Microtubule dynamics in cultured cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording by a digital camcorder. In the cell lamella, thepositive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode around 0. The maximum rate of lengthening and shortening reaches 30 and 50 microns/min, respectively. The positive ends of microtubules in PtK1 cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were displaced, usually, to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK1 cells appeared, mostly due to spontaneous assembly in the cytoplasm, not in the relationship with the preexisting microtubules, and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1-2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts, as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place, mostly, at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.     

Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.     

Evaluation of various methods of measurement of the microtubule length in the cytoplasm of cultured cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 +/- 3.69 microns. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 +/- 0.72 microns long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 microns, averaging 3.33 +/- 2.43 microns; the average length of centrosomal microtubules was 1.49 +/- 0.82 microns. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of free microtubules in Vero cells actually have a length of 2-5 microns; i.e., they are much shorter than the cell radius (about 25 microns). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 m. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 ± 0.72 m long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 m, averaging 3.33 ± 2.43 m; the average length of centrosomal microtubules was 1.49 ± 0.82 m. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of the free microtubules in Vero cells actually have a length of 2–5 m; i.e., they are much shorter than the cell radius (about 25 m). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

Spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts

The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after 30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15% of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained, we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome; rather, their orientation is preset by local factors.     

Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 μm/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione–2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that ~80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.     

Centrosome reorientation in wound-edge cells is cell type specific

The reorientation of the microtubule organizing center during cell migration into a wound in the monolayer was directly observed in living wound-edge cells expressing gamma-tubulin tagged with green fluorescent protein. Our results demonstrate that in CHO cells, the centrosome reorients to a position in front of the nucleus, toward the wound edge, whereas in PtK cells, the centrosome lags behind the nucleus during migration into the wound. In CHO cells, the average rate of centrosome motion was faster than that of the nucleus; the converse was true in PtK cells. In both cell lines, centrosome motion was stochastic, with periods of rapid motion interspersed with periods of slower motion. Centrosome reorientation in CHO cells required dynamic microtubules and cytoplasmic dynein/dynactin activity and could be prevented by altering cell-to-cell or cell-to-substrate adhesion. Microtubule marking experiments using photoactivation of caged tubulin demonstrate that microtubules are transported in the direction of cell motility in both cell lines but that in PtK cells, microtubules move individually, whereas their movement is more coherent in CHO cells. Our data demonstrate that centrosome reorientation is not required for directed migration and that diverse cells use distinct mechanisms for remodeling the microtubule array during directed migration.     

Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases

To get insight into the action of Rho GTPases on the microtubule system we investigated the effects of Cdc42, Rac1, and RhoA on the dynamics of microtubules in Swiss 3T3 fibroblasts. In control cells microtubule ends were dynamic: plus ends frequently switched between growth, shortening and pauses; the growth phase predominated over shortening. Free minus ends of microtubules depolymerized rapidly and never grew. Free microtubules were short-lived, and the microtubule network was organized into a radial array. In serum-starved cells microtubule ends became more stable: although plus ends still transited between growth and shortening, polymerization and depolymerization excursions became shorter and balanced each other. Microtubule minus ends were also stabilized. Consequently lifespan of free microtubules increased and microtubule array changed its radial pattern into a random one. Activation of Cdc42 and Rac1 in serum-starved cells promoted dynamic behavior of microtubule plus and minus ends, while inhibition of these GTPases in serum-grown cells suppressed microtubule dynamics and mimicked all effects of serum starvation. Activation of RhoA in serum-grown cells had effects similar to Cdc42 /Rac1 inactivation: it suppressed the dynamics of plus and minus ends, reduced the length of growth and shrinking episodes, and disrupted the radial organization of microtubules. However, in contrast to Cdc42 and Rac1 inactivation, active RhoA had no effect on the balance between microtubule growth and shortening. We conclude that Cdc42 and Rac1 have similar stimulating effects on microtubule dynamics while RhoA acts in an opposite way.     

