Biomedical subjects

I B Alieva

Publications and source records attributed to I B Alieva.

At least 19 recordsLinked to original sources

PubMed2004

[The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules].

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.

Animals↗

PubMed2004

[Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment].

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.

Actins↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Dimerization↗

PubMed1999

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.

3T3 Cells↗

PubMed1997

Polyclonal antibodies against human gamma-tubulin stain centrioles in mammalian cells from different tissues.

Rabbit polyclonal antibodies were raised against the C-terminal fragment (amino acid residues 318-451) of human gamma-tubulin. These antibodies were used to stain cultured cells of various tissues (epithelium, nervous tissue, fibroblasts) from different animals (human, monkey, pig, rat, kangaroo rat, mouse, hamster, chicken, triton). The antibodies specifically stained centrioles in the interphase and mitotic cells of mammals, but not birds (chicken) or amphibians (newt). In the interphase cells, centrioles were stained as a pair of dots (or as a double dot) in 96-97% of the cells. The distances between the maternal and filial centrioles varied in different cultures. Procentrioles were stained in certain cells, but with less intensity than mature centrioles. In mitotic cells, the antibodies revealed two spots corresponding to two mitotic poles. The spots in mitosis were significantly larger than the interphase dots, but the staining was more faint. In spontaneous tripolar mitoses, only two poles were stained. Thus, it was shown that, on the one hand, gamma-tubulin is associated with centrioles irrespective of whether or not they serve as the microtubule organizing centres and, on the other hand, gamma-tubulin might not be an essential component of the microtubule organizing centres.

Animals↗

PubMed1997

Stereoscopic analysis of microtubule pattern around the centrosome in interphase PK cells after treatment with taxol and nocodazole.

In the interphase PK cells, more than 85% of microtubules radiating from the centrosome were not longer than 1.5 microns. A half of microtubules had their proximal ends free. After nocodazole treatment (20 microM), the number of microtubules attached to the centrosome decreased by 20% after 10 min of treatment, remained the same after 20 min of treatment, and increased after 60 min of nocodazole treatment slightly above the control level. After 5 and 60 min of treatment, the number of attached microtubules with the length over 0.7 micron increased twice as compared to the control level. During the first 20 min of nocodazole treatment, the immunofluorescent staining of cells with antibodies to gamma-tubulin was the same as in the control cells. The number of free microtubules decreased fourfold during the first 5 min, then it decreased slowly (for 20 min) and remained at the same level after 60 min. After 10 min of taxol (12 microM) treatment, the number of attached and free microtubules increased more than two times, whereas the number of attached microtubules with the length over 0.7 micron increased more than tenfold. After 15 min of treatment, the number of attached microtubules was slightly higher, and the number of free microtubules was half of the control level. After 20-60 min of treatment, the number of microtubules of all types decreased. Thus, upon the nocodazole treatment, the microtubules attached to the centrosome were more resistant to depolymerization: however, these microtubules were the most reactive to taxol treatment. The data obtained suggest that (a) in PK cells, the centrosome-attached microtubules occupy not all of the existing templates; (b) during prolonged treatment with inhibitors, the centrosome performed the compensatory reaction-the inhibition of microtubule assembly results in the decrease in the number of active templates on the centrosome; the inhibition of microtubule depolymerization results in the inactivation of hitherto active reserve templates. The microtubules formed on the centrosome within the first minutes disengage from it and then leave the chromosomal region.

Animals↗

PubMed1996

[The effect of the increase in the calcium concentration induced by action of the ionophore A-23187 on mitosis in cultured ESK cells].

After addition of 20 mM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells, [Ca++] in cytosol increased by more than 10 times. The maximum [Ca++] concentration was observed 1-2 min after drug introduction. Later on [Ca++] gradually decreased, and after 30 min of incubation with A23187 [Ca++] its concentration was 3-5 times higher than in the norm. 1 min after introduction of the calcium ionophore, mitotic spindles shortened for 1/3 and the angle of divergence of spindle microtubules from the centrosome extended. These changes remained for 5 min of treatment. After nocodazole treatment the length of the mitotic spindle reduced (2 min), then mitotic spindle and the metaphase plate were disrupted. The rate of mitotic spindle shortening after addition of the ionophore is about the same as after addition of nocodazole, but after ionophore treatment the metaphase plate remained for more than 5 min. Based on the results obtained we suggest that the maximum distance between spindle poles at metaphase is in the intact cell, and after any perturbation of normal microtubule dynamics the spindle may rapidly collapse. The collapsed mitotic spindle becomes more stable and its size is determined predominantly by kinetochore fibers. The metaphase spindle is completely and rapidly destroyed when the microtubule growth is prohibited, but it is preserved when this growth is restricted.

