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.     

An intact centrosome is required for the maintenance of polarization during directional cell migration

Background

Establishing and maintaining polarization is critical during cell migration. It is known that the centrosome contains numerous proteins whose roles of organizing the microtubule network range include nucleation, stabilization and severing. It is not known whether the centrosome is necessary to maintain polarization. Due to its role as the microtubule organizing center, we hypothesize that the centrosome is necessary to maintain polarization in a migrating cell. Although there have been implications of its role in cell migration, there is no direct study of the centrosome''s role in maintaining polarization. In this study we ablate the centrosome by intracellular laser irradiation to understand the role of the centrosome in two vastly different cell types, human osteosarcoma (U2OS) and rat kangaroo kidney epithelial cells (PtK). The PtK cell line has been extensively used as a model for cytoskeletal dynamics during cell migration. The U2OS cell line serves as a model for a complex, single migrating cell.

Methodology/Principal Findings

In this study we use femtosecond near-infrared laser irradiation to remove the centrosome in migrating U2OS and PtK2 cells. Immunofluorescence staining for centrosomal markers verified successful irradiation with 94% success. A loss of cell polarization is observed between 30 and 90 minutes following removal of the centrosome. Changes in cell shape are correlated with modifications in microtubule and actin organization. Changes in cell morphology and microtubule organization were quantified revealing significant depolarization resulting from centrosome irradiation.

Conclusions/Significance

This study demonstrates that the centrosome is necessary for the maintenance of polarization during directed cell migration in two widely different cell types. Removal of the centrosome from a polarized cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. These results demonstrate that the centrosome plays a critical role in the maintenance of cytoskeletal asymmetry during cell migration.     

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.     

The Role of Microtubules and Their Dynamics in Cell Migration

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.     

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor–induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.     

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.     

Locomotion of CV-1 cytoplasts with or without a centrosome

The movement of cultured cells along the substratum is a convenient model for studying cell movement in the organism, occurring during embryogenesis, angiogenesis, metastasis, wound closure, etc. The moving cells must control their pseudopodial activity along the perimeter, regulate the attachment and reattachment to the substratum, and pull their body following pseudopodium during their movement along the substratum. As proven by numerous investigations, these processes directly depend on the actomyosin system of cells. The role of microtubules as components of cytoskeleton in cell locomotion still remains obscure. The role of microtubules in cell movement is commonly investigated using microtubule-destructive drugs. Therefore in the final results the accessory drug effect on, for example, signal cascades cannot be excluded. Another mode of action on the microtubule dynamics is centrosome removal from the cells, which is easily realized by their removal together with the nucleus. It has been shown that in cytoplasts of centrosome containing fibroblasts, dynamic instability of microtubules remains. Unlike, in non-centriolar cytoplasts tread milling is observed. Some literature evidence suggests that cytoplasts of cultured cells move along the substratum not worse that intact cells do. In this study cytoplasts with and without centrosome were obtained and identified, and parameters of movement along the substratum (speed, direction) were registered for both these two populations of cytoplasts, and for control intact cells and cells involved in the experiment. The model of experimental wound of monolayer was used, because it guaranteed cell synchronization in respect to movement direction and speed. Centrosome-containing CV-1 cytoplasts displayed radial microtubule array, and centrosome-lacking cytoplasts exhibited chaotic distribution of microtubules, which is characteristic of microtubule tread milling. In addition, both kinds of cytoplasts appeared to move along the substratum much slower than the whole cells. No difference was found is speed and keeping direction between centriolar and non-centriolar cytoplasts.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

The mammalian interphase centrosome: two independent units maintained together by the dynamics of the microtubule cytoskeleton.

In mammalian cells the centrosome or diplosome is defined by the two parental centrioles observed in electron microscopy and by the pericentriolar material immunostained with several antibodies directed against various centrosomal proteins (gamma-tubulin, pericentrin, centrin and centractin). Partial destabilization of the microtubule cytoskeleton by microtubule-disassembling substances induced a splitting and a slow migration of the two diplosome units to opposite nuclear sides during most of the interphase in several mammalian cell lines. These units relocated close together following drug removal, while microtubule stabilization by nM taxol concentrations inhibited this process. Cytochalasin slowed down diplosome splitting but did not affect its relocation after colcemid washing. These results account for the apparently opposite effects induced by microtubule poisons on centriole separation. Moreover, they provide new information concerning the centrosome cycle and stability. First, the centrosome is formed by two units, distinguished only by the number of attached stable microtubules, but not by pericentrin, gamma-tubulin, centrin and centractin and their potency to nucleate microtubules. Second, the centrosomal units are independent during most of the interphase. Third, according to the cell type, these centrosomal units are localized in close proximity because they are either linked or maintained close together by the normal dynamics of the microtubule cytoskeleton. Finally, the relocalization of the centrosomal units with their centrioles in cells possessing one or two centrosomes suggests that their relative position results from the overall tensional forces involving at least partially the microtubule arrays nucleated by each of these entities.     

