The Orientation of Actin Filaments within Dendrites Is Essentially Random
Light immunofluorescence micrographs of melan-a melanocytes (D
) stained with fluorescent phalloidin (Fig. , C
) typically reveal sparse staining in the central cytoplasm, intense edge staining along the length of the dendrite, and prominent staining at dendritic tips (see also Lacour et al., 1992
; Wu et al., 1997
). Confocal microscopy revealed that the edge staining in dendrites is due to a cortical shell of F-actin under the plasma membrane (data not shown). To determine the orientation of actin filaments within this cortical shell, melan-a melanocytes were permeabilized with detergent, labeled with rabbit skeletal muscle myosin subfragment 1, fixed, processed for transmission electron microscopy, and sectioned such that the cell body and as much of the dendritic extension as possible were present in a single section (Fig. A
). As in the light micrographs, the electron micrographs show that actin filaments are scarce in the central cytoplasm and positioned in dendrites almost exclusively just under the plasma membrane, forming a cortical shell that surrounds a microtubule-rich, actin-poor central zone. Actin polarity in dendrites was scored in a series of 1-μm-wide cross-sectional bands that spanned the short axis of the dendrite and that ranged from the base of the dendrite to where the dendrite left the plane of the section. A total of 28 such bands from seven dendrites, each from a different melanocyte, and each spanning a distance of between 20–30 μm from the nucleus, were scored. Decorated filaments were grouped into one of three categories, as described in the schematic in Fig. B
. Those in which the arrowhead pointed within a 90° arc whose center points directly away from the nucleus were scored as pointed end–out. Those in which the arrowhead pointed within a 90° arc whose center points directly towards the nucleus were scored as barbed end–out. Those that fell into the two remaining 90° arcs were scored as “other.” Three main points summarize what was found. First, the number of barbed end–out filaments per 1-μm cross-sectional band (4 ± 2.3) was not significantly different from the number of pointed end–out filaments (3.3 ± 2.2) (n
= 28). Second, in several instances where the number of barbed end–out filaments exceeded pointed end–out filaments by a factor of greater than 3:1, the filaments in the adjacent 1-μm cross-sectional areas always exhibited an inverse ratio (data not shown). Third, of the 367 filaments scored, 112 (31%) were barbed end–out, 92 (25%) were pointed end–out, and 163 (44%) fell into the “other” category. These three results indicate that actin filament orientation in dendrites is essentially random. Using other thin sections that contained a total of 19 melanosome-rich dendritic tips, we also found that the number of barbed end–out filaments per tip (5.3 ± 3.5) was not significantly different from the number of pointed end–out filaments per tip (4.2 ± 2.9) (n
= 19). Of 227 filaments scored in tips, 100 (44%) were barbed end–out, 80 (35%) were pointed end–out, and 47 (21%) were “other.” Therefore, as in the dendrite proper, the actin in dendritic tips exhibits a largely random orientation. Together, these results indicate that if myosin Va is responsible for the long-range centrifugal transport of melanosomes, it would be via a mechanism wherein random walks of melanosomes catalyzed by myosin Va on actin filaments of random orientation drive the outward spreading or “facilitated diffusion” of the organelle.
Figure 2 Orientation of F-actin within melanocyte dendrites. (A) Low-magnification transmission electron micrograph of the dendrite of a melan-a melanocyte labeled with subfragment 1. (B) Schematic indicating how F-actin orientation was scored in dendrites. (more ...)
