Phenylthiourea treatment allows quantitative characterization of individual melanosome motion
To understand the role of individual motors in organelle transport and how transport is regulated during aggregation versus dispersion, we combined tracking of individual melanosomes with quantitative analysis of movement. We compared the motion in wild-type, kinesin II dominant negative and myosin V dominant negative backgrounds.
It is impossible to follow individual melanosomes in regular melanophores because of the high spatial density of organelles (
A). To solve this problem, we reduced the number of pigment granules by treating cells with the tyrosinase inhibitor phenylthiourea (PTU). Because tyrosinase is a key enzyme in melanin biosynthesis, PTU inhibited formation of new melanosomes: after several passages in the presence of PTU, cells become completely devoid of pigment ( B). PTU is not toxic, and melanophores can proliferate indefinitely (≥100 passages) in its presence.
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 (more ...)
The effect of PTU is completely reversible, since removal of the inhibitor from the medium allows recovery of melanin synthesis and melanosome biogenesis. After 24–48 h without PTU, typical cells contain only ~100 melanosomes ( C). These melanosomes undergo normal aggregation and dispersion () and can be easily tracked by eye ( F) and automatically using image processing software. All the quantitative studies reported here examined motion in such cells.
Myosin V and kinesin II are required for pigment dispersion
Knowing that pigment dispersion requires both kinesin II and myosin V, we sought first to determine the role of these motors in the process. Since microtubules are relatively straight and radially oriented (
A) and actin filaments tend to be more randomly organized ( B), one would intuitively expect that in vivo motion driven by microtubule motors would be linear and radial, whereas motion driven by myosin V would appear less linear and more random. This appears to be the case; in general, trajectories of individual melanosomes are more linear in latrunculin-treated cells or cells expressing the myosin V dominant negative construct than in nocodazole-treated cells ()
. We confirmed that long linear tracks correspond to microtubule-based motion by tracking melanosome movement in cells microinjected with fluorescent tubulin and stimulated to disperse pigment 60 min after injection. Overlay of melanosomes trajectories over one of the microtubule images ( C) shows that long linear movements of organelles correspond to microtubule profiles, confirming that these movements are indeed microtubule based. As expected, outward linear trajectories are absent predominantly in the kinesin II dominant negative cells. Inward linear trajectories are observed in both the kinesin II and the myosin V dominant negative cells, suggesting that the use of the dominant negative constructs to inhibit kinesin II or myosin V activity does not impair cytoplasmic dynein.
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 (more ...)
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 (more ...)
We first determined how well the dominant negative constructs block the activity of their targets. In kinesin II/myosin V double dominant negative cells, most melanosomes are aggregated in the cell center. The few individual granules not in the aggregated “clump” also show dramatically reduced motion (unpublished data). Thus, the majority of outward motion is blocked by the combined dominant negative constructs. This is consistent with our previous characterization (Tuma et al., 1998
; Rogers et al., 1999
Because we can use nocodazole to depolymerize all the microtubules, it is possible to directly observe motion due entirely to myosin V activity ()
. Thus, we could directly quantify the magnitude of the myosin V dominant negative's effect: we tracked the motion of individual melanosomes and calculated the average distance from the point we started, tracking the melanosome as a function of time, that is, r2(t) (see Materials and methods for details). In nocodazole-treated cells, the r2(t) analysis indicates robust myosin V activity, but this activity completely disappears in myosin V dominant negative cells treated with nocodazole (). Thus, the dominant negative construct fully blocks myosin V function. In addition, the negligible residual r2(t) indicates both that nocodazole does indeed eliminate microtubule-based motion and that passive diffusion of melanosomes is insignificant. Identical results were obtained with a different dominant negative myosin V construct that encodes only the globular portion of myosin V tail and is thus incapable of dimerization (unpublished data). This result suggests that inhibition of myosin V motility by dominant negative constructs is due to competition of the truncated protein with the full-length myosin V for binding to organelles.
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 (more ...)
Regulation occurs by changes in dynein- and myosin-based transport
The ability to independently quantify the actin- or microtubule-based components of motion allowed us to investigate how the cell regulates motor activity resulting in net aggregation and dispersion. To quantify the microtubule component of motion, we used analysis software (Gross et al., 2000
) to resolve the long linear paths into individual “runs,” that is, periods of uninterrupted plus or minus end motion. This data was analyzed using two complementary approaches: the mean travel distance was determined using a modified averaging procedure ( and )
, whereas the properties of long runs were examined by fitting the distribution histogram (
and ; see Materials and methods). Initially, we used the dominant negative myosin V construct (Rogers et al., 1999
) to block myosin V activity and looked at the lengths and velocities of plus and minus end runs in cells treated with MSH or melatonin. Comparing these measurements, we found that the mean outward (plus end) run length is constant, regardless of the state of the cells (i.e., MSH versus melatonin stimulation) ( A and ), whereas the mean inward (minus end) run length changes significantly between MSH- and melatonin-treated cells ( A). Thus, one of the mechanisms that cells use to shift transport from net aggregation to net dispersion is to alter the length of minus end (dynein-powered) runs, while keeping plus end runs constant.
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 (more ...)
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 (more ...)
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 (more ...)
Physical parameters of melanophore motion
To quantify the regulation of myosin V–based motion, we used the r2(t) statistic, which measures the average travel distance of a melanosome from the point at which we started tracking it. We depolymerized microtubules with nocodazole, stimulated cells with melatonin or MSH, and computed r2(t) from tracking many melanosomes chosen at random in each class of cells (). Myosin V–driven motion is decreased ~50% in melatonin- versus MSH-treated cells as judged by comparing the slope of r2(t) under these two conditions (). Thus, a second component of the switch from dispersion to aggregation includes a down-regulation of myosin V–driven motion.
Other work (Reese and Haimo, 2000
) suggested that the change from net aggregation to net dispersion resulted from changes in the ability of both kinesin II and dynein to interact with microtubules. Their biochemical analysis found that during dispersion, binding of dynein to microtubules was somewhat decreased after 15 min of treatment with MSH and completely lost after 60 min with the hormone. We compared the microtubule component of motion in cells 15 and 60 min after addition of MSH and found minus end movements are present even after 60 min in MSH. Furthermore, the length of both plus end and minus end runs increased at 60 min so the minus end motion clearly was not impaired ( A), whereas the mean travel velocity did not change. Because melanosomes show bidirectional motion even 60 min after the cells receive the dispersion or aggregation signal, the data of (Reese and Haimo, 2000
) are inconsistent with our observations.
There is a formal possibility that although the motion of individual melanosomes appears similar, the number of plus or minus moving organelles is different. To investigate this possibility, we identified all melanosomes that appeared moving on microtubule tracks in a given cell in a 30-s period, 15 and 60 min after addition of MSH. At 15 min, we observed that 53 ± 10% (four cells; mean ± SEM) of the moving melanosomes included a minus end run, whereas at 60 min 75 ± 4% (four cells; mean ± SEM) of the moving melanosomes included a minus end run. Thus, there is no evidence of a decrease in dynein activity as a function of time in dispersing cells.
Finally, in biochemical experiments directly repeating the protocol published by Reese and Haimo (2000)
we were unable to reproduce the change in microtubule binding. In our experiments, the amount of kinesin II and cytoplasmic dynein that binds to microtubules in the presence of AMP-PNP did not change after 60 min treatment with melatonin or MSH (
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 (more ...)