Symmetry, stability, and reversibility properties of idealized confined microtubule cytoskeletons

Many cell cytoskeletons include an aster of microtubules, with the centrosome serving as the focal point. The position of the centrosome within the cell is important in such directional activities as wound closure and interactions of immune cells. Here we analyzed the centrosome positioning as it is dictated by microtubule elasticity alone in a mechanical model of an intrinsically fully symmetric microtubule aster. We demonstrate that the symmetry and the central position of the centrosome are unstable. The equilibrium deviation of the centrosome from the center is approximately proportional to the difference of the microtubule length and cell radius. The proportionality coefficient is 1 in flat cells and 2 in three-dimensional cells. The loss of symmetry is irreversible, and in general, the equilibrium form of the aster exhibits memory of past perturbations. The equilibrium position of the centrosome as a function of the microtubule length exhibits hysteresis, and the history of the length variation is reflected in the aster form. These properties of the simple aster of elastic microtubules must be taken into account in the analysis of more comprehensive theoretical models, and in the design and interpretation of experiments addressing the complex process of cytoskeleton morphogenesis.     

Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In “frustrated” conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.     

Infection with replication-deficient adenovirus induces changes in the dynamic instability of host cell microtubules

Adenovirus translocation to the nucleus occurs through a well characterized minus end-directed transport along microtubules. Here, we show that the adenovirus infection process has a significant impact on the stability and dynamic behavior of host cell microtubules. Adenovirus-infected cells had elevated levels of acetylated and detyrosinated microtubules compared with uninfected cells. The accumulation of modified microtubules within adenovirus-infected cells required active RhoA. Adenovirus-induced changes in microtubule dynamics were characterized at the centrosome and at the cell periphery in living cells. Adenovirus infection resulted in a transient enhancement of centrosomal microtubule nucleation frequency. At the periphery of adenovirus-infected cells, the dynamic instability of microtubules plus ends shifted toward net growth, compared with the nearly balanced growth and shortening observed in uninfected cells. In infected cells, microtubules spent more time in growth, less time in shortening, and underwent catastrophes less frequently compared with those in uninfected cells. Drug-induced inhibition of Rac1 prevented most of these virus-induced shifts in microtubule dynamic instability. These results demonstrate that adenovirus infection induces a significant stabilizing effect on host cell microtubule dynamics, which involve, but are not limited to, the activation of the RhoGTPases RhoA and Rac1.     

Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity

Scratch-induced disruption of cultured monolayers induces polarity in front row cells that can be visualized by spatially localized polymerization of actin at the front of the cell and reorientation of the centrosome/Golgi to face the leading edge. We previously reported that centrosomal reorientation and microtubule polarization depend on a Cdc42-regulated signal transduction pathway involving activation of the Par6/aPKC complex followed by inhibition of GSK-3beta and accumulation of the adenomatous polyposis coli (APC) protein at the plus ends of leading-edge microtubules. Using monolayers of primary rodent embryo fibroblasts, we show here that dishevelled (Dvl) and axin, two major components of the Wnt signaling pathway are required for centrosome reorientation and that Wnt5a is required for activation of this pathway. We conclude that disruption of cell-cell contacts leads to the activation of a noncanonical Wnt/dishevelled signal transduction pathway that cooperates with Cdc42/Par6/aPKC to promote polarized reorganization of the microtubule cytoskeleton.     

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.     

Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end–directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150^Glued subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150^Glued and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150^Glued to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150^Glued expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150^Glued suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.     

Nucleokinesis in neuronal migration

Neuronal migration is a critical phase of nervous system development and can be divided into two distinct phases: extension of the leading process and movement of the cell body and nucleus (nucleokinesis). Nucleokinesis appears to require many of the same cytoskeletal and signaling molecules used in cell mitosis. Converging studies suggest it requires cytoplasmic dynein, cell polarity genes, and microtubule-associated proteins that coordinate microtubule remodeling. These coordinate first the positioning of the centrosome (microtubule organizing center) in the leading process in front of the nucleus and then the movement of the nucleus towards the centrosome. The positioning of the centrosome and the dynamic regulation that couples and uncouples the nucleus underlies directed migration of neurons.     

How cellular membranes can regulate microtubule network

Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing “tracks” for their subsequent transport.     

The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules.

gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Centrosome reorientation in regenerating endothelial monolayers requires bFGF

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti-bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.