Melanosomes in Dilute Melanocytes Move Rapidly and Bidirectionally between the Cell Center and the Periphery
In contrast to the mechanism described above, melanosomes in primary dilute
null melanocytes were found to be undergoing rapid, bidirectional translocation between the cell center and the periphery. Fig. A
shows a still image of one such melanocyte (dl20J
) possessing a broad, well-spread cell extension projecting to the upper right. As is typical of heavily melanized dilute
melanocytes (Wei et al., 1997
), the majority of melanosomes are perinuclear, although some are clearly present in the broad flat cell extension. Time lapse video microscopy revealed that many of these latter melanosomes are moving rapidly (>1 μm/s) and bidirectionally between the cell center and the periphery. Two examples of these melanosome movements, which are very obvious when viewed at ~15–30 times real speed, are shown in successive video stills (1-s time intervals) in Fig. , B1–B10
(centrifugal) and C1–C10
show the paths (1-s time intervals) for some of the melanosomes whose centrifugal (D
) and centripetal (E
) movements within the boxed area in A
were obvious to the eye over a 3-min time span (see video segment No. 1).2
While not all melanosomes traverse the entire cell extension in one continuous movement, some do (for example, see melanosome numbers 5
, respectively). Such long-range persistent movements are even more obvious in the lightly melanized mutant cell shown in Fig. F
, where melanosome numbers 2–4
, closed circles
) move from near the nucleus to near the margin of the cell without significant pause, while melanosome numbers 7
, open circles
) move from near the cell margin to near the nucleus with only a few short pauses. In typical fashion, essentially all of the melanosomes in the periphery of this cell are undergoing rapid bidirectional movement (or are temporarily pausing; see video segment No. 2), and individual melanosomes can be seen to move by this fast component in rapid succession from the center to the periphery and back again. One of these melanosomes is shown in successive video stills in Fig. , I1–I11
, while H
shows the paths for four different melanosomes undergoing such bidirectional movements. Fig. , J–L
, shows that this bidirectional melanosome movement is also evident within longer cell extensions that are more typical of melanocyte dendrites in vivo (see video segment No. 3). Finally, Fig. , M
, shows very striking bidirectional melanosome movements within a large dendritic extension emanating from a mutant cell (see video segment No. 4). These movements are notable in that they persist for long distances (melanosome 1
, 48 μm; melanosome 6
, 24 μm).
Figure 3 Rapid, bidirectional melanosome translocation in the periphery of dilute melanocytes. (A) A typical dilute null melanocyte in primary culture. (B1–B10 and C1–C10) Successive video stills (1-s intervals) of typical centrifugal (B1–B10 (more ...)
From analysis of these and other dilute melanocytes, we obtained the following information regarding this bidirectional component of melanosome motility. First, individual melanosomes move on average 13.3 ± 6.9 μm (n = 34) and 11.9 ± 8.5 μm (n = 34) between stops for centrifugal and centripetal movements,3 respectively (Table ). Second, stops may be short pauses on the order of seconds or longer pauses lasting for more than a minute, and pauses may or may not be associated with reversals in direction. Third, melanosomes that reverse direction sometimes appear to follow the same track in reverse (for example, see the path in the upper left corner of the Fig. H). Fourth, while long (i.e., 20–30 μm) movements can occur without significant pauses, it is more common to see such movements interrupted by several pauses lasting on the order of a few seconds. Fifth, the average and maximum speeds of melanosomes undergoing this movement are 1.04 ± 0.29 and 1.67 ± 0.64 μm/s (n = 34), respectively, for centrifugal movements, and 1.13 ± 0.35 and 1.86 ± 0.67 μm/s (n = 34), respectively, for centripetal movements (Table ). These values for centrifugal and centripetal movements are not significantly different. Finally, while we did not attempt to quantitate the numbers of centrifugal and centripetal movements per unit time, their frequencies appear to be essentially the same. Indeed, they must be, given the phenotype of dilute null melanocytes, where net accumulation of melanosomes in the cell's periphery does not occur. Therefore, while this component of melanosome motility provides an efficient means to move melanosomes to the periphery, it does not by itself generate the net peripheral accumulation of melanosomes characteristic of wild-type melanocytes.
