Gross SP , Tuma MC , Deacon SW , Serpinskaya AS , Reilein AR , Gelfand VI .

GM-52111 NIGMS NIH HHS , R01 GM052111 NIGMS NIH HHS

	Figure 1. Tracking organelles in cells with a limited number of melanosomes. (A) Cultured Xenopus melanophore. The large number of melanosomes makes tracking individual organelles impossible. (B) Cell after 4 wk of culture in 1 mM PTU. No melanosomes are seen in the cytoplasm. (C) Melanophore after 24 h recovery from PTU has a small number of melanosomes. (D and E) Melanophore after 24 h recovery from PTU before (D) and 1000 s after (E) addition of MSH, showing that the melanosomes in PTU-treated cells disperse in response to MSH. (F) Sequence of images taken during pigment dispersion of the cell shown in D and E. Arrows show positions of two melanosomes in subsequent frames. Time after addition of MSH (in seconds) is shown on each frame. Bright-field microscopy (A and D–F); phase–contrast microscopy (B and C). Bars: (A–E), 10 μm; (F) 2.5 μm.
	Figure 2. Distribution of microtubules and actin filaments in Xenopus melanophores. (A) Microtubules stained by a tubulin antibody. Note radial pattern of distribution. (B) Actin filament distribution as revealed by rhodamine-phalloidin staining. In addition to thin bundles, most of the staining is distributed diffusely. Since rhodamine-phalloidin does not bind nonpolymerized actin, diffuse staining suggests random arrangement of actin filaments. Bar, 20 μm.
	Figure 3. Linear melanosome tracks correlate with the position of microtubules. Movement of melanosomes in cells transfected with dominant negative myosin V (A) or treated with nocodazole (B). Note that in A melanosomes move along linear tracks, whereas movement in B is more random. Position of melanosomes was registered at 30 Hz, but for clarity only every sixth frame is displayed. Bar, 1 μm. (C) Overlay of melanosome tracks and fluorescently labeled microtubules. Microtubules were labeled by injecting cells with Alexa 488 tubulin. Movement of melanosomes was tracked using a series of bright-field images taken every 3 s. The resulting tracks were overlayed onto a fluorescent image showing the microtubule distribution. Note that linear movements correlate with the position of microtubules. The track labeled with an asterisk does not exactly overlap with the image of the underlying microtubule because this microtubule moved substantially in the course of the tracking. Bar, 5 μm.
	Figure 4. Quantification of displacement generated by myosin V. The r2(t) plots show the square of the average displacement of the pigment granules as a function of time. Each curve represents an average of ∼100 plots for individual moving granules (∼25 melanosomes chosen at random in four different cells). The indicated error bars are the standard error of the mean. Nocodazole was used to depolymerize microtubules in order to quantify myosin V (M-V)–driven motion. The decrease in motion in aggregating versus dispersing cells indicates that myosin V–driven motion is down-regulated as part of the dispersion to aggregation transition. The lowest curve shows that in the absence of microtubules expression of the myosin V dominant negative construct completely eliminates motion, demonstrating the efficacy of the construct. The magnitude of dispersion in a wild-type background driven by kinesin II, dynein, and myosin V (top curve) is similar to dispersion driven only by myosin V (second curve).
	Figure 5. Regulation of microtubule-based motion. To investigate regulation of microtubule motors, their activity was studied in the absence of functional myosin V. Shown are (modified) averages of travel distances (as described in Materials and methods) and velocities of plus and minus end runs. A run was defined as a period of uninterrupted motion along the microtubule (as described in Materials and methods). (A) The change from dispersion to aggregation coincides with a change in the length of (long-type) minus end runs, whereas the length of plus end runs remains constant. (B) The average velocity of (short-type) plus and minus end runs. There is no significant change in run velocity in either direction due to the change from dispersion to aggregation. These data show an average of ∼400 individual runs from four or more cells. M-V, myosin V. The error bars are the standard error of the mean.
	Figure 6. Microtubule motion as a function of time and as altered by the expression of the dominant negative constructs. Average travel distances (modified as described in Materials and methods) and velocities were measured during dispersion (A and B) and aggregation (C and D). Run lengths, dispersion (A); velocities, dispersion (B); run lengths, aggregation (C); velocities, aggregation (D). The myosin V and kinesin II constructs both alter the length of minus end runs (A), but myosin V does not alter the length of plus end runs. Blocking myosin V function results in increased velocities in both plus and minus end travel (B), but decreasing kinesin II function does not alter minus end velocities (B). During aggregation, blocking myosin V activity has no effect on either run lengths (C) or velocities (D). M-V, myosin V. Error bars are the standard error of the mean.
	Figure 7. Distribution of run lengths for minus end motion in wild-type (A) and myosin V (B) cells stimulated with MSH. Individual run lengths were determined as described in Materials and methods. In all cases examined, the distribution was well described by the sum of two decaying exponentials (Table I). The original histogram (bars) together with the double-decaying exponential fit (solid line) is shown in the inserts. Note that there are relatively more long runs in the myosin V cells (B) than in wild-type cells (A). The full graphs show the natural logarithm of the number of runs versus run length. The solid line is the complete fit, whereas the dotted and dashed lines indicate the contributions of the short and long runs, respectively. The long runs (tail of the distribution) are significantly altered due to myosin V activity, but the short runs are not (compare the slope of the long-runs line in A and B).
	Figure 8. The ability of motors to interact with melanosomes and microtubules does not change between aggregation and dispersion. (A) Melanosomes were purified from cells treated with the aggregating stimulus, melatonin, or the dispersing stimulus, MSH, and Western blots were performed with antibodies against myosin V (M-V), kinesin II (K-II), and cytoplasmic dynein (Dy). Note that the amount of microtubule motors on melanosomes does not change due to aggregation (A) or dispersion (D), whereas the amount of myosin V is higher on melanosomes purified from cells dispersing the pigment than from cells aggregating the pigment. (B) Binding of kinesin II (K-II) or cytoplasmic dynein (Dy) to microtubules in the presence of AMP-PNP. Bovine brain microtubules were added to extracts from cells aggregating (A) or dispersing (D) pigment, the mixture was spun through a glycerol cushion, and the pellets were analyzed for the presence of motors by Western blotting (experimental protocol from Reese and Haimo (2000).
	Figure 9. Model for transport of melanosomes by actin- and microtubule-based motors. A pigment organelle can be moved in three different ways: along the microtubule by kinesin II (A and D) or dynein (B and E), or along the actin filaments by myosin V (C and F). Direction of movement is shown by double arrows. Although at any moment motion is dominated by one of the two transport systems (i.e., microtubules or actin), the motors of the other transport system are transiently active and interacting with their substrate (i.e., actin or microtubules). During dispersion, these transient interactions (indicated by T) are significant and allow myosin V to reduce the velocity of microtubule-based transport (A and B). The interactions also play a role in the switch between the two transport systems, that is, between B and C. Because myosin V activity predominantly decreases the length of minus end microtubule-based motion, we hypothesize that the switch from microtubule- to actin-based transport occurs only between state B and C (from dynein movement to myosin V movement) and that transitions from kinesin II movement (A) to myosin V movement (C) are rare. Because kinesin II but not dynein appears to win in tug-of-wars with myosin V, we suggest that the C to A transition is possible but not the C to B transition; however, we have no direct evidence on this point. During aggregation, myosin V activity is decreased, which results in reduced weak transient interactions (indicated by weak T) in D and E, and myosin V is no longer able to interfere with microtubule-based motion. Due to the weakness of the interactions, any time the microtubule motors are in contact with the microtubules they win the tug-of-war with myosin V and there is a transfer (F to D or F to E). Similarly, the reverse transfer from microtubules to actin-based transport (D to F or E to F) does not occur. M-V, myosin V; K-II, kinesin II; Dy, dynein; Ms,melanosome.

