Abstract
SummaryIn animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11].
Highlights
Melanocytes adopted a uniform circular shape determined by the micropattern, with the nucleus positioned near the center and the melanosomes distributed throughout the surrounding cytoplasm
We report melanosome distribution in standardized cells in two ways that convey complementary information about the results of our experiments: (1) the average pigment distribution map and radial pigment profile for each population of cells (e.g., Figures 1A and 1B) and (2) pigment dispersion distance (PDD) for each cell within a population (e.g., Figure 1C)
Confocal immunofluorescence microscopy (CIFM) analysis using alpha-tubulin-specific antibodies confirmed the efficacy of our nocodazole treatment in depleting microtubules in melanocytes (Figure S1C available online)
Summary
Comparison of nocodazole versus solvent-treated melan-a cells indicated that microtubule depletion had little effect on pigment distribution (mean PDD; DMSO = 19.94 6 0.6940 mm versus nocodazole = 19.18 6 0.8312 mm; Figures 1A–1C). This revealed that microtubule depletion significantly reduced jasplakinolide-triggered clustering compared with control cells (mean PDD; Noc/JK = 17.86 6 1.908 mm versus DMSO/JK = 14.16 6 2.034 mm; Figures 2Ai and 2Aii).
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