The Bidirectional Translocation of Melanosomes in the Periphery of Dilute Melanocytes Is Microtubule Dependent
The fact that the component of melanosome motility described above runs parallel to the long axis of cell extensions, exhibits linear trajectories often exceeding 15 μm in length without pausing, and possesses speeds characteristic of microtubule-dependent motor proteins (Hirokawa, 1998
), together with the fact that in melanocytes stained with an α tubulin antibody, microtubules were found to run the length of dendritic extensions (Fig. B
), stopping just shy of the dendritic tip (Fig. , F
), all suggest that this component of melanosome motility is microtubule dependent. To verify this, melanosome dynamics within dilute
melanocytes were recorded before and after the addition of nocodazole. Fig. B
shows the paths for a portion of the fast centrifugal (left
) and centripetal (right
) melanosome movements that occurred over a 2-min time span within the dendritic extension of the dilute
melanocyte in A
just before the addition of nocodazole. C
shows this same cell 5 min after the addition of 16 μM nocodazole to the media (37°C), while D
shows the paths for every melanosome in this dendrite over the ensuing 1-min time interval (see video sequences Nos. 5A and 5B). Clearly, most if not all fast melanosome movements have disappeared (the slow displacements of a few melanosomes are due to local contractions of the cytoplasm). Occasional fast movements that were sometimes seen in dendrites of other similarly treated melanocytes completely disappeared when nocodazole treatment was supplemented with a 30-min incubation at 4°C, and fast bidirectional melanosome movements resumed when cells were returned to 37°C in the absence of nocodazole (see below). These observations, together with immunofluorescence staining of melanocytes incubated at 37°C in 16 μM nocodazole for 5 min, which indicated that this treatment largely eliminated microtubules from the periphery of melanocytes (data not shown), support the conclusion that the fast bidirectional component of melanosome movement is microtubule dependent. In addition, the nearly complete abrogation of this movement within cell extensions produced by nocodazole addition alone (i.e., without a 4°C incubation) suggests that microtubules within dendritic extensions are highly dynamic.
Figure 4 Microtubule dependence of the fast peripheral and central melanosome movements. (A) A dilute melanocyte before the addition of nocodazole. (B) Some of the fast centrifugal (left) and centripetal (right) movements that occurred over a 2-min period within (more ...)
Melanosomes in the Center of Dilute Melanocytes Are Also Undergoing Rapid, Microtubule-dependent Movements
Time lapse video microscopy of untreated and nocodazole-treated dilute melanocytes revealed that essentially all of the melanosomes in the central cytoplasm of these cells are also moving rapidly on microtubules. Fig. E shows a still image of the same lightly melanized dilute melanocyte described in Fig. , F–I, while Fig. F shows the paths for a portion of the fast melanosome movements seen in the central cytoplasm of this cell (boxed area in E) over a 2-min time span before the addition of 16 μM nocodazole. These fast movements exhibit nearly the same speed as fast movements in the periphery (1.05 ± 0.44 μm/s, n = 15 from this cell), but differ in that their average excursions are much shorter (3.1 ± 1.3 μm, n = 15 from this cell). G shows this same cell after incubation at 37°C for 10 min, at 4°C for 30 min, and at 37°C for 10 min, all in the presence of 16 μM nocodazole, while H shows the paths for all melanosomes in the boxed area over the ensuing 1 min. Clearly, most if not all of the short, fast movements have disappeared (see video segments Nos. 6A and 6B). We also found that these central, short-range movements reappeared first upon nocodazole washout, with peripheral movements following some time later once cell extensions have reformed. This latter point is demonstrated in I and J, which show this same cell 30 min after nocodazole wash out (see video segment No. 7). We conclude, therefore, that the majority of melanosomes in dilute melanocytes are undergoing microtubule-dependent translocation, with most moving on the large number of short microtubules (see Fig. ) that terminate in the central cytoplasm, while the rest ride on the smaller number of long microtubules that extend to the periphery of the cell.
