Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://cellmotility.genebee.msu.ru/html/articles/PNAS_2001.pdf Äàòà èçìåíåíèÿ: Tue Dec 11 18:43:18 2001 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 20:03:28 2012 Êîäèðîâêà: Ïîèñêîâûå ñëîâà: granules

Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules
Ivan Vorobjev*, Viacheslav Malikov*, and Vladimir Rodionov*
*Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06032-1507; and Laboratory of Cell Motility, A. N. Belozersky Institute, Moscow State University, Moscow 119899, Russia Communicated by V. I. Keilis-Borok, Russian Academy of Sciences, Moscow, Russia, July 11, 2001 (received for review May 15, 2001)

Polarized radial arrays of cytoplasmic microtubules (MTs) with minus ends clustered at the cell center define the organization of the cytoplasm through interaction with microtubule motors bound to membrane organelles or chromosomes. It is generally assumed that the radial organization results from nucleation of MTs at the centrosome. However, radial MT array can also be attained through self-organization that requires the activity of a minusend-directed MT motor, cytoplasmic dynein. In this study we examine the role of cytoplasmic dynein in the self-organization of a radial MT array in cytoplasmic fragments of fish melanophores lacking the centrosome. After activation of dynein motors bound to membrane-bound organelles, pigment granules, the fragments rapidly form polarized radial arrays of MTs and position pigment aggregates at their centers. We show that rearrangement of MTs in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial pattern is generated by continuous disassembly and reassembly of MTs and concurrent minus-end-directed transport of pigment granules bearing the nucleation sites.

n an imal cells, c y toplasmic MTs are c ommonly organ ized into polarized radial arrays (1). Minus ends of MTs are clustered, whereas plus ends are f ree and display the behav ior k nown as dynamic inst abilit y (2), alternating phases of grow th and shorten ing. Therefore radial organ ization allows MTs to ef ficiently ex plore the intracellular space. In interphase, a radial array of MTs directs membrane traf fick ing and deter mines steady-st ate positions of organelles (3) through interaction w ith organellebound microtubule motors of the k inesin family [translocation mostly to MT plus ends or to the cell peripher y (4)] or c y toplasmic dynein [translocation to the minus ends or to the center (5)]. During mitosis, the focal points of MT asters ser ve as the poles of the mitotic spindle. Interaction of MTs extending f rom the poles w ith the k inetochores of chromosomes, w ith each other, and w ith the cell c ortex prov ides the str uctural basis for for mation and position ing of the mitotic spindle (6, 7). Therefore polarized radial MT arrays play a key role in the organ ization of c y toplasm in interphase and during mitosis. Focusing of the minus ends into a radial array is generally assumed to be the result of MT outgrow th f rom the centrosome, which prov ides st able nucleation templates (6). Polarized MT assemblies can also for m through a self-organ ization mechan ism, which requires the activ it y of MT motors (8). Infor mation about the role of MT motors in the organ ization of focused MT arrays was prov ided to a large extent by the studies of the for mation of MT asters and mitotic spindle poles in mitotic cell extracts (9 ­13). In the extracts, clustering of the MT minus ends was per for med by a minus-end-directed motor, c y toplasmic dynein, which for med a large multisubun it c omplex w ith its activator dynactin and a high-molecular-weight protein, NuMa (9, 10, 13). To ex plain the dynein-dependent self-organ ization of MTs into focused arrays, a model was presented based on the inherent capacit y of MT motors to rec ogn ize the intrinsic polarit y of MTs (9, 14). The model required organ ization of dynein molecules into multivalent c omplexes, which were capable of interacting w ith more than one MT, and assumed that dynein molecules remained att ached to MTs when they reached the minus end.
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The assembly of asters was ex plained by the un idirectional transport of MTs w ith plus ends leading, which resulted in the clustering of the minus ends (9, 14). In support of the MT transport model, multimolecular assemblies of MT motors generated radial MT arrays in purified systems in vitro (15, 16). Polarit y of MTs in the for med asters was indeed deter mined by the polarit y of a MT motor (15, 16). Further more, in mitotic cell extracts short MT seeds were moved by c y toplasmic dynein to the mitotic spindle poles (11). Therefore, the results of in vitro studies indicate that MT motors are capable of arranging MTs into focused arrays by producing force for the un idirectional MT movement. However, such MT selforgan ization as a result of MT transport requires v igorous movement of MTs in the c y toplasm and therefore is apparently inc onsistent w ith the role of MTs as immobile ``rails'' that direct the transport of membrane organelles and chromosomes. In this study, we have attempted to elucidate the role of c y toplasmic dynein in the self-organ ization of a polarized radial MT array in vivo by examin ing the for mation of MT asters in c y toplasmic f ragments of melanophores. In melanophores thousands of membrane-bounded organelles, pigment granules, are rapidly transported to the cell center to for m a tight aggregate or redisperse un ifor mly throughout the c y toplasm (17, 18). The granules move by means of the minus-end-directed MT motor c y toplasmic dynein (aggregation; ref. 19) or a plus-end-directed k inesin-related motor (dispersion; refs. 20 and 21). Microsurgically produced c y toplasmic f ragments of melanophores lack ing the centrosome rapidly for m a polarized radial array af ter stimulation of minus-end-directed movement w ith adrenaline and position the pigment aggregate to a focal point of c onverging MTs (22, 23). For mation of the focused array depends on the activ it y of c y toplasmic dynein bound to the pigment granules (23). Therefore, melanophore f ragments prov ide an excellent model system for the study of the role of c y toplasmic dynein in the est ablishment of radial MT organ ization. Materials and Methods as described in (24). Aggregation of pigment granules was induced w ith 10 5 adrenaline and pigment dispersion w ith 5 mM caf feine.
Cell Culture. Cultures of black tetra melanophores were prepared

