Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro

被引:187
作者
Rogers, SL [1 ]
Tint, IS [1 ]
Fanapour, PC [1 ]
Gelfand, VI [1 ]
机构
[1] UNIV ILLINOIS, DEPT CELL & STRUCT BIOL, CHEM & LIFE SCI LAB B107, URBANA, IL 61801 USA
关键词
D O I
10.1073/pnas.94.8.3720
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Although many types of membrane-bound organelles rely upon microtubule-based transport for their proper placement within the cytoplasm, the molecular mechanisms that regulate intracellular motility remain largely unknown. To address this problem, we have studied the microtubule-dependent dispersion and aggregation of pigment granules from an immortalized Xenopus melanophore cell line. We have reconstituted pigment granule motility along bovine brain microtubules in vitro using a microscope-based motility assay. Pigment granules, or melanosomes, move along single microtubules bidirectionally; however, analysis of the polarities of this movement shows that melanosomes that have been purified from dispersed cells exhibit mostly plus end-directed motility, while movement of organelles from aggregating cells is biased toward the minus end. Removal of all soluble proteins from the melanosome fractions by density gradient centrifugation does not diminish organelle motility, demonstrating that all the components required for transport have a stable association with the melanosome membranes. Western blotting shows the presence of the plus end-directed motor, kinesin-II, and the minus end-directed motor, cytoplasmic dynein in highly purified melanosomes. Therefore, purified melanosomes retain their ability to move along microtubules as well as their regulated state. Direct biochemical comparison of melanosomes from aggregated and dispersed cells may elucidate the molecular mechanisms that regulate organelle transport in melanophores.
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页码:3720 / 3725
页数:6
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