Centrosomal Control of Microtubule Dynamics

The Journal of Cell Biology, 1980

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.

Journal of Cell Biology, 1997

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931–937).To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends ...

Journal of Cell …, 2007

Tsitologiia, 2008

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 grow...

Cell and Tissue Biology, 2007

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.

Biology of the Cell, 2002

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 µm), and the elongation stage (from 1 to 7 µm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.

Doklady Biological Sciences, 2005

The Journal of Cell Biology, 2003

Current Biology, 2003

until late prometaphase when a bipolar spindle was present. The results demonstrate that peripheral bundles and clusters are incorporated into the forming spindle in two ways. In some cases, clusters of peripheral microtubules moved inward along astral microtubules;

Current Opinion in Cell Biology, 2017

The process of cellular differentiation requires the distinct spatial organization of the microtubule cytoskeleton, the arrangement of which is specific to cell type. Microtubule patterning does not occur randomly, but is imparted by distinct subcellular sites called microtubule-organizing centers (MTOCs). Since the discovery of MTOCs fifty years ago, their study has largely focused on the centrosome. All animal cells use centrosomes as MTOCs during mitosis. However in many differentiated cells, MTOC function is reassigned to non-centrosomal sites to generate non-radial microtubule organization better suited for new cell functions, such as mechanical support or intracellular transport. Here, we review the current understanding of non-centrosomal MTOCs (ncMTOCs) and the mechanisms by which they form in differentiating animal cells.