Divergent Impact of Actin Isoforms on Division of Epithelial Cells.

We investigated distribution and functions of beta- and gamma-cytoplasmic actins (CYAs) at different stages of non-neoplastic epithelial cell division using laser scanning microscopy (LSM). Here, we demonstrated that beta- and gamma-CYAs are spatially segregated in the early prophase, anaphase, telophase, and cytokinesis. Small interfering RNA (siRNA) experiments revealed that in both beta-CYA- and gamma-CYA-depleted cells, the number of cells was significantly reduced compared with the siRNA controls. Beta-CYA depletion resulted in an enlargement of the cell area in metaphase and high percentage of polynuclear cells compared with the siRNA control, indicating a potential failure of cytokinesis. Gamma-CYA depletion resulted in a reduced percentage of mitotic cells. We also observed the interdependence between the actin isoforms and the microtubule system in mitosis: (i) a decrease in the gamma-CYA led to impaired mitotic spindle organization; (ii) suppression of tubulin polymerization caused impaired beta-CYA reorganization, as incubation with colcemid blocked the transfer of short beta-actin polymers from the basal to the cortical compartment. We conclude that both actin isoforms are essential for proper cell division, but each isoform has its own specific functional role in this process.

Advances and Challenges of Nanoparticle-Based Macrophage Reprogramming for Cancer Immunotherapy.

Despite the progress of modern medicine, oncological diseases are still among the most common causes of death of adult populations in developed countries. The current therapeutic approaches are imperfect, and the high mortality of oncological patients under treatment, the lack of personalized strategies, and severe side effects arising as a result of treatment force seeking new approaches to therapy of malignant tumors. During the last decade, cancer immunotherapy, an approach that relies on activation of the host antitumor immune response, has been actively developing. Cancer immunotherapy is the most promising trend in contemporary fundamental and practical oncology, and restoration of the pathologically altered tumor microenvironment is one of its key tasks, in particular, the reprogramming of tumor macrophages from the immunosuppressive M2-phenotype into the proinflammatory M1-phenotype is pivotal for eliciting antitumor response. This review describes the current knowledge about macrophage classification, mechanisms of their polarization, their role in formation of the tumor microenvironment, and strategies for changing the functional activity of M2-macrophages, as well as problems of targeted delivery of immunostimulatory signals to tumor macrophages using nanoparticles.

Structural Features of Actin Cytoskeleton Required for Endotheliocyte Barrier Function.

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.

The Centrosome as the Main Integrator of Endothelial Cell Functional Activity.

The centrosome is an intracellular structure of the animal cell responsible for organization of cytoplasmic microtubules. According to modern concepts, the centrosome is a very important integral element of the living cell whose functions are not limited to its ability to polymerize microtubules. The centrosome localization in the geometric center of the interphase cell, the high concentration of various regulatory proteins in this area, the centrosome-organized radial system of microtubules for intracellular transport by motor proteins, the centrosome involvement in the perception of external signals and their transmission - all these features make this cellular structure a unique regulation and distribution center managing dynamic morphology of the animal cell. In conjunction with the tissue-specific features of the centrosome structure, this suggests the direct involvement of the centrosome in execution of cell functions. This review discusses the involvement of the centrosome in the vital activity of endothelial cells, as well as its possible participation in the implementation of barrier function, the major function of endothelium.

Dysfunction of Kidney Endothelium after Ischemia/Reperfusion and Its Prevention by Mitochondria-Targeted Antioxidant.

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen-glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.

[The reorganization of actin cytoskeleton and microtubule system of human endothelial vein in the intercellular contacts formation].

Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, ß- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against ß-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 µM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of ß-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always higher than in free lamella region.

[Endothelial cell cytoskeleton reorganization during functional monolayer formation in vitro].

The endothelium lining the inner surface of blood vessels regulates vascular permeability, permitting the exchange between the blood circulating in vessels and tissue fluid, and performs thereby the barrier function. Endothelial cells cultured in vitro retain the ability to perform a barrier function that is inherent in vascular endothelial cells in vivo. Endothelial monolayer in vitro is a unique model system that allows studying the interaction of cytoskeletal and adhesive structures of endothelial cells from the earliest stages of its formation. In this paper we describe and characterize quantitatively the changes in the cytoskeleton of endothelial cells from the time of endothelial cells spreading on the glass and formation of the first contacts between neighboring cells un- til the formation of a functional confluent monolayer. The main type of intermediate filaments of endothelial cells were vimentin filaments. The location of vimentin filaments and their number did not change at different stages of the endothelial monolayer formation, they occupied more than 80% of the cells. The system of actin filaments in endothelial cells was represented by the cortical actin at the cell periphery and bundles of actin stress fibers arranged in parallel. Upon the formation of contacts with neighboring cells the number and thickness of actin filaments increased. In addition, the formation of the endothelial monolayer led to changes in microtubule network, which was evident from the increase in the number of microtubules at the cell edge. Further, at all stages of assembling the endothelial monolayer, the number of microtubules formed at the cell margin in the area of cell-cell contacts exceeded the number of microtubules in the area of the free lamellae.