Bridgman, Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex. 1999, Pubmed

Bridgman, Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex. 1999, Pubmed
Brown, Cooperation between microtubule- and actin-based motor proteins. 1999, Pubmed
Cole, Novel heterotrimeric kinesin-related protein purified from sea urchin eggs. 1993, Pubmed , Xenbase
Daniolos, Action of light on frog pigment cells in culture. 1990, Pubmed , Xenbase
Evan, Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. 1985, Pubmed , Xenbase
Gross, Dynein-mediated cargo transport in vivo. A switch controls travel distance. 2000, Pubmed
Hancock, Processivity of the motor protein kinesin requires two heads. 1998, Pubmed
Hirschberg, Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells. 1998, Pubmed
Huang, Direct interaction of microtubule- and actin-based transport motors. 1999, Pubmed
Karcher, Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II. 2001, Pubmed , Xenbase
Keating, Microtubule release from the centrosome. 1997, Pubmed
Krendel, Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules. 1998, Pubmed
Kuznetsov, Actin-dependent organelle movement in squid axoplasm. 1992, Pubmed
Langford, Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. 1995, Pubmed
Maples, Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells. 1997, Pubmed
Mermall, Transport of cytoplasmic particles catalysed by an unconventional myosin in living Drosophila embryos. 1994, Pubmed
Morris, Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. 1995, Pubmed
Reese, Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport. 2000, Pubmed , Xenbase
Rodionov, Functional coordination of microtubule-based and actin-based motility in melanophores. 1998, Pubmed
Rogers, Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. 1997, Pubmed , Xenbase
Rogers, In vitro motility assay for melanophore pigment organelles. 1998, Pubmed , Xenbase
Rogers, Myosin cooperates with microtubule motors during organelle transport in melanophores. 1998, Pubmed , Xenbase
Rogers, Regulation of melanosome movement in the cell cycle by reversible association with myosin V. 1999, Pubmed , Xenbase
Tabb, Transport of ER vesicles on actin filaments in neurons by myosin V. 1998, Pubmed
Tuma, Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores. 1998, Pubmed , Xenbase
Welte, Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics. 1998, Pubmed
Wu, Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function In vivo. 1998, Pubmed
Wu, Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor. 1997, Pubmed