lewis MC, Arnaoutova I, Loh YP. Dense-core secretory granule biogenesis. Physiology (Bethesda) , 2006, 21(2): 124-133.
[2] Crivellato E, Nico B, Ribatti D. The chromaffin vesicle: advances in understanding the composition of a versatile, multifunctional secretoryorganelle. Anat Rec (Hoboken) , 2008, 291(12): 1587-1602.
[3] Hook V, Funkelstein L, Lu D, Bark S, Wegrzyn J, Hwang SR. Proteases for processing proneuropeptides into peptide neurotransmitters and hormones. Annu Rev Pharmacol Toxicol , 2008, 48: 393-423.
[4] Fei H, Grygoruk A, Brooks ES, Chen A, Krantz DE. Trafficking of vesicular neurotransmitter transporters. Traffic , 2008, 9(9): 1425-1436.
[5] Matsuoka H, Harada K, Nakamura J, Fukuda M, Inoue M. Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells. Cell Tissue Res , 2011, 344(1): 41-50.
[6] Chang CW, Hui EF, Bai JH, Bruns D, Chapman ER, Jackson MB. A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores. J Neurosci , 2015, 35(14): 5772-5780.
[7] Li TM, Lan WJ, Huang C, Zhang C, Liu XM. Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines. Hereditas (Beijing) , 2016, 38(5): 427-435. 李泰明, 蓝文俊, 黄灿, 张春, 刘晓玫. 近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定. 遗传, 2016, 38(5): 427-435.
[8] Mantegazza AR, Barrio MM, Moutel S, Bover L, Weck M, Brossart P, Teillaud JL, Mordoh J. CD63 tetraspanin slows down cell migration and translocates to the endosomallysosomal-MIICs route after extracellular stimuli in human immature dendritic cells. Blood , 2004, 104(4): 1183- 1190.
[9] Zhang Z, Li W. Formation and function of Weibel- Palade bodies. Hereditas (Beijing) , 2009, 31(9): 882-888. 张喆, 李巍. Weibel-Palade小体形成和功能研究进展. 遗传, 2009, 31(9): 882-888.
[10] Vischer UM, Wagner DD. CD63 is a component of Weibel-Palade bodies of human endothelial cells. Blood , 1993, 82(4): 1184-1191.
[11] Nishibori M, Cham B, McNicol A, Shalev A, Jain N, Gerrard JM. The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin. J Clin Invest , 1993, 91(4): 1775-1782.
[12] Pols MS, Klumperman J. Trafficking and function of the tetraspanin CD63. Exp Cell Res , 2009, 315(9): 1584-1592.
[13] Wei AH, Li W. Hermansky-Pudlak syndrome: pigmentary and non-pigmentary defects and their pathogenesis. Pigment Cell Melanoma Res , 2013, 26(2): 176-192.
[14] Hao ZH, Li W. Cellular functions of the lysosomal transport. Chin Bull Life Sci , 2010, 22(11): 1138-1146. 郝振华, 李巍. 内体—溶酶体运输及其细胞功能. 生命科学, 2010, 22(11): 1138-1146.
[15] Hao ZH, Wei LS, Feng YQ, Chen XW, Du W, Ma J, Zhou Z, Chen LY, Li W. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells. J Cell Sci , 2015, 128(7): 1365-1374.
[16] Zhang Q, Li W, Novak EK, Karim A, Mishra VS, Kingsmore SF, Roe BA, Suzuki T, Swank RT. The gene for the muted ( mu ) mouse, a model for Hermansky-Pudlak syndrome, defines a novel protein which regulates vesicle trafficking. Hum Mol Genet , 2002, 11(6): 697-706.
[17] Flannery AR, Czibener C, Andrews NW. Palmitoylation-dependent association with CD63 targets the Ca 2+ sensor synaptotagmin VII to lysosomes. J Cell Biol , 2010, 191(3): 599-613.
[18] van Niel G, Charrin S, Simoes S, Romao M, Rochin L, Saftig P, Marks MS, Rubinstein E, Raposo G. The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis. Dev Cell , 2011, 21(4): 708-721.
[19] Källquist L, Hansson M, Persson AM, Janssen H, Calafat J, Tapper H, Olsson I. The tetraspanin CD63 is involved in granule targeting of neutrophil elastase. Blood , 2008, 112(8): 3444-3454.
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