Regulation of myosin V involves changes in the number of cargo-bound motors
The tracking experiments demonstrate that regulation alters both the dynein-driven component of motion and the myosin V component of motion. To determine if these changes are due to an alteration of the number of motors bound to the cargo, we measured the amount of melanosome-bound motors on purified melanosomes using quantitative Western blot analysis. We found no change in the overall amount of microtubule motors bound to melanosomes purified from dispersing versus aggregating cells ( A). Thus, for the microtubule motors alteration of motor cargo binding does not appear to play a significant role in regulation of net transport. This is consistent with previously published work (Reese and Haimo, 2000
However, the situation is clearly different in the case of myosin V. Melanosomes from aggregating cells have substantially less myosin V than melanosomes from dispersing cells ( A, M-V). We quantified the number of molecules of myosin V on the surface of melanosomes from aggregating and dispersing cells, using Western blotting with a recombinant fragment of myosin V as a standard. This procedure yields an estimate of 65 ± 10 and 88 ± 7 molecules of myosin V per melanosome in aggregating and dispersing cells, respectively. This 35% change is comparable to the decrease in myosin V–based movement (); therefore, an attractive model is that the decrease in myosin V–based motion is the result of a change in melanosome-bound myosin V (see Discussion).
Interactions between the microtubule and actin transport systems
How does myosin V alter microtubule-based transport? To understand interactions between the two systems during melanosome transport, we studied how the function of one system is altered by the inactivation of the other. Inactivation of myosin V alters microtubule-based motion: the travel velocity along microtubules in each direction is increased ( B) as is the mean length of minus end runs ( A).
Because a distribution's mean is an incomplete description of the underlying process, we determined how the loss of myosin V activity altered the distribution of microtubule-based run lengths. The length of runs was well described in all cases by the sum of two decaying exponentials (, reduced chi-squared values, and ), suggesting that there are two classes of motion, that is, short runs and long runs. From the fit, we determine distance constants characterizing both types of runs (DS and DL) and the relative proportion of short versus long runs (NSL). DL provides an estimate for the average length of long runs (). During dispersion, impairment of myosin V activity due to latrunculin or the myosin V dominant negative construct resulted in an increase in DL ( and ) and an increase in NSL. The effect was much greater on minus end motion than plus end motion (). DS remained approximately constant (unpublished data). Thus, during dispersion myosin V has two opposite effects: it improves microtubule-based transport by suppressing short runs but also impairs microtubule transport by shortening long runs.
Further comparing motion in the wild-type and myosin V dominant negative cells, we observed that in wild-type cells there appear to be fewer melanosomes that remained stationary. To quantify this effect, we tracked ≥80 randomly chosen melanosomes in three different backgrounds: wild-type, myosin V dominant negative, and latrunculin-treated cells. We operationally defined as stationary any melanosome that did not move at least 500 nm away from the starting point at least once over the course of 33 s of observation (this corresponds to an average velocity of <15 nm/s). In the wild-type cells, only 6.8% of the melanosomes fall into the stationary category compared with 17.1% in a latrunculin-treated cells and 21.6% in myosin V dominant negative cells.
However, inactivation of myosin V activity either by latrunculin or the myosin V construct resulted in a subset of the microtubule-driven motion that was particularly effective. In the wild-type (88 granules tracked), there were no displacements of more than 4,000 nm over the course of 33 s (0%), whereas there were 5 such events (out of 97) in the myosin V dominant negative background (5.1%) and 11 such events (out of 82) in the latrunculin background (13.4%).
To find if the interaction between the two systems is regulated, we compared the microtubule-based motion in control cells (aggregating and dispersing) and myosin V dominant negative cells (aggregating and dispersing). In aggregating cells, the microtubule-based motion is not altered by the myosin V inactivation (, and ) in contrast to the observed effect during dispersion (, ). Thus, cells can regulate the extent to which myosin V interferes with the microtubule-based motion.
Interactions between the microtubule motors
How does kinesin II alter dynein-based motion? To investigate, we compared minus end motion in wild-type and kinesin II dominant negative cells. We find no difference in travel velocity in wild-type versus the kinesin II dominant negative background ( B); however, loss of kinesin II activity does result in an increase in the mean minus end travel distance ( A). Further work remains to more fully understand the effects of disabling kinesin II on the minus end motion.