Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.     

A stereoscopic analysis of the centrosome structure in the cells of continuous and primary cell cultures]

A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells. It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends. The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres.     

A stereoscopic analysis of centrosome structure in the cells of a tissue culture under the action of energy metabolism inhibitors. I. 2,4-Dinitrophenol, deoxyglucose, sodium azide and the calcium ionophore A23187]

A 30-min action of energy transfer inhibitors (2,4-dinitrophenol, deoxyglucose, azide and calcium ionophore A23187) on tissue culture cells results in a significant increase in the quantity of microtubules around the centrosome. After the action of all the inhibitors, mostly increases the number of long microtubules with free proximal end oriented towards the centrosome. It is suggested that energy transfer inhibitors may stimulate foundation of microtubules on the centrosome and stabilize free microtubules, while they exert no effect on the frequency of detachment of microtubules from the centrosome.     

Ultrastructure of the centrosome in L cells

Three types of microtubule-organizing centers are present in the interphase L-cells: centriolar matrix, pericentriolar satellites, and electron-dense bodies that are not attached to the centrioles. Different types of microtubule-organizing centers may be present simultaneously in the same centrosome. In most of the cells some microtubules have their proximal ends free, rather than attached to the microtubule-organizing center. A network of intermediate filaments is condensed around the centrosome. The intermediate filaments run from the centrosome parallel to the microtubules. Although the filaments are often in close proximity to the centrioles and microtubules, direct contacts between them are rare. The intermediate filaments have convergence foci of their own in the centrosome.     

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.     

The centrosome reaction in tissue-culture cells to depolarization of the cell membranes

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ouabain and calcium ionophore A23187 caused a centrosome restructuring expressed in mean number increase of satellites on the active (mother) centriole, in an increase of the mean slope of centrioles to the substrate surface and in the more frequent occurrence of primary cilia. In the presence of FCCP the effect appeared only in 10 min and retained for 2 h. The ouabain caused the separation of active and inactive centrioles in more than in a half of the cells. The data obtained permit to conclude that depolarization of the plasmatic membrane only is needed for the initiation of centrosome restructuring. The authors propose that this reaction of centrosome is a component of the cell overall response to nonspecific lesions.     

Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. I. The structure and behavior of centrosomes before fusion of the pronuclei

During fertilization the sperm brings two centrosomes into the egg. One centrosome contains a centriole of normal length originally seen as the basal body of the sperm flagellum. Characteristically, the proximal half is enwrapped in osmiophilic material. This centrosome is attached to the centrosomal fossa, a bowl-shaped depression of the nuclear envelope of the male pronucleus. Microtubules radiate out from the osmiophilic half characterizing this structure as a centrosome and microtubule organizing center (MTOC). The second centrosome which also acts as an MTOC is attached to the mitochondrion of the sperm. At the beginning it appears as an unstructured accumulation of osmiophilic material out of which later on centriolar microtubules grow. Though this centrosome is marked by an immature centriole it is capable of organizing microtubules and of reproducing itself. This centrosome becomes loosely associated with the female pronucleus by means of microtubules. Then it separates from the mitochondrion which finally is lost. When the two pronuclei fuse, the centrosome derived from the basal body remains firmly attached to the centrosomal fossa, which has persisted in the envelope of the zygote nucleus after pronuclear fusion. Using the fossa as a marker of the position of this centrosome on the nuclear surface, we conclude that it is a stationary centrosome in the process of bipolarization for the first mitosis.     

A composite model for establishing the microtubule arrays of the neuron

Neurons generate two distinct types of processes, termed axons and dendrites, both of which rely on a highly organized array of microtubules for their growth and maintenance. Axonal microtubules are uniformly oriented with their plus ends distal to the cell body, whereas dendritic microtubules are nonuniformly oriented. In neither case are the microtubules attached to the centrosome or any detectable structure that could establish their distinct patterns of polarity orientation. Studies from our laboratory over the past few years have led us to propose the following model for the establishment of the axonal and dendritic microtubule arrays. Microtubules destined for these processes are nucleated at the centrosome within the cell body of the neuron and rapidly released. The released microtubules are then transported into developing axons and dendrites to support their growth. Early in neuronal development, the microtubules are transported with their plus ends leading into immature processes that are the common progenitors of both axons and dendrites. This sets up a uniformly plus-end-distal pattern of polarity orientation, which is preserved in the developing axon. In the case of the dendrite, the plus-end-distal microtubules are joined by another population of microtubules that are transported into these processes with their minus-ends leading. Implicit in this model is that neurons have specialized machinery for regulating the release of microtubules from the centrosome and for transporting them with great specificity.     

