Interphase microtubules in cultured cells: long or short? (original) (raw)
Related papers
Microtubules in the metaphase-arrested mouse oocyte turn over rapidly
Proceedings of the …, 1990
After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5-(and -6)-carboxy-x-rhodamine], was microhijected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging '=22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.
Cellular regulation of microtubule organization
The Journal of cell biology, 1984
Microtubules are constituents of axonemes, mitotic spindles, and elaborate arrays in interphase cells, and, with intermediate filaments and microfilaments, are among the most prevalent structures visualized in the cytomatrix (22, 44). With the exception ofthe A microtubule ofcilia and flagella, the lattice geometry ofmicrotubules is highly conserved. However, each of the major subunits of microtubules, a-and a-tubulin, shows heterogeneity. The number ofa-and f3-tubulin subspecies differs among tissues and organisms, and a number of types of analysis are used to examine how these tubulin variants are related to specific cell functions (1, 9-11, 33, 40). Investigations of the number and complexity of genes coding for these polypeptides have also been initiated (see reference 13 for review). However, the mechanisms that regulate the posttranslational compartmentalization of subunits, the spatial and temporal assembly of subunits into microtubules, and the integration of microtubules in various cellular events are still largely unknown. There are many levels at which the formation and organization of microtubules might be determined. A postulate originating from early analyses of mitotic spindle formation (32) was that a pool of subunits existed in equilibrium with formed microtubules; increases in the subunit concentration could therefore result in a net increase in polymer. With few exceptions, however, a rapid increase in the total tubulin pool does not appear to occur before the elaboration of more extensive microtubule arrays. For example, our studies (42, 50) have demonstrated that mouse neuroblastoma cells possessing microtubule-filled neurites contain four to five times more tubulin polymer than rounded, nondifferentiated cells, but the total tubulin content of these two cell types is the same. On the basis of volume calculations, the equilibrium concentration of subunits in the nondifferentiated cells is at least twice that in differentiated cells. Data such as this indicate that a simple equilibrium between subunit and polymer cannot account for the changes in microtubule formation coordinated with certain cellular events. In addition, recent findings show that an increase in the subunit concentration in cells, brought about either by drug treatment (15) or injection of tubulin (16), results in a depression of tubulin synthesis and the loss of tubulin mRNA. These data suggest that cells autoregulate the total tubulin pool and that this may be effected by "monitoring" of the monomer concentration (14).
Increased visualization of microtubules by an improved fixation procedure
Journal of Histochemistry & Cytochemistry, 1977
We have found that when a buffer utilized for in vitro polymerization of microtubules, i.e., 1 mM guanosine triphosphate, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9 polymerization mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in a number of microtubules greater than or equal to 1 mu long, (c) an enhanced association of microtubules with cytoplasmic organelles, and (d) an increased clustering of 100 A filaments located in a perinuclear region of the cell. Furthermore, we found that after we incubated purified chick brain microtubules on a Sephadex G-25 column pre-equilibrated with polymerization mi...
Lattice defects in microtubules: protofilament numbers vary within individual microtubules
Journal of Cell Biology, 1992
We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.
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.
Membrane & cell biology
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 a...