Bidirectional Microtubule-dependent Melanosome Traffic Also Exists in Wild-Type Melanocytes but Does Not Involve Melanosomes Concentrated along the Edge or at the Tip of Dendrites
Fig. A shows a primary wild-type melanocyte, which exhibits the typical bias in peripheral distribution of melanosomes characteristic of myosin Va+ melanocytes, while B shows a portion of the fast centrifugal and centripetal melanosome movements that occurred within this cell over a 2-min time span. As in many wild-type melanocytes, these fast movements, which in well-spread cells like this one are most evident in the region between the nucleus and the melanosome-rich periphery, are considerably less obvious than in dilute melanocytes (see video sequence No. 8). This appears to be due in part to the fact that the peripheral cytoplasm of wild-type melanocytes, unlike dilute melanocytes, contains significant numbers of melanosomes that are not undergoing this fast movement and that block the view of those that are. Striking bidirectional melanosome movements in wild-type melanocytes are not uncommon, however, especially within dendritic extensions. D and G show the paths for a portion of the fast centrifugal (closed circles) and centripetal (open circles) melanosome movements that occurred over a 3-min time span within the boxed area of the dendritic extensions in C and F, respectively. In addition to the melanosomes that are undergoing these fast movements (which occur mostly in the central, microtubule-rich zone of the dendrite), wild-type cells possess large numbers of melanosomes that are at the tip (C) and along the edge (F) of their dendrites and that are undergoing slow, local motions (see video sequences Nos. 9 and 10 and Figs. and ). Importantly, while microtubule-dependent melanosome movements in these actin-rich zones are rare, melanosomes can be removed from the tip (E) and lateral edge (H) of the dendrite by the fast component, and new melanosomes can be delivered to the tip (E) and lateral edge (H) by the fast component. The extent to which the balance of these dynamics can change the degree of peripheral accumulation of melanosomes was evident in several instances where a surfeit of centrifugal or centripetal movements led to relatively rapid (on the order of minutes) increases or decreases, respectively, in the degree of peripherally accumulated melanosomes. For example, just before the capture of images used for the data in F–H, a surfeit of centrifugal melanosome movements spanning a period of ~10 min lead to an approximate doubling of the number of melanosomes at the tip (I and J).
Figure 5 Rapid, bidirectional melanosome translocation in wild-type melanocytes. (A) A well-spread wild-type melanocyte. (B) The paths for a portion of the fast centrifugal and centripetal (arrowheads) movements that occurred over a 2-min span within the boxed (more ...)
Figure 9 The dynamics of melanosomes in microtubule-depleted wild-type and dilute null melanocytes. (A and C) Microtubule-depleted wild-type and dilute null melanocytes, respectively. (B and D) The paths for all of the melanosomes present within the boxed (more ...)
Figure 10 Histograms of the speeds of melanosomes in microtubule-depleted wild-type and dilute melanocytes. Speeds for wild-type (filled bars) and dilute (hashed bars) melanocytes were binned in 0.025-μm/s increments (except for the first bin, which represents (more ...)
From analysis of these and other wild-type melanocytes (e.g., melan-a cells, which are essentially indistinguishable from primary wild-type melanocytes), we obtained the following information regarding this fast component in the context of wild type cells. First, as in dilute melanocytes, it is dependent on the presence of intact microtubules (see below). Second, the average and maximum speeds of melanosomes undergoing these movements in wild-type cells are 0.68 ± 0.17 and 0.95 ± 0.41 μm/s (n = 38), respectively, for centrifugal movements, and 0.76 ± 0.19 and 1.03 ± 0.33 μm/s (n = 34), respectively, for centripetal movements (Table ). While these values for centrifugal and centripetal movements are not significantly different from each other, they are all significantly slower than the values for dilute melanocytes (P < 0.001). Third, individual melanosomes move on average 4.6 ± 2.5 μm (n = 38) and 5.4 ± 2.7 μm (n = 34) between stops for centrifugal and centripetal movements, respectively (Table ). While these values are not significantly different from each other, they are both significantly shorter than the values obtained for dilute melanocytes (P < 0.001). These differences may be due to a myosin Va–dependent damping of both the rate and persistence of microtubule-dependent melanosome movements in vivo (see Discussion).