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.     

Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.     

Radial-organized microtubules provide cell shape support and more effective intercellular transport then free microtubules

Microtubules take part in various cell processes, including cell polarization, migration, intercellular transport, and some others. Therefore, the spatial organization of microtubules is crucial for normal cell behavior. Fibroblasts have radial microtubule arrays that consist of microtubules that run from the centrosome. Two components compose this microtubule array, i.e., (1) minus ends attached to the centrosome microtubules with their plus ends radiating to the cell periphery and (2) free microtubules with ends not attached to the centrosome. Distinctions in the dynamic properties, intercellular organization, and structure of centrosome-attached and free microtubules allow us to assume that their cellular functions are also different. To study centrosome-attached and free microtubules functions, we used cytoplasts, i.e., nucleus-lacking cellular fragments that, under certain conditions, also lose their centrosomes. In these cytoplasts, there are only free microtubules. The shape, general morphology, and size of cytoplasts that retain their centrosomes differ only slightly from whole cells. Cytoplasts who have lost their centrosomes have an extremely thin network of microtubules located in their central region; furthermore, they lose the shape that is typical for fibroblast and become rough lamellae with protrusions. The internal architecture of the cytoplasm and organoid arrangement are also broken. Saltatory movements in cytoplasts with centrosomes are similar to those in whole cells; in cytoplasts without centrosomes, saltatory movements occur with velocities that are twofold less and by shorter distances. Saltatory movements of granules in centrosome-lacking cytoplasts took place basically in the central region of cytoplast and were less ordered than in whole cells and in cytoplasts with centrosomes. We believe that radial organized microtubules ensure the effective transport and dynamical interaction of microtubule plus ends with cellular cortical structures, which is sufficient to support the common fibroblast-like shape, whereas the disorganized free microtubules are not able to maintain the external fibroblast shape and its intercellular organization.     

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.     

Centrosome Behavior under the Action of a Mitochondrial Uncoupler and the Effect of Disruption of Cytoskeleton Elements on the Uncoupler-Induced Alterations

Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) induced in pig kidney embryo cells a loss of rhodamine 123 staining of mitochondria in 2-3 min. Within 5 min after FCCP inoculation of cells prestained with rhodamine 123, the diffuse staining of the cytoplasm was absent. FCCP did not induce changes in the cytoplasmic microtubule complex, but induced nonrandom (preferentially perpendicular to the substrate surface) orientation of maternal centrioles. Nonrandom orientation of maternal centrioles occurred 10 min after treatment and remained for 2 hr. At 30 min after introduction of the drug, FCCP treatment increased the mean number of pericentriolar satellites on maternal centrioles and the frequency of primary cilia. The percentage of centrioles perpendicular to the substrate induced by FCCP treatment was slightly increased by disruption of microtubules and slightly diminished by disruption of microfilaments. In both cases centrioles were oriented significantly differently from random (P < 0.01). These results suggest that microtubules are neither involved in the signaling pathway from plasma membrane to the centriole, nor do they anchor the centrioles perpendicular to the substrate, as proposed by Albrecht-Buehler and Bushnell (Experimental Cell Research 120, 1979).     

Free and centrosome-attached microtubules: Quantitative analysis and modeling of two-component system

In cultivated in vitro interphase animal cells, microtubules form a network whose density is highest in the central cell area, in the region of centrosome, and decreases towards the cell periphery. Since identification of individual microtubules in the central cell area is significantly difficult and more often is impossible, there are several approaches to studying microtubules in the internal cell cytoplasm. These approaches are based on a decrease of microtubule density—both real, due to their partial depolymerization (by the action of cold temperatures or cytostatics), or apparent, due to a decrease of cell thickness (by photobleaching of preexisting microtubules and analysis of newly formed ones). In the present work, we propose a method based on the determination of optical density which allows evaluation of the state of the cytoplasmic microtubule system as a whole. The method consists of a comparison of the dependences describing changes of the microtubule optical density from the cell center to the periphery in controls and in experiments. Analysis of living cells by the proposed method has shown that the character of curves describing the decrease of optical density from the cell center to its periphery is different for various cell types; the dependence can be described both as an exponential regression (the CHO cell line) and as a linear regression (the NIH-3T3 and REF cell lines). Our previous studies have allowed the suggestion that the character of the dependence is determined by the ratio of free and centrosome-attached microtubules and by the position of their ends in the cell cytoplasm. To test this hypothesis, we considered model systems with all microtubules assumed to be in a straight orientation and divergent radially from the centrosome, but with different arrangements of plus-and minus-ends. In the model system, in which all the microtubule minus-ends are attached to the centrosome while the plus-ends are at different distances from it, the microtubule density is described by the exponential (f(x) = ae ^?bx ). Introduction of free microtubules into the system leads to a change of the character of this dependence, and the system in which the concentration of free microtubules with minus ends located at different distances from the cytoplasm is 5 times higher than that of the centrosome-attached microtubules is described by the linear regression equation (f(x) = k ^* x + b), which corresponds to the experimentally obtained dependences for 3T3 and REF cells. Thus, we believe that even in cells with a radial microtubule system, free microtubules may constitute the majority.     