Preparation of Cy3-Tubulin. Porcine brain tubulin depleted of

microtubule-associated proteins was labeled w ith Cy3-reactive dye (Amersham Phar macia) as described (25) and used at a needle c oncentration of 10 ­15 mg ml. For f luorescence speckle

Abbreviation: MTs, microtubules.


To whom reprint requests should be addressed at: University of Connecticut Health Center, Department of Physiology and Center for Biomedical Imaging Technology, 263 Farmington Avenue, MC-1507, Farmington, CT 06032-1507. E-mail: rodionov@ nso.uchc.edu.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Fig. 1. Organization of a polarized radial array does not involve MT transport. (a) Live fluorescence images of MTs in a fragment before and after stimulation with adrenaline. (b) A model proposed to describe the organization of a radial array through the transport of MTs (black thick lines; arrows indicate plus ends) by multivalent minus-end-directed MT motors (black circles). Gray lines indicate the initial position of MTs. The diagram at the bottom illustrates the direction of movement of a MT end during the formation of a radial array through a transport mechanism. The attainment of a radial organization depends on the axial and lateral displacement of MT ends. (c) Behavior of fluorescent speckles produced by injection of melanophores with fluorescently tagged tubulin subunits at a low (0.5 mg ml) concentration. (Upper) MTs with speckles at low magnification. (Lower) Time sequences of speckles on MTs in the regions indicated in the upper panel. Numbers indicate the time in seconds for all three time sequences [scale bars, 10 m(Upper) and 2 m(Lower)]. (d and e) Fishtailing of a MT in a fragment stimulated with adrenaline. (d) Time sequence. Contours of a fishtailing MT are shown as a dotted line. The plus end is at the top and the minus end is at the bottom. The time in seconds is indicated at the lower right corner of each panel (scale bar, 5 m). (e) Life histories of a plus end (Left) and a minus end (Right) of a MT shown in d.( f) Processivity of the lateral displacement of the minus ends of fishtailing MTs. The ratio of lateral to axial displacement of a MT minus end (shift at right angles to a MT axis vs. shift along the axis) was plotted against the total displacement (total distance covered by an end) at increasing time intervals (for the definition of total, axial, and lateral displacement see also bottom part of b).

microsc opy (26) needle c oncentration of tubulin was reduced to 0.3­ 0.5 mg ml.
Production and Purificaton of Dynein Inhibitors. Monoclonal anti-

body 74.1 against an inter mediate chain of c y toplasmic dynein (ref. 27; a gif t f rom Kev in Pfister, Un iversit y of Virgin ia School of Medicine) was purified f rom ascitic f luid by chromatography on protein A­Agarose (Sigma). The rec ombinant 50-kDa subun it of dynactin c omplex, dynamitin (28), was ex pressed in bacteria (the ex pression vector was prov ided by Richard Vallee, Un iversit y of Massachusetts Medical School) and purified by ammon ium sulfate precipit ation and Mono Q chromatography (29). Dynein antibody and dynamitin were used at needle c oncentrations of 3.3 and 15 mg ml, respectively.

