Centrosome fine ultrastructure of the osteocyte mechanosensitive primary cilium.

The centrosome is the principal microtubule organization center in cells, giving rise to microtubule-based organelles (e.g., cilia, flagella). The aim was to study the osteocyte centrosome morphology at an ultrastructural level in relation to its mechanosensitive function. Osteocyte centrosomes and cilia in tibial cortical bone were explored by acetylated alpha-tubulin (AαTub) immunostaining under confocal microscopy. For the first time, fine ultrastructure and spatial orientation of the osteocyte centrosome were explored by transmission electron microscopy on serial ultrathin sections. AαTub-positive staining was observed in 94% of the osteocytes examined (222/236). The mother centriole formed a short primary cilium and was longer than the daughter centriole due to an intermediate zone between centriole and cilium. The proximal end of the mother centriole was connected with the surface of daughter centriole by striated rootlets. The mother centriole exhibited distal appendages that interacted with the cell membrane and formed a particular structure called "cilium membrane prolongation." The primary cilium was mainly oriented perpendicular to the long axis of bone. Mother and daughter centrioles change their original mutual orientation during the osteocyte differentiation process. The short primary cilium is hypothesized as a novel type of fluid-sensing organelle in osteocytes.

The centrosome is a polyfunctional multiprotein cell complex.

Contemporary knowledge about centrosome proteins and their ensembles, which can be divided into several functional groups--microtubule-nucleating proteins, microtubule-anchoring proteins, centriole-duplication proteins, cell cycle control proteins, primary cilia growth regulation proteins, and proteins of regulation of cytokinesis--is reviewed. Structural-temporal classification of centrosomal proteins and the scheme of interconnection between the different centrosomal protein complexes are presented.

[The centrosome--a riddle of the "cell processor"].

In the present review the description of history of the centrosome investigation is given and the current state of knowledge of this cellular structure in morphological, biochemical, and functional aspects is presented. Besides of the classical functions of the centrosome as a MT nucleating, MT ancoriging, and MT organizing center, the idea about the centrosome as a cellular regulating center and as a structural part of the mechanism operating dynamic morphology of a cell is discussed.

[A comparative level of expression of some proteins in XL2 cell synchronized on different phases of cell cycle]. / Issledovanie sravnitel'nogo urovnia ékspressii belkov v sinkhronizirovannykh na razlichnykh stadiiakh kletochnogo tsikla kletkakh XL2.

Cells of cultured line XL2 (Xenopus laevis) were synchronized by a combine effect of serum deprivation, aphidicolin, nocodazole and ALLN treatments. Four fractions were prepared, with maximum percentage of cells being in G1, S and G2 phases of cell cycle, and in mitosis, respectively. Comparative levels of six different proteins (beta-tubulin, DNA topoisomerase IIa, Xenopus Aurora A kinase pEg2, kinesin-like motor protein X1Eg5, and two members of condensis family proteins pEg7 (XCAP D2) and XCAP E were detected by quantitative Western blot analysis of these fractions. We used a new method of mathematic processing of data that commonly provides a possibility to calculate a comparative quantity of proteins in hypothetically "clean" fraction composed of cells being in the same phase of the cell cycle. This method makes it possible to use even partly synchronized cell cultures for analysis of changes in protein quantity, provided a precede determination of cell population composition is made.

Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle.

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.

[Intracellular localization of XCAP-E protein in XL2 (Xenopus laevis) cells under normal conditions and during inhibition of pRNA transcription and processing]. / Issledovanie vnutrikletochnoi lokalizatsii belka XCAP-E v kletkakh linii XL2 (Xenopus laevis) v norme i pri ingibirovanii transkriptsii i protsessinga pRNK

The interaction of condensin subunit XCAP-E with various nucleolar subcompartments in XL2 cells was studied. In the interphase cells, XCAP-E was associated with a granular component of nucleoli (as shown by double staining with antibodies against B23) and with small nucleolus-like structures in the nucleoplasm. Inhibition of transcription by actinomycin D does not disrupt interaction of XCAP-E with the granular compartment of segregated nucleoli. Treatment with DRB 5,6-dichloro-1 beta-ribofuranozide-benzimidazole causes disintegration of nucleolar fibrillar complexes, but does not affect nucleolar localization of XCAP-E. The data suggest that nucleolar association of XCAP-E is independent on the functional state of the nucleolus, and imply a possible role of this protein in rRNA processing and pre-fibosome assembly.

[Intracellular localization of XCAP-E and pEg7 condensins in normal mitosis and after the treatment inducing artificial changes in structural organization of mitotic chromosomes]. / Vnutrikletochnaia lokalizatsiia kondensinov XCAP-E i pEg7 v norme i pri vozdeistviiakh, vyzyvaiushchikh iskusstvennoe izmenenie strukturnogo sostoