K. M. Smurova

Novel role of microtubules in thrombin-induced endothelial barrier dysfunction

Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin‐induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin‐ induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin‐induced activation of Rho and Rho‐kinase. Expression of activated Gα12/13 subunits involved in thrombin‐mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF‐specific negative regulator RGS preserved MT from thrombin‐induced disassembly. Consistent with these results, expression of activated RhoA and Rho‐kinase induced MT disassembly. Conversely, thrombin‐induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho‐kinase mutants or by pharmacological inhibition of Rho‐kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin‐induced EC barrier dysfunction and indicate G‐protein‐dependent mechanisms of thrombin‐induced MT alteration.—Birukova, A. A., Birukov, K G., Smurova, K., Adyshev, D., Kaibuchi, K., Alieva, I., Garcia, J. G. N., Verin, A. D. Novel role of microtubules in thrombin‐induced endothelial barrier dysfunction. FASEB J. 18, 1879‐1890 (2004)

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti‐cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND‐induced activation of Rho GTPase. Rho‐mediated activation of its downstream target, Rho‐kinase, induced phosphorylation of Rho‐kinase effector EC MLC phosphatase (MYPT1) at Thr696 and Thr850 resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho‐MLC, which induced acto‐myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho‐kinase by Y27632 abolished ND‐induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)‐Rho, or DN‐Rho‐kinase) attenuated ND‐induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho‐dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho‐kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND‐induced MT disassembly. J. Cell. Physiol. 201: 55–70, 2004.

The LIM-domain protein Zyxin binds the homeodomain factor Xanf1/Hesx1 and modulates its activity in the anterior neural plate of Xenopus laevis embryo

The question of how subdivision of embryo into cell territories acquiring different fates is coordinated with morphogenetic movements shaping the embryonic body still remains poorly resolved. In the present report, we demonstrate that a key regulator of anterior neural plate patterning, the homeodomain transcriptional repressor Xanf1/Hesx1, can bind to the LIM‐domain protein Zyxin, which is known to regulate cell morphogenetic movements via influence on actin cytoskeleton dynamics. Using a set of deletion mutants, we found that the Engrailed‐type repressor domain of Xanf1 and LIM2‐domain of Zyxin are primarily responsible for interaction of these proteins. We also demonstrate that Zyxin overexpression in Xenopus embryos elicits effects similar to those observed in embryos with downregulated Xanf1. In contrast, when the repressor‐fused variant of Zyxin is expressed, the forebrain enlargements typical for embryos overexpressing Xanf1 develop. These results are consistent with a possible role of Zyxin as a negative modulator of Xanf1 transcriptional repressing activity. Developmental Dynamics 237:736–749, 2008.

Microtubule system in endothelial barrier dysfunction: Disassembly of peripheral microtubules and microtubule reorganization in internal cytoplasm

Endothelial cell barrier dysfunction is associated with dramatic cytoskeletal reorganization, the activation of actomyosin contraction, and, finally, gap formation. Although the role of microtubules in the regulation of endothelial cell barrier function is not fully understood, a number of observations allow for the assumption that the reaction of the microtubule is an extremely important part in the development of endothelial dysfunction. These observations have forced us to examine the role of microtubule reorganization in the regulation of the endothelial cell barrier function. In quiescent endothelial cells, microtubule density is the highest in the centrosome region; however, microtubules are also present near the cell margin. The analysis of microtubule distribution after specific antibody staining using the method of measurement of their fluorescence intensity showed that, in control endothelial cells, the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of exponential regression. The edemagenic agent, thrombin (25 nM), caused the rapid increase of endothelial cell barrier permeability accompanied by a fast decrease in quantity of the peripheral microtubules and reorganization of the microtubule system in the internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress within as little as 5 min after the beginning of treatment). Both effects are reversible; within 60 min after the beginning of treatment, the microtubule network does not differ from the standard one. Thus, the microtubule system is capable of adapting to the influence of a natural regulator, thrombin. The reorganization of microtubules develops more quickly than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in the circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

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.

Dose-dependent effect of nocodazole on endothelial cell cytoskeleton

The endothelium lining the inner surface of blood vessels fulfils an important barrier function and specifically, it controls vascular membrane permeability as well as nutrient and metabolite exchange in circulating blood and tissue fluids. Disturbances in vascular endothelium barrier function (vascular endothelium dysfunction) are coupled to cytoskeleton rearrangements, actomyosin contractility, and as a consequence, formation of paracellular gaps between endothelial cells. Microtubules constitute the first effector link in the reaction cascade resulting in vascular endothelium dysfunction. Increased vascular permeability associated with many human diseases is also manifested as a side effect in anticancer mitosis-blocking therapy. The aim of this study was to examine the possibility of preventing side effects of mitostatic drugs in patients with vascular endothelium dysfunction and to establish effective doses able to disrupt the microtubular network without interfering with the endothelial barrier function. Previously, it was found that the population of endothelial cell microtubules is heterogeneous. Along with dynamic microtubules, cell cytoplasm contains a certain amount of post-translationally modified microtubules that are less active and less susceptible to external influences than dynamic microtubules. We have shown that the area occupied with stable microtubules is relatively large (approx. one third of the total cell area). We assume that it can account for a higher resistance of the endothelial monolayer to factors responsible for vascular endothelium dysfunction. This hypothesis was validated in this study, in which nocodazole was used to induce vascular endothelium dysfunction in lung endothelial cells. The effect of nocodazole on endothelial cell cytoskeleton was found to be dose-dependent. Nocodazole in micromolar concentrations not only irreversibly changed the barrier function, but also upset the viability of endothelial cells and induced their death. Nanomolar concentrations of nocodazole also increased the permeability of the endothelial monolayer; this effect was reversible at the drug concentration ranging from 100 to 200 nM. At 100 nM, nocodazole induced partial disruption of the microtubule network near the cell margin without any appreciable effect on acetylated microtubules and actin filaments. At 200 nM, nocodazole exerted a pronounced effect on the system of dynamic (but not acetylated) microtubules and increased the population of actin filaments in the central region of the cell. Our data suggest that disruption of peripheral microtubules triggers a cascade of reactions culminating in endothelial barrier dysfunction; however, the existence of a large population of microtubules resistant to nanomolar concentrations of the drug provides higher viability of endothelial cells and restores their functional activity.

Inhibition of RHO-kinase depends on factors that modify endothelial permeability

The endothelium that lines the inner surface of all vessels plays the role of a barrier and regulates the permeability of vascular walls that control the exchange between circulating blood and tissue fluids. The disturbance of normal functions (endothelial dysfunction) can be caused both internal and external factors. Endothelial dysfunction is characterized by the increasing permeability of the vascular wall, as is observed in many human diseases. The dysfunction is also a side effect observed during the treatment of cancer with mitosis-blocking drugs. The depolymerization of microtubules is the first step in the cascade of reactions that lead to the dysfunction of the endothelial barrier. This stage is general and does not depend on the nature of factors that provoke the dysfunction. To develop the strategy to prevent barrier dysfunction, the purpose of the present work was to elucidate the extent to which the endothelial cell cytoskeleton reaction will be universal in the barrier dysfunction. We found that cascade events that followed microtubule depolymerization and associated with the activity of Rho-Rho-kinases have features that depend on the factor that provokes barrier dysfunction. With Rho-kinase activity, suppressed actin filament behavior is independent of the agent that caused the dysfunction. Conversely, the microtubule system reacts differently to the treatment and depends on the factor that provokes the dysfunction. Under the suppression of Rho-kinase activity, unlike thrombin, nocodazole destroys both dynamic and stable microtubules. Thus, independent of the dysfunction-provoking factor, initial stages of the dysfunction associated with the depolymerization of microtubules appeared to be unchangeable. Consequently, the endothelial cell defense strategy should be based on the application of cytoplasmatic microtubule protectors, rather than the use of factors involved in the cascade at later stages, as we had previously assumed.