pany, Mansfield, OH) to reduce photodamage and photobleaching (25). Dishes w ith Ox y rase-treated cells were c overed w ith a layer of mineral oil (Squibb) to ret ard gas exchange. Injected cells were obser ved on a Nik on Diaphot 300 inverted microsc ope equipped w ith a Plan 100 1.25 NA objective. Images were c ollected w ith a slow-scan back-illuminated c ooled chargec oupled dev ice camera (CH350; Roper Scientific, Trenton, NJ) driven by MET AMORPH imaging sof t ware (Un iversal Imaging, Media, PA). The values for the rates of grow th and shorten ing of MTs were calculated as displacements of MT ends w ithin 3-s time inter vals traced w ith a mouse-driven cursor w ith SCION IMAGE sof t ware. The proportion of MT poly mer in c y toplasmic f ragments of melanophores was deter mined by a f luorescence ratiometric procedure as described (30, 31). Results Microsurgically produced c y toplasmic f ragments of melanophores rapidly ( 10 min) for m a radial MT array and aggregate pigment granules at the center af ter stimulation w ith adrenaline, which activates dynein motors located on the pigment granules (ref. 23; Fig. 1a). Conceivably, the radial arrangement of MTs in the f ragments c ould be achieved by the transport of MTs, by remodeling of a MT array through c oncurrent disassembly
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Microsurgery and Microinjection. To prepare f ragments, melanophore processes were dissected w ith microneedles w ith a 0.1- m tip diameter. Microinjection was per for med as described (24). Af ter microinjection w ith f luorescently t agged tubulin subun its, cells were incubated for at least 1 h at 30°C to allow inc orporation of labeled tubulin into microtubules. Imaging and Data Analysis. Cells injected w ith Cy3-tubulin were

treated w ith the ox ygen-depleting agent Ox y rase (Ox y rase Com-

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and reassembly, or through a synergistic activ it y of the t wo mechan isms. Vectorial transport of MTs by dynein motors located on pigment granules c ould result in the clustering of minus ends similar to the focusing of MTs in mitotic cell extracts (9, 10, 13). This mechan ism (9, 14) requires sign ificant movement of c y toplasmic MTs (Fig. 1b). To test for MT transport, we followed the behav ior of f luorescently t agged MTs during self-organ ization by high-resolution digit al f luorescence microsc opy (24). MTs were t agged w ith a f luorescent dye by injection of int act melanophores w ith labeled tubulin subun its (24). In the f ragments stimulated w ith adrenaline, the ends of MTs c ontinuously moved, displaying behav ior c onsistent w ith MT gliding along their tracks. To deter mine whether the movement of MT ends was indeed due to such transport, we injected melanophores w ith labeled tubulin subun its at a low ( 1 mg ml) needle c oncentration. Stochastic association of labeled tubulin subun its w ith grow ing MT ends produced speckled variations of f luorescence intensit y along MT (26), which ser ved as internal reference marks. Af ter stimulation w ith adrenaline, f luorescence speckles remained st ationar y on most MTs (Fig. 1c). A x ial displacement was extremely rare (seven MTs in n ine examined f ragments), and shif ts were small ( 0.5 m). Inf requently, the MTs that c ont acted local pigment aggregates displayed v igorous movement-- fisht ailing--which appeared to result f rom the application of external force by MT motors bound to pigment granules (Fig. 1d; the fisht ailing MT is shown as a dotted line). A lthough the f raction of MTs that exhibited such behav ior was small ( 10%), we sought to deter mine whether the fisht ailing movement might c ontribute to the organ ization of a radial MT array. Clustering of minus ends through movement in the c y toplasm requires progressive lateral displacement of MTs (Fig. 1a). Therefore, to examine the c ontribution of fisht ailing to the est ablishment of the radial organ ization, we attempted to deter mine whether the ends of a given MT moved c ontinuously away f rom the in itial ax is. L ocations of plus (grow ing) and minus (shorten ing) ends were deter mined at increasing time inter vals af ter stimulation w ith adrenaline, and the dat a were plotted as life histories of indiv idual MT ends (Fig. 1e). A nalysis of the plots indicated that plus ends advanced essentially along the in itial MT ax is (Fig. 1e Left). Minus ends displayed both lateral and ax ial movement, but the lateral shif ts appeared to be nonprocessive (Fig. 1e Right). Therefore these dat a strongly argued against the involvement of MT transport in the est ablishment of radial organ ization. To c ompletely r ule out the transport mechan ism we per for med for mal analysis of the processiv it y of lateral displacement for the minus ends. We calculated the values for ax ial displacement (shif t along a MT ax is), lateral displacement (shif t at right angles to the ax is), and tot al displacement (tot al dist ance c overed by a minus end), as shown in Fig. 1b (Lower). Averaging of the dat a for 52 minus ends of fisht ailing MTs in eight f ragments clearly showed that the ratio of lateral and ax ial displacements declined when the tot al displacement increased (Fig. 1f ). Therefore lateral shif ts in the positions of the minus ends were indeed not processive. Taken together, these dat a clearly indicate that the movement of MTs does not c ontribute to the organ ization of a radial array. Limited nonprocessive movement of MTs invokes a mechan ism that depends on c oncurrent disassembly and reassembly at the ends. We found that in the f ragments stimulated w ith adrenaline, MTs c ontinuously grew f rom the plus ends and shortened f rom the minus ends. Further more, in c ontrast to the f ragments w ith est ablished radial array, where most of the minus ends were st abilized at the pigment aggregate (23), during aggregation the f raction of st able minus ends was insign ificant (not shown). Therefore the dynamic properties of MTs were c onsistent w ith the organ ization of a radial array through a disassembly­reassembly mechan ism. However, the generation of
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Fig. 2. Pigment aggregates are capable of MT nucleation. (a) Live images of MTs during recovery after cold treatment. Numbers in the lower right corner of each panel indicate the time in minutes and seconds after transfer of a chilled fragment to room temperature (scale bar, 10 m). (b) MTs in a fragment before and after pigment aggregation. (Upper) Live images of MTs (scale bar, 5 m). (Lower) Change in MT length after aggregation. (c) The ratio of MT densities before and after stimulation with adrenaline in control (left bar) or pigment-free (right bar) fragments. MT densities were determined by measuring the total MT length in the same fragments before and 20 min after stimulation with adrenaline.