Expression of the Myosin Va Tail Domain in a Wild-Type Background Creates a Dilute-like Phenotype
The fact that the fast, bidirectional, microtubule-dependent component of melanosome transport identified above can move melanosomes to the periphery efficiently but cannot concentrate them there, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggested a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in peripheral regions of the cell prevents these organelles from returning to the cell center via the centripetal aspect of this fast component, thereby causing their peripheral accumulation. To test this “capture” model, we expressed the myosin Va tail domain within wild-type melanocytes. If, as conjectured (Mooseker and Cheney, 1996
), this portion of the myosin Va molecule contains the site that mediates myosin Va/ melanosome interaction, then it should compete with endogenous myosin Va for binding to the melanosome. This competition should, in turn, serve to uncouple the organelle from the peripheral actin cytoskeleton. Once uncoupled, the melanosome should redistribute to the cell center, the site of greatest microtubule density (see Fig. B
), by the action of bidirectional, microtubule-dependent traffic. Melan-a melanocytes (D
) were transiently transfected with plasmid EGFP-MC-ST (see Materials and Methods), which drives the expression of a fusion protein containing EGFP fused to the COOH-terminal 619 amino acids of the melanocyte-specific isoform of myosin Va. This portion of the myosin Va heavy chain contains the last ~40% of the central stalk domain (including both melanocyte-specific exons [exons D and F in Seperack et al., 1995
]), and all of the COOH-terminal 410-residue globular tail domain. Fig. , A
, shows the appearance of several such transfected cells, each surrounded by a number of untransfected cells. The transfected cells, with their striking perinuclear distribution of melanosomes, are very reminiscent of dilute
melanocytes, and contrast sharply with the surrounding untransfected cells in which melanosomes are concentrated in the periphery. Similar results were obtained with plasmid FLAG-MC-LT, which drives the expression of a fusion protein containing the nine-residue FLAG epitope tag fused to the COOH-terminal 786 amino acids of the melanocyte-specific isoform of myosin Va (Fig. E
). Conversely, cells transfected with the GFP or FLAG vectors alone did not demonstrate this dilute
-like phenotype (data not shown). In agreement with the mechanism proposed above, GFP and FLAG fluorescence in cells exhibiting the dilute
-like phenotype was largely coincident with the mass of aggregated melanosomes in the cell center. This coincidence was seen not only in fixed cells (B
), but in living cells expressing the GFP-tagged myosin Va tail chimera as well (D
), indicating that the colocalization is not an artifact of fixation.
Figure 6 Expression of myosin Va tail domain fusion proteins in wild-type melanocytes creates a dilute-like phenotype. (A, C, and E) Fields of melan-a melanocytes showing untransfected cells (nonfluorescent) and cells transfected with plasmids EGFP-MC-ST (more ...)
Evidence that tail expression actually generates the dilute-like phenotype, and further insight into the mechanism by which it does so, came from analysis of GFP fluorescence in transfected cells that do not show the dominant-negative phenotype. Fig. shows one of these cells, which are common between ~24 and 36 h of transfection but rare after ~50 h of transfection, and which are characterized by having peripherally distributed melanosomes (Fig. A) and GFP fluorescence that largely colocalizes with these melanosomes (B, arrows). Given that this pattern is precisely what one would predict must precede the generation of the dilute-like phenotype if the mechanism is as described above, we sought to demonstrate by time lapse microscopy that transfected cells like the one in Fig. do indeed turn into cells exhibiting the dilute-like phenotype. Fig. A shows a transfected melan-a cell that was identified by eye as having peripheral, melanosome-associated fluorescence, while B–F show this cell at five successive 30-min intervals. Over this 150-min time span, melanosomes can be seen to undergo a dramatic redistribution, beginning predominantly at the margin of the cell (Fig. A), and ending predominantly in the cell center (F) (see video sequence No. 11). The distribution of melanosomes and GFP fluorescence in this living cell ~5 min after the time point corresponding to F are shown in G and H, respectively. Once again, GFP fluorescence is largely coincident with the mass of melanosomes in the cell center. These results, which were replicated in each of three similar transfected cells (all of which took ~120 min to generate this dominant-negative phenotype), lend strong support to the idea that tail expression initiates the generation of the dilute-like phenotype by displacing endogenous myosin Va from melanosomes in the periphery. The microtubule-based melanosome traffic that drives the redistribution of these melanosomes, which are now uncoupled from the peripheral actin cytoskeleton, is evident in high-magnification time lapse images of transfected cells (data not shown), where melanosomes are seen to undergo rapid, bidirectional movements that gradually result in their accumulation in central regions of the cell, where the density of microtubules is greatest (Fig. ).