Animals↗

PubMed1995

Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.

2,4-Dinitrophenol↗

PubMed1995

[An analysis of centriolar orientation in cultured ESK cells under the action of the calcium ionophor A23187].

Addition of 20 microM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells gave an increase of Ca2+ in cytosol by more than 10 times. The maximum of [Ca2+] was achieved in 1-2 min after introduction of the drug. Later on [Ca2+] gradually decreased, and after 30 min of incubation with A23187 [Ca2+] was 3-5 times above normal level. Immunofluorescent and electron microscope studies showed no alterations in the microtubule system of interphase cells after 1-30 min treatment. The electro microscope study showed that-following 1.5 min introduction of the drug the random orientation of material and daughter centrioles changed: most of them settled down at an angle more, than 74 degrees to the substrate surface. After 3 min of A23187 treatment more than half of maternal centrioles were oriented perpendicular to the substrate surface. After 5 min of A23187 treatment, the percentage of maternal centrioles with perpendicular orientation was the same and this orientation remained for 30 min. The percentage of perpendicular daughter centrioles decreased after 3 min of treatment, and after 30 min their orientation was random. We suggest that the perpendicular orientation of centrioles to the substrate surface is mediated through centrosome-associated calcium-binding proteins.

Animals↗

PubMed1992

[A stereoscopic analysis of centrosome structure in the cells of a tissue culture under the action of energy metabolism inhibitors. I. 2,4-Dinitrophenol, deoxyglucose, sodium azide and the calcium ionophore A23187].

A 30-min action of energy transfer inhibitors (2,4-dinitrophenol, deoxyglucose, azide and calcium ionophore A23187) on tissue culture cells results in a significant increase in the quantity of microtubules around the centrosome. After the action of all the inhibitors, mostly increases the number of long microtubules with free proximal end oriented towards the centrosome. It is suggested that energy transfer inhibitors may stimulate foundation of microtubules on the centrosome and stabilize free microtubules, while they exert no effect on the frequency of detachment of microtubules from the centrosome.

2,4-Dinitrophenol↗

PubMed1992

[A stereoscopic analysis of the centrosome structure in tissue culture cells under the action of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ouabain].

The action of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and ouabain results in significant increase of the quantity of microtubules with attached and free proximal end around the centrosome. The majority of free microtubules are oriented with their proximal ends towards the heads of pericentriolar satellites or towards the walls of centriolar cylinders. The increasing of total number of microtubules is the result of the increasing of microtubules attached to or oriented towards the pericentriolar satellites. Comparing the action of FCCP and ouabain from one side and taxol from the other side it is possible to conclude that FCCP and ouabain promote the initiation of microtubule growth in the centrosome of they have an influence on the frequency of separation of the microtubules from microtubule nucleating centers.

Animals↗

PubMed1991

Induction of multipolar mitoses in cultured cells: decay and restructuring of the mitotic apparatus and distribution of centrioles.

During recovery after a long (up to 12 h) treatment of pig embryo culture cells (PK) with nocodazole at concentrations of 0.02 microgram/ml and 0.2 microgram/ml all c-metaphase cells divide normally into two daughter cells. During recovery after a short (1-4 h) treatment with 0.6 microgram/ml nocodazole only multipolar mitoses (as a rule tripolar) arise. At the ultrastructural level, the increasing nocodazole concentration leads to progressive disruption of the mitotic spindle. At a nocodazole concentration of 0.2 microgram/ml kinetochores are not associated with microtubules. At a nocodazole concentration of 0.6 microgram/ml there are no microtubules around the centrosomes, and in every cell one of the two diplosomes disintegrates. In tripolar telophase centrioles are distributed among the spindle poles generally in a 2:2:0 pattern. Mother and daughter centrioles are always disoriented but not separated. The centriole-free pole contains a cloud of electron-dense material. During tripolar division two of the three daughter cells mainly fuse shortly after telophase forming one binucleate cell. Thus a multipolar mitosis arises as a result of the uncoupling of mother centrioles and spindle microtubules, but not of the duration of the c-mitotic arrest. Centriole-free poles account for the divergence of chromosomes, but mainly they are unable to ensure the normal cytokinesis of daughter cells.