a radial pattern by this mechan ism would require nucleation at local sites that eventually ac cumulate in the center of a f ragment. Our prev ious work (32) documented c ontinuous outgrow th of MTs f rom the aggregates of pigment granules. Therefore we hypothesized that pigment aggregates c ould ser ve as primar y sites for MT nucleation in the absence of the centrosome. The st andard test for nucleation involves rec over y of MTs af ter disr uption w ith c old or noc odazole. In int act cells, regrow th of MTs oc curs predominantly f rom the centrosome. To test the nucleating capacit y of pigment aggregates, we labeled MTs w ith f luorescent tubulin subun its. Fragments were treated w ith adrenaline for 15 min to allow for pigment aggregation and the for mation of a radial array and then chilled on ice for 30 min. Incubation in the c old c ompletely depoly merized MTs, but aggregates of pigment granules remained essentially int act (Fig. 2a). Sequences of f luorescence images acquired af ter the f ragments were transferred to room temperature indicated that MTs in itially appeared inside or in close prox imit y to the pigment aggregates (Fig. 2a). Grow th through the c y toplasm to the peripher y of a f ragment gradually reest ablished the radial organ ization (Fig. 2a). We c oncluded that the pigment aggregates in the f ragments had a capacit y for MT nucleation similar to that of the centrosomes in int act cells. MT nucleating activ it y c ould be an inherent propert y of pigment granules or might be att ained because of the stimulation
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Fig. 3. Dynein inhibitors disorganize radial MT arrays in the fragments. (a) Immunoblot of the extract of black tetra melanophores with antibody against the intermediate chain of cytoplasmic dynein (antibody 74.1). (b) Live images of MTs after injection of antibody 74.1 (3.3 mg ml needle). Numbers in the lower right corner indicate the time in minutes after injection (scale bar, 5 m). (c) Kinetics of the decrease in the level of MT polymer after injection of 74.1 antibody. The level of MT polymer at each time point was calculated as the difference between mean fluorescence intensities in a region containing and a region lacking MT. (d) Effect of injecting dynein inhibitors on the level of MT polymer. Error bars indicate the 99% confidence interval. MT polymer level was determined by single-cell fluorescence ratiometric assay. Pigment-free fragments were dissected from cells with aggregated pigment granules. Needle concentrations of nonimmune mouse IgG, dynein antibody 74.1 (14), and recombinant subunit of dynactin (p50, dynamitin) were 3.5, 3.3, and 15 mg ml respectively.