Free and centrosome-attached microtubules: quantitative analysis and the modeling of two-component system

In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult.     

Assembly of centrosomal proteins and microtubule organization depends on PCM-1

The protein PCM-1 localizes to cytoplasmic granules known as "centriolar satellites" that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1-containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization.     

Organization of microtubules in centrosome-free cytoplasm

Many different cell types possess microtubule patterns which appear to be polarized and oriented, in part, by cytoplasmic factors not directly associated with a centrosome. Recently, we demonstrated that cytoplasmic extensions ("arms") of teleost melanophores will reorganize their microtubule population outward from their centers after surgical isolation (McNiven, M. A., M. Wang, and K. R. Porter. 1984. Cell. 37:753-765). In the study reported here, we examine microtubule dynamics within the centrosome-free fragments and find that, after severing, microtubule reorganization is initiated at the proximal (cut) end of an arm and migrates distally with the aggregated pigment mass until it becomes permanently positioned at the middle of the arm. Computer-aided image analysis demonstrates that this middle position is located at the arm centroid, implicating the action of a cytoplasmic gel in this process. Morphological studies of arms devoid of pigment reveal that microtubules do not emanate from a single site or structure within the centroid area, but from a more generalized region. Taken together, these findings suggest that factors distributed throughout cytoplasm participate in microtubule assembly and organization.     

In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules

FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules are disrupted by nocodazole. A similar delay was required for the recovery of mitochondrial function as assessed by rhodamine 123 labelling. The effect of FCCP on microtubules was partially inhibited by preincubation of the cells with NaN3, suggesting that FCCP acts on microtubules through mitochondria. FCCP did not depolymerize microtubules of cells permeabilized with Triton X-100. In vitro polymerisation of microtubule protein was only slightly diminished by concentrations of FCCP which provoke complete disassembly in vivo. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the microtubules polymerized in vitro in the presence of FCCP showed a reduced amount of high mol. wt. proteins, mainly MAP 2, associated with them. In an attempt to reproduce the mitochondrial effects of FCCP in vitro, we checked the effects of alkaline pH and calcium on microtubule protein polymerization in the presence of FCCP. FCCP did not influence the calcium inhibitory effect but did significantly increase the inhibitory effect of alkaline pH. We conclude that FCCP could depolymerise microtubules in vivo through a dual operation: increasing the intracellular pH by the disruption of the mitochondrial H+ gradient and decreasing the stability of microtubules by impairing the binding of microtubule-associated proteins.     

An essential role for katanin in severing microtubules in the neuron

Several lines of evidence suggest that microtubules are nucleated at the neuronal centrosome, and then released for transport into axons and dendrites. Here we sought to determine whether the microtubule-severing protein known as katanin mediates microtubule release from the neuronal centrosome. Immunomicroscopic analyses on cultured sympathetic neurons show that katanin is present at the centrosome, but is also widely distributed throughout the neuron. Microinjection of an antibody that inactivates katanin results in a dramatic accumulation of microtubules at the centrosome, indicating that katanin is indeed required for microtubule release from the centrosome. However, the antibody also causes an inhibition of axon outgrowth that is more immediate than expected on this basis alone. It may be that katanin severs microtubules throughout the cell body to keep them sufficiently short to be efficiently transported into developing processes. Consistent with this idea, there were significantly fewer free ends of microtubules in the cell bodies of neurons that had been injected with the katanin antibody compared with controls. These results indicate that microtubule-severing by katanin is essential for releasing microtubules from the neuronal centrosome, and also for regulating the length of the microtubules after their release.     

Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.