Proc Natl Acad Sci U S A
April 15, 1997;
Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro.
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.
Proc Natl Acad Sci U S A
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Figure 1 (A) Electron micrograph of a thin-sectioned pellet of purified melanosomes. The purified melanosome fractions are uncontaminated by other organelles. (Bar = 0.5 μm.) (Inset) A melanosome at higher magnification showing bilayer membrane encapsulating the electron-dense melanin core. (Bar = 0.1 μm.) (B) A Coomassie-stained polyacrylamide gel comparing melanophore extract (lane 1) with purified melanosomes (lane 2). The positions of the molecular weight markers are indicated to the right of lane 2.
Figure 2 Bidirectional motility of a single melanosome along an axoneme-nucleated microtubule observed by video-enhanced DIC microscopy. Portions of a video sequence showing (A) minus end-directed motility and (B) plus end-directed motility of the same melanosome (arrowheads). The elapsed time between frames is 2 s. (Bar = 6 μm.)
Figure 4 Comparison of the movement of crude (shaded bars) and Percoll-purified melanosomes (open bars). The samples were assayed on a continuous carpet of microtubules, and the total number of motile melanosomes per field was tallied over 10-min periods. At least three different samples were assayed per preparation. The results for each treatment were averaged, and both results were normalized for melanosome concentration using the OD550 of each sample. Results of three individual experiments are shown.
Figure 5 Microtubule motors in melanophores and on melanosomes. Lanes 1–10 show immunoblots probed with antibodies against motor proteins. Kinesin was probed with polyclonal HD-kin5 in cell extract (lane 1) and melanosomes (lane 2). Kinesin heavy chain is marked with “K.” Kinesin-related proteins were detected with the pan-kinesin antibody HIPYR in cell extract (lane 3), melanosomes (lane 4), and proteins that cosediment with microtubules in the presence of AMP-PNP (lane 5) or ATP (lane 6). The 85- and 95-kDa subunits of kinesin-II are indicated. Kinesin-II was probed with the monoclonal K2.4 in cell extract (lane 7) and melanosomes (lane 8). The position of the 85-kDa chain is indicated. Cytoplasmic dynein was detected with the monoclonal 74.1 against dynein intermediate chain in cell extract (lane 9) and melanosomes (lane 10). The position of the 83-kDa intermediate chain is indicated with “D.” The positions of the molecular weight standards are marked to the right of each blot.
Cell cycle control of microtubule-based membrane transport and tubule formation in vitro.