Figure 8 Time lapse video micrographs of a transfected melan-a melanocyte during the generation of the dilute-like phenotype. (A–F) Video stills at 30-min intervals of a single melan-a melanotype transfected with plasmid EGFP-MC-ST (center). At time zero, (more ...)
Intermittent, Microtubule-independent, 0.14 μm/s Melanosome Movements Are Seen Only in Wild-Type Melanocytes
The capture mechanism described above requires a myosin Va–dependent interaction of melanosomes with the actin cytoskeleton. We sought to identify such interactions indirectly by comparing the dynamics of melanosomes in wild-type and dilute melanocytes. To focus on these interactions, the microtubule-dependent component of melanosome transport was eliminated by treating cells with nocodazole at 4°C for 30 min followed by incubation at 37°C in the presence of nocodazole. This treatment was also necessary to get reasonable numbers of melanosomes in dilute melanocytes that were not microtubule associated. To quantify melanosome dynamics, the positions of every melanosome within ~120 μm2 areas were digitized over a period of 100 s (at 1-s intervals). Care was taken to choose cells in which there were no obvious changes in cell shape or position over this 100-s time span, and where melanosomes were well spread (a prerequisite for quantitating individual melanosome movements). Fig. shows video stills of the wild-type (A) and dilute null (C) melanocytes used in this analysis, while B and D show the entire paths for all of the melanosomes present in the boxed areas. The difference between these paths, which is reasonably obvious from visual inspection (also see video sequences Nos. 12A and 12B), was borne out by quantitative analysis. Fig. shows histograms in which the speeds of these melanosomes (41 for wild-type, 52 for dilute) were binned in 0.025-μm/s increments and plotted as a percentage of total steps (2,432 for wild-type, 5,174 for dilute). Three main points can be made from this plot. First, the percentage of steps in which melanosomes did not move (zero bin) is slightly more than twice as big in the dilute sample (75.3%) as in the wild-type sample (33.4%). Second, both wild-type and dilute samples have a peak (peak 1) centered around ~0.05 μm/s. While we do not know the precise nature of these movements, they clearly are not myosin Va dependent. Third, the wild-type sample, but not the dilute sample, has another peak (peak 2) that falls roughly between the 0.075–0.1-μm/s bin and the 0.175–0.2-μm/s bin. This portion of the wild-type histogram contains 17.4% of the total steps. By contrast, only 0.17% of the total steps for the dilute sample fall in these bins, suggesting that the melanosome movements corresponding to peak 2 are myosin Va dependent. The average speed of these movements is 0.14 μm/s. Analysis of individual path plots from the wild-type sample revealed that 24 of the 41 paths (~60%) exhibited at least three contiguous 1-s steps with a speed exceeding 0.1 μm/s. Whether these events represent myosin Va–dependent movements of melanosomes on actin filaments or the movements of actin filaments to which melanosomes are tethered by myosin Va is unclear. Nevertheless, because both situations require a myosin Va– dependent interaction of melanosomes with F-actin, these results support the capture mechanism.
We also examined the fate of melanosomes in microtubule-depleted cells over a longer time interval to estimate the ability of these movements to drive longer-range translocations of melanosomes. Fig. E shows a melan-a melanocyte in which melanosomes were concentrated in the peripheral cortex (see arrows), and were undergoing rapid, bidirectional movements in the zone between the cortex and the nucleus before microtubule depletion (data not shown). Fig. F shows this cell 5 min after being returned to 37°C (in the presence of nocodazole) following a 30-min incubation at 4°C in the presence of nocodazole, while G shows this same cell 90 min later. Comparison of F and G shows that the melanosomes concentrated in the peripheral cortex of this cell immediately after microtubule depletion (arrows) did not spread back into the cell, but rather remained highly concentrated in the cortex for at least 90 min. These results indicate that myosin Va– dependent melanosome movements, whatever their exact nature, do not drive obvious spreading of the organelles over 90 min.