Animals↗

PubMed1991

[A stereoscopic analysis of the centrosome structure in the cells of continuous and primary cell cultures].

A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells. It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends. The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres.

Animals↗

PubMed1990

[The centrosome reaction in tissue-culture cells to depolarization of the cell membranes].

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ouabain and calcium ionophore A23187 caused a centrosome restructuring expressed in mean number increase of satellites on the active (mother) centriole, in an increase of the mean slope of centrioles to the substrate surface and in the more frequent occurrence of primary cilia. In the presence of FCCP the effect appeared only in 10 min and retained for 2 h. The ouabain caused the separation of active and inactive centrioles in more than in a half of the cells. The data obtained permit to conclude that depolarization of the plasmatic membrane only is needed for the initiation of centrosome restructuring. The authors propose that this reaction of centrosome is a component of the cell overall response to nonspecific lesions.

Animals↗

PubMed1990

[The effect of the disruption of the cytoskeletal elements on uncoupler-induced changes in the centrosome].

The disruption of microtubules with nocodazole or microfilaments with cytochalasin B did not prevent mother centrioles from nonrandom, preferentially perpendicular orientation with respect to the substrate plane after FCCP treatment. The microtubules affect negatively the reorientation of centrioles, because after their disruption by nocodazole the percentage of centrioles with the perpendicular orientation (the angle is tipped to the substrate plane by over 74 degrees) is seen to increase. The microfilaments have the positive effect, because after their disruption by cytochalasin B the share of centrioles with the perpendicular orientation decreases. Thus, our observations do not support the hypothesis that the long microtubules can provide the perpendicular orientation of centrioles anchoring them in the cytoplasm.

Animals↗

PubMed1989

[Reaction of the cell center to exposure to the calcium ionophore A-23187].

Calcium ionophore A23187, taken at a concentration of 0.1 microgram/ml, quickly uncouples mitochondria in PE culture cells in medium 199. The cell ultrastructure undergoes reversible changes (especially that of mitochondria): maximum changes occur 2 hours after the start of the treatment; in 8 hours they become less pronounced. The adaptation of cells does not involve the ionophore inactivation in the medium. 10 micrograms/ml of A23187 induces gradual but irreversible alterations. Microtubules in PE cells are not destroyed when incubated in medium 199 containing 10 micrograms/ml of A23187 and 11 mM Ca2+. The addition of 10 micrograms/ml ionophore to the normal 199 medium (1.26 mM Ca2+) results in the formation of electron dense bodies in the cell center 30 minutes after the start of incubation. These bodies disappear in the course of a subsequent incubation. The number of cells with primary cilia decreases. The percentage of centrioles located perpendicularly to the substrate increases 30 minutes following treatment with 0.1 microgram/ml A23187 in medium 199. 2 hours after the start of treatment with 0.1 microgram/ml ionophore no such changes are detected; an electron dense halo appears around the centriolar cylinders. 8 hours after the start of treatment the structure of the cell center does not differ from the normal one.

Animals↗

PubMed1989

[Morphofunctional characteristics of the striated centrosome rootlets of cultured embryonic pig kidney cells].

The analysis of the centrosome structure in PK-cells has shown that the striated rootlets occur in 25-30% of the cells. Most frequently, the centrosome contains 1-2 rootlets, oriented either to the active or to the nonactive centriole. Occasionally, they occur in both the centrioles simultaneously. The presence of striated rootlets does not correlate with the presence of a primary cilium in the centrosome: there occur centrosomes with the primary cilium but without striated rootlets, and conversely: with striated rootlets but without the primary cilium.

Animals↗