Anton V. Burakov

Centrosome positioning in interphase cells

The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

By comparing computer modeling predictions with observations, we conclude that strong dynein and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center.

Cytoplasmic Dynein is Involved in the Retention of Microtubules at the Centrosome in Interphase Cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid (t1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein‐inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT‐anchoring proteins.

Dynactin Subunit p150Glued Isoforms Notable for Differential Interaction with Microtubules

Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N‐terminal region that contains a globular cytoskeleton‐associated protein (CAP)‐Gly domain and basic microtubule‐binding domain of unknown structure. The p150Glued gene has a complicated intron–exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued‐1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued‐1B with a basic domain of 21 aa because of the lack of exons 5–7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase‐polymerase chain reaction (RT‐PCR) and western blot data, p150Glued‐1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP‐p150Glued‐1A and ‐1B fusion proteins and immunostaining of cultured cells with 1A‐specific antibodies show that the p150Glued‐1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus‐ends. The higher affinity of the p150Glued‐1A isoform for microtubules is confirmed by a co‐pelleting assay. In fibroblast‐like cells, the interaction of p150Glued‐1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued‐1B. In polarized cells, p150Glued‐1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued‐1A and p150Glued‐1B interaction with microtubules and their tissue‐specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.

The centrosome keeps nucleating microtubules but looses the ability to anchor them after the inhibition of dynein-dynactin complex.

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.

Association of nucleus and centrosome: magnet or velcro?

A structural link between cells nucleus and centrosome was proposed years ago. Such a link was suggested to maintain nucleus–centrosome axis, determine polarity of interphase cells and ensure spindle assembly in mitotic cells. The idea of structural link is supported by the facts that centrosomes are usually located in close proximity to the nuclei and remain attached to the nuclei in mildly homogenated cells. However, juxtaposed location can result rather from the tendency of both organelles to occupy central position in cell than from the existence of a specific structural link. Moreover, the nucleus was shown to be transported towards the centrosome along microtubules by dynein bound to nuclear envelope; inhibition of dynein results in the increase of nucleus–centrosome distance. The interaction of both organelles is disturbed in response to actin depolymerisation, although the exact role of actin filaments in this process remains unknown. The link between the nucleus and the centrosome can support simultaneous migration of nuclei and centrosomes in large cells and in syncytia, but its existence in interphase fibroblast‐like and epithelia‐like cells was not confirmed yet. Further studies include direct visualisation of a specific link between centrosome and nucleus and elucidation of actin role in its formation.

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.

LOSK (SLK) protein kinase activity is necessary for microtubule organization in the interphase cell centrosome.

Microtubules, polymers of tubulin, are one of the main components of cytoskeleton. In many types of animal cells, microtubules form a radial network whose center is the centrosome. The microtubule network is very dynamic and can be rearranged rapidly, e.g., during the transition of a cell to mitosis. The mobility of the network and its geometry is important for many cellular functions and determined by inherent properties of the microtubules and the centrosome.

SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization

Cell motility is an essential complex process that requires actin and microtubule cytoskeleton reorganization and polarization. Such extensive rearrangement is closely related to cell polarization as a whole. The serine/threonine kinase SLK/LOSK is a potential regulator of cell motility, as it phosphorylates a series of cytoskeleton‐bound proteins that collectively participate in the remodeling of migratory cell architecture. In this work, we report that SLK/LOSK is an indispensable regulator of cell locomotion that primarily acts through the small GTPase RhoA and the dynactin subunit p150Glued. Both RhoA and dynactin affect cytoskeleton organization, polarization, and general cell locomotory activity to various extents. However, it seems that these events are independent of each other. Thus, SLK/LOSK kinase effectively functions as a switch that links all of the processes underlying cell motility to provide robust directional movement.

Disturbance of the radial system of interphase microtubules in the presence of excess serum in cell culture medium

The work is focused on the intracellular role of microtubule organization. The population of Vero cells with chaotic microtubules was obtained after cultivation with the excess of serum. Such cells were characterized by the increased area and exhibited slightly dispersed Golgi, whereas growth rate remained unaltered. Thus we have shown that radial organization of microtubules is not essential even for cells where it is well pronounced.