Assembly and turnover of detyrosinated tubulin in vivo

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.     

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.     

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(- 8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half- life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.     

Centrosome behavior in motile HGF-treated PtK2 cells expressing GFP-gamma tubulin.

In response to locomotory cues, many motile cells have been shown to reposition their centrosome to a location in front of the nucleus, towards the direction of cell migration. We examined centrosome position in PtK(2) epithelial cells treated with hepatocyte growth factor (HGF), which stimulates motility but, unlike chemotactic agents or wounding of a monolayer, provides no directional cues. To observe centrosome movement directly, a plasmid encoding human gamma tubulin fused to the green fluorescent protein was expressed in HGF-treated cells. In cells whose movements were unconstrained by neighboring cells, we found that the position of the centrosome was not correlated with the direction of cell locomotion. Further, in cells where the direction of locomotion changed during the observation period, the centrosome did not reorient toward the new direction of locomotion. Analysis of centrosome and nuclear movement showed that motion of the centrosome often lagged behind that of the nucleus. Analysis of 249 fixed cells stained with an antibody to gamma tubulin confirmed our observations in live cells: 69% of the cells had centrosomes behind the nucleus, away from the direction of locomotion. Of these, 41% had their centrosome in the retraction tail. Confocal microscopy showed that the microtubule array in HGF treated PtK(2) cells was predominantly non-centrosomal. Because microtubules are required for efficient cellular locomotion, we propose that non-centrosomal microtubules stabilize the direction of locomotion without a requirement for reorientation of the centrosome.     

Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold- induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.     

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.     

The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules

According to the current view, the microtubule system in animal cells consists of two components: microtubules attached to the centrosome (these microtubules stretch radially towards the cell margin), and free microtubules randomly distributed in the cytoplasm without visible association with any microtubule-organizing centers. The ratio of the two sets of microtubules in the whole microtubule array is under discussion. Addressing this question, we have analysed the recovery of microtubules in cultured Vero nucleated cells and cytoplasts, with and without centrosomes in these. Cells were fixed at different time points, and individual microtubules were traced on serial optical sections. During a slow recovery after cold treatment (4 degrees C, for 4 h; recovery at 30 degrees C) polymerization of microtubules started mainly from the centrosome. At early stages of recovery the share of free microtubules made about 10% of all microtubules, and their total length increased slower than the lenght of centrosome-attached microtubules. During a rapid recovery after nocodazole treatment (10 microg/ml, 2 h; recovery in drug-free medium at 37 degrees C), the share of free microtubules was about 35%, but their total length increased slower than the length of centrosome-attached microtubules. In 6-8 min (rapid recovery) or 12-16 min (slow recovery), tips of centrosomal microtubules reached the cell margin, and their increased density made it impossible to recognize individual microtubules. However, under the same conditions in cytoplasts without centrosomes the normal number of microtubules recovered only in 60 min, which enabled us to suppose that the complete recovery of microtubule system in the whole cells may be also rather long. When the first centrosomal microtubules reached the cell margin, the optical density of microtubules started to decrease from the centrosome region towards the cell margin, according to the exponential curve. Later on, the optical density in the centrosome region and near the cell margin remained at the same level, but microtubule density increased in the middle part of the cell, and in 45-60 min the plot of the optical density vs the distance from the centrosome became linear, as in control cells. Since no significant curling of microtubules occurs near the cell margin, the density of microtubules in the endoplasm may increase due only to polymerization of free microtubules. We suppose that in cultured cells the microtubule network recovery proceeds in two stages. At the initial stage, a rapid growth of centrosomal microtubules takes place in addition to the turnover of free microtubules with unstable minus ends. At the second stage, when microtubule growth from the centrosome becomes limited by the cell margin, a gradual extension of free microtubules occurs in the internal cytoplasm.     

Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells

A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nuclear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.