Role of microtubule cytoskeleton in regulation of endothelial barrier function.

Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.

[The effect of Rho-kinase inhibition depends on the nature of factors that modify endothelial permeability].

Endothelium lining the inner surface of all vessels plays barrier role and regulates permeability of vascular walls controling the exchange between circulating blood and tissue fluids. Disturbance of normal functions (endothelial dysfunction) can be caused by both internal, and external factors. Endothelial dysfunction is characterized by increased vascular wall permeability observed in many human diseases. Dysfunction is also a drug side effect of oncological diseases treatment by mitosis-blocking medications. Depolymerization of microtubules is the first step in the cascade of reactions leading to endothelial barrier dysfunction, and this stage is universal, it does not depend upon the nature of a factor provoking dysfunction. To develop the strategy of barrier dysfunction prevention, we are supposed here to find out to what stage the endothelial cell cytoskeleton reaction during the development of barrier dysfunction is universal. It has been found that the cascade stages, which follow the microtubule depolymerization and are connected with Rho-Rho-kinases activity, have the features depending on the factor provoking barrier dysfunction. Under suppression of Rho-kinase activity, the reaction of actin filaments does not depend on what substance caused dysfunction. But the microtubule system responds to the treatment varies depending on the dysfunction-provoking factor. Unlike thrombin, under the conditions of Rho-kinase activity suppression, nocodazole renders more strong effect, as much as possible destroying both dynamic, and stable microtubules. Thus, regardless of the dysfunction provoking factor, the initial stages of dysfunction connected with the depolymerization of microtubules appear to be unalterable. Consequently, endothelial cell defence strategy should be based on cytoplasmatic microtubules protectors application instead of employment of the factors involved in the cascade at later stages as we assumed earlier.

[Radial-organized microtubules provide maintenance of the cell shape and more effective intercellular transport than in the case of free microtubules].

Microtubules take part in very different cell processes including cell polarization and migration, intercellular transport and some others. Therefore the microtubules spatial organization is crucial for normal cell behaviour. Fibroblasts have radial microtubule array consisting of microtubules running from the centrosome. This microtubule array includes two components: (1) centrosomal microtubules with their minus ends attached to the centrosome and with their plus ends radiating to the cell periphery and (2) free microtubules with the ends non-attached to the centrosome. Distinction in the dynamic properties, intercellular organization and structure of centrosome-attached and free microtubules allow us to assume that their functions in the cell are also different. In order to investigate centrosome-attached and free microtubules functions we used the cytoplasts--experimentally denucleated cellular fragments and under certain condition lacking of the centrosome as well--which contain only free microtubules. Centrosome-containing cytoplasts do not differ significantly in the form, general morphology and the size from the intact cells. At the same time centrosome-lacking cytoplasts keep extremely thinned out network of microtubules located in the central area of the cytoplast. These cytoplasts lose the original cell shape usual for fibroblasts and get rough, with protrusions, lamella; the internal architecture of the cytoplasm and organoids arrangement is also broken. Saltatory movements in the centrosome-containing cytoplasts are similar to those in the intact cells, and saltatory movements in centrosome-lacking cytoplasts show half the speed and smaller distances compared with intact cells. Besides, the saltatory movements of granules in the centrosome-lacking cytoplasts occur mainly in the central regions of the cytoplasts and they are less ordered than in the intact cells and in the cytoplasts kept the centrosome. We believe that radial organization of the microtubules provide effective transport and dynamical interactions of microtubules plus ends with cortical structures of the cell, which are sufficient for maintenance of typical fibroblast-like shape, whereas disorganized free microtubules by themselves cannot keep up the shape and intercellular organization characteristic of fibroblasts.

The centrosome is a polyfunctional multiprotein cell complex.

Contemporary knowledge about centrosome proteins and their ensembles, which can be divided into several functional groups--microtubule-nucleating proteins, microtubule-anchoring proteins, centriole-duplication proteins, cell cycle control proteins, primary cilia growth regulation proteins, and proteins of regulation of cytokinesis--is reviewed. Structural-temporal classification of centrosomal proteins and the scheme of interconnection between the different centrosomal protein complexes are presented.

[The centrosome--a riddle of the "cell processor"].

In the present review the description of history of the centrosome investigation is given and the current state of knowledge of this cellular structure in morphological, biochemical, and functional aspects is presented. Besides of the classical functions of the centrosome as a MT nucleating, MT ancoriging, and MT organizing center, the idea about the centrosome as a cellular regulating center and as a structural part of the mechanism operating dynamic morphology of a cell is discussed.

[The microtubule system in endothelial barrier dysfunction: disassembly of peripheral microtubules and microtubules reorganization in internal cytoplasm].

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

[The spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts].

Microtubules spatial organization is essential for different cellular processes to proceed normally. It is supposed traditionally, that the fibroblasts have radial microtubule array consisting of long microtubules running from the centrosome. However, the detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study we used laser photobleaching for the analysis of the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, and then in the bleached zone growth of microtubules in the centrosome region and in the peripheral parts of cytoplasm was analyzed. In most cases microtubules growth in the bleached zone occurred rectilinearly, on the distance up to 5 microm they seldom bend more than 10-15 degrees. We considered a growing fragment of the microtubule as a vector with the beginning in the point of occurrence and with the end in a point where growth terminated (or the end point after 30 s if microtubule's persistent growth proceeded longer). We defined the direction of microtubules 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 the vector of comparison inside of the bleached zone from the centrosome to the beginning of the growing microtubule segment; in fibroblast lamella and in fibroblast trailing part we used, the vector of comparison was directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing immediately from the centrosome grew along the cell radius. However at a distance of 10 microm from the centrosome radially growing microtubules gave 40% from the overall number, and at a distance of 20 microm--only 25%. The rest of microtubules grew in different directions, with the preferred angle between their growth direction and cell radius around 90 degrees. Fibroblast lamella and trailing part 80% of all microtubules grew along the cell long axis or at the angle no more than 20 degrees, and 10-15% of microtubules grew along cell axis but towards the centrosome. Thus, in 3T3 fibroblasts the radial system of microtubules is perturbed starting from the 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 cell axis. From the results obtained we conclude that orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome, but their orientation is preset by local factors.

[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.

[Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment]. / Reorganizatsiia sistemy mikrotrubochek v kletkakh legochnogo éndoteliia v otvet na vozdeistvie trombina.

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.

[The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules]. / Dinamika repolimerizatsii mikrotrubochek v kletke: bystryi rost ot tsentrosomy i medlennoe vosstanovlenie svobodnykh mikrotrubochek.

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.

[Evaluation of various methods of measurement of the microtubule length in the cytoplasm of cultured cells]. / Analiz razlichnykh metodicheskikh podkhodov k izmereniiu dliny mikrotrubochek v tsitoplazme kul'tiviruemykh kletok.

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.

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules.

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.

Interphase microtubules in cultured cells: long or short?

Presently, the question about the length of microtubules in the interphase cell became actual, since the parameters of dynamic instability of the plus end measured in vivo do not allow one to explain the rapid turnover of the long microtubule system. The problem may be solved if one of the following suppositions is assumed: either microtubules undergo rapid depolymerization from the minus end or they are on the average much shorter than it is usually considered. To check the last hypothesis, we have reconstructed microtubules using stereophotography of electron microscopic sections. Microtubules around the cell center in cultures of epithelial cells (kidney of pig embryo (PK) and bovine trachea (FBT)) and fibroblasts (MEF, primary mouse embryo fibroblasts, and L cells), as well as at the periphery of PK cells were studied. All in all, no less than 200 microtubules were found near the centrosome in each cell culture. From 2.5 to 8% microtubules were beyond the studied volume (4.0 x 5.5 x 1.5 microm). Most of microtubules in all studied cell lines were up to 1 microm and about 1/3 of them were 0.2-0.4 microm long. The mean length of microtubules surrounding the centrosome in different cell lines differed insignificantly and equalled 0.4-0.8 microm. In this case, the microtubules attached to the centrosome were on the average slightly shorter than the free ones. Thus, almost all microtubules around the centrosome are short, and the majority of those attached to it do not reach the cell periphery. A similar reconstruction of a part of the PK cell cytoplasm (10 x 35 microm) has shown that at the periphery, the mean length of microtubules is about 1.6 microm and most of them are 0.5 to 1.5 microm long. Thus, our data confirm the recent hypothesis of Vorobjev et al. (I. A. Vorobjev, T. M. Svitkina, and G. G. Borisy, J. Cell Sci. 110:2635-2645 (1997)) that most of microtubules in the cells are not connected with the centrosomes.