w ith adrenaline. To discriminate bet ween these possibilities we have c ompared the amount of MT poly mer in the same f ragments before and af ter pigment aggregation. Nucleation of MTs on a MT organ izing center, such as the centrosome, allows poly merization at a lower min imal c oncentration of f ree tubulin subun its (``critical c oncentration'') and therefore increases the amount of MT poly mer in the c y toplasm (31). Thus, if the granules acquired nucleating capacit y in response to aggregating stimuli, the level of MT poly mer should be sign ificantly higher in the aggregated c ompared w ith the dispersed st ate. To quantif y poly mer levels we took advant age of the fact that in small (10 ­15 m) f ragments we c ould trace ever y MT and measure tot al MT length in the same f ragment before and af ter pigment aggregation. We found that in dispersed st ate the MT poly mer level defined by the tot al MT length was low, as ex pected in the case of self-nucleation of MTs in the c y toplasm (31). In c ontrast, in the aggregated st ate the tot al MT length was about 2-fold higher (Fig. 2b). The increase in the poly mer level clearly depended on pigment granules, because in pigment-f ree f ragments produced by microdissection of cells w ith aggregated pigment granules, MT poly mer level did not increase in response to treatment w ith adrenaline (Fig. 2c). These results strongly suggested that nucleation of MTs on pigment granules was provoked by the treatment w ith adrenaline. The most noticeable ef fect of adrenaline is the activation of dynein motors essential for the organ ization of MTs. A lthough the primar y role of dynein motors appeared to be transport of pigment granules (23), they c ould also be involved in MT nucleation. To test for the role of c y toplasmic dynein in MT nucleation, we injected f ragments w ith est ablished radial arrays w ith dynein inhibitors, a monoclonal antibody to dynein intermediate chain (74.1; ref. 27), or a rec ombinant (29) 50-kDa
Vorobjev et al.

subun it of the dynein activator dynactin (dynamitin; ref. 28). The t wo inhibitors, at needle c oncentrations of 3.3 and 15 mg ml, respectively, c ompletely inhibited aggregation of pigment granules in int act melanophores (not shown). Injection of the f ragments w ith dynein inhibitors resulted in suppression of MT outgrow th f rom the pigment aggregates, immediate release of MT minus ends, gradual randomization of the MT net work (Fig. 3b), and rapid decrease in MT densit y (Fig. 3c). The level of MT poly mer, estimated w ith a single cell ratiometric f luorescence assay (30, 31), decreased by about 40% (Fig. 3d) w ithin 20 min af ter injection of the dynein inhibitors, return ing to the level characteristic for the dispersed st ate. The rates of grow th (8.6 SD) and shorten ing (7.6 1.6 m min, 1.1 m min, mean mean SD) in the f ragments injected w ith dynein inhibitors were similar to the rates of grow th and shorten ing in c ontrol non injected f ragments (7.7 0.5 and 5.1 0.6, mean SD, respectively). Therefore the ef fects of dynein inhibitors were c onsistent w ith inhibition of MT nucleation and uncapping of MT minus ends associated w ith pigment aggregates. Taken together, these results indicated that c y toplasmic dynein was directly or indirectly involved in MT nucleation and capping of the MT minus ends. The dynein-dependent nucleation of MTs on pigment granules in the f ragments c ould ref lect a mechan ism that c omplements nucleation at the centrosome in int act melanophores. However, in cells the steady-st ate c oncentration of f ree tubulin subun its should be lower than in the f ragments, because centrosomes presumably have a higher nucleating capacit y than pigment aggregates and therefore induce the for mation of a larger amount of MTs (31). To deter mine whether pigment granules were capable of MT nucleation at the low c oncentration of f ree tubulin subun its t ypical of int act melanophores, we tested
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Fig. 4. Outgrowth of microtubules from a local pigment aggregate formed on the noncentrosomal side of a wound in a whole melanophore. (a) Phasecontrast image of a melanophore with dispersed pigment with a U-shaped wound produced with a glass microneedle. (b) Magnified phase-contrast image of a region outlined in a 10 min after induction of aggregation with adrenaline. The pigment aggregate has been formed on the noncentrosomal side of the wound. (c and d) Live fluorescence images of microtubules that surround the pigment aggregate shown in b.(c) Distribution of microtubules around the pigment aggregate. (d) Time sequence of microtubules at a high magnification in the boxed region shown in c. MTs continuously emerged from the pigment aggregate and grew to the wound or to the cell margin by the addition of subunits to the plus ends (indicated by arrowheads). Numbers indicate the time in seconds [scale bars, 20 m (a), 10 m (b and c), and 5 m (d)].

for the for mation of a pigment aggregate on the noncentrosomal side of a wound, which isolated a portion of the c y toplasm f rom the inf luence of the centrosomal radial array. Melanophores were injected w ith f luorescent tubulin subun its, and a wound was produced w ith a sharp glass microneedle (Fig. 4a). Remark ably, adrenaline resulted in the for mation of a local pigment aggregate on the noncentrosomal side of a wound (Fig. 4b). Digit al f luorescence microsc opy demonstrated that MTs c ontinuously emerged f rom a noncentrosomal pigment aggregate and grew toward the preex isting cell margin or toward the wound (Fig. 4d). Thus, the behav ior of MTs surrounding a local pigment aggregate that for med in cells was ver y similar to the behav ior of MTs in the f ragments. These results strongly suggest that the presence of the centrosome does not preclude nucleation on pigment aggregates. Therefore MT nucleation on pigment granules, which ac cumulated in the centrosomal region in response to the treatment w ith adrenaline, c ontributes to the organ ization of the radial MT array in int act melanophores. Discussion c y toplasmic dynein and MT dynamics in the organ ization of a polarized radial MT array in the absence of the centrosome in vivo. The for mation of a radial array is achieved through c oncurrent disassembly and reassembly of other w ise immobile MTs. Grow th of MTs is in itiated on noncentrosomal sites that prov ide nucleation templates but, unlike the centrosome, do not anchor the minus ends tightly. Frequent release and depoly merization f rom minus ends allows for rapid reorgan ization of a MT array. The spatial distribution of MTs at any given moment is deter mined by the location of noncentrosomal nucleation sites. However, the nucleation sites themselves never remain st ationar y, but are rapidly transported to MT minus ends. Nucleation and f requent release of MTs superimposed on minus-enddirected transport of nucleation sites eventually results in the for mation of a radial array. We hypothesize that the follow ing sequence of events leads to the organ ization of random MTs into a radial array in melano10164 www.pnas.org cgi doi 10.1073 pnas.181354198

Fig. 5. A model for the formation of a radial MT array in melanophore fragments. (a) In the dispersed state, pigment granules and microtubules are distributed randomly in a fragment. (b) After stimulation with adrenaline, transport of pigment granules to the MT minus ends results in the formation of local pigment aggregates. (c) Nucleation of MTs on the pigment aggregates produces microasters. (d) Fusion of microasters results in the formation of a single radial array of microtubules with the pigment aggregate at the center.

phore f ragments (Fig. 5). At an early st age of the selforgan ization, rapid transport of pigment granules to the MT minus ends induces the for mation of local pigment aggregates (Fig. 5b). MT nucleation on local aggregates and grow th in all directions produce microasters (Fig. 5c). Transport of pigment granules along MTs extending bet ween the local pigment aggregates results in the fusion of microasters and the for mation of a single radial array w ith the pigment aggregate at the center (Fig. 5d). In our model, c y toplasmic dynein plays a dual role in the self-organ ization mechan ism by participating in MT nucleation and by supporting minus-end-directed transport of nucleation sites.
Assembly of MT Arrays by Cytoplasmic Dynein. Organ ization of MTs

Self-Organization of a Radial Microtubule Array in Melanophore Fragments. Our results shed light on the role of the activ it y of

into interphase radial arrays (33, 34), mitotic spindle poles (13, 28), or bundles in axonal shaf ts (35) requires the activ it y of c y toplasmic dynein. Given that c y toplasmic dynein is a forceproducing enz y me, it seems reasonable to assume that it functions in MT organ ization by prov iding the driv ing force for un idirectional transport of MTs. It is not clear, however, if the force produced by dynein motors att ached to membrane organelles or anchored in the c y toplasm is suf ficient to overc ome the drag force for the MT movement. A lthough some live obser vations document movement of indiv idual MTs (25, 36), others indicate that MTs generally remain st ationar y in the c y toplasm (37, 38). In our ex periments MTs never displayed persistent lateral or ax ial displacement characteristic of the transport mechan ism. Radial organ ization was att ained through c oncurrent disassembly and reassembly of MTs. Our results indicate that mechan ical force produced by c y toplasmic dynein is used in the transport of MT nucleation and capping sites to the MT minus ends. Remark ably, the same mechan ism seems to operate in int act cells, w ith radial organ ization already est ablished through the MT nucleation at the
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Possible Mechanisms of Dynein Involvement in MT Nucleation. We

env ision three possible mechan isms of MT nucleation on noncentrosomal sites that require c y toplasmic dynein. It may promote nucleation indirectly, by binding protein c omplexes that ser ve as templates for the MT nucleation. A possible candidate for the nucleation template located on the pigment granules is
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The authors are grateful to K. K. Pfister for prov iding 74.1 antibody and R. B. Vallee for a gif t of p50 cDNA. We thank Timothy Mitchison and anony mous rev iewers for helpful c omments and A. S. Kashina, S. M. K ing, and A. E. Cowan for critical reading of the manuscript. This work was supported by National Institues of Health Grant GM62290-01 and National Science Foundation Grant MCB 9996320 (to V.R.) and Russian Basic Science Foundation Grant 99-04-49436 (to I.V.).
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Vorobjev et al.

PNAS

August 28, 2001

vol. 98

no. 18

10165

CELL BIOLOGY

centrosome. In melanophores, ac cumulation at the cell center of the pigment granules bearing noncentrosomal nucleating sites apparently increases the densit y of MTs in centrosomal radial arrays and therefore enhances the ef ficienc y of pigment aggregation. Surprisingly, the assembly of the centrosome itself also seems to depend on the dynein-dependent transport of large particles c ont ain ing the integral centrosomal c omponents pericentrin (39), PCM1 (40), and -tubulin (41), a protein directly involved in the organ ization of MT nucleating templates. Therefore transport of c omponents of microtubule-organ izing centers by c y toplasmic dynein seems to ref lect a general mechan ism for the c onstr uction of radial MT arrays. Our results indicate that c y toplasmic dynein may play an even more critical role in MT nucleation than merely transporting the nucleation sites. It also seems to ac c ount for the MT nucleation and minus-end capping activ it y displayed by pigment granules, which are critical for the att ainment of radial MT organ ization but separate f rom the production of the mechan ical force. Frequent release of MTs f rom the pigment granules indeed suggests that the molecular mechan ism of dynein-dependent minus-end capping is fundament ally dif ferent f rom the capping of the minus ends at the centrosome, which apparently involves tight att achment to the -tubulin ring c omplex. Therefore, in addition to participation in the minus-end-directed transport of membrane organelles or chromosomes, c y toplasmic dynein may play an import ant role in the c ontrol of MT assembly by capping of MT minus ends or MT nucleation.

the -tubulin ring c omplex responsible for MT nucleation at the centrosome. Binding of the -tubulin ring c omplex to the pigment granules may be mediated by pericentrin, which is k nown to interact w ith a light inter mediate chain (LIC1) of c y toplasmic dynein (42, 43). We feel, however, that mediation by pericentrin is an unlikely possibilit y because immunost ain ing of melanophores w ith -tubulin antibody did not detect -tubulin on the pigment granules (23). A lternatively, c y toplasmic dynein may be involved in the transport of a dif ferent, as yet un identified factor that induces MT capping or nucleation. However, we favor a third possibilit y, that the dynein dynactin c omplex may be directly involved in MT nucleation. Indeed, in melanophores aggregation stimuli dramatically enhance the abilit y of dynein dynactin to bind MTs (43). Further more, partially purified c y toplasmic dynein has been obser ved to stimulate MT assembly in vitro (J. R. McIntosh, personal c ommun ication). Each dynein dynactin c omplex possesses three MT-binding sites located on the dynein heav y chains (44) and on the p150Glued subun it of the dynactin (45). Several dynein dynactin c omplexes closely spaced on the sur face of a pigment granule c ould prov ide a suf ficient number of binding sites for the 12 tubulin dimers that for m a nucleus for the MT grow th (46). A lthough the det ails of the molecular mechan ism remain to be deter mined, we believe that dynein-dependent nucleation of MTs may prov ide a mechan ism that c ooperates w ith the centrosome in the organ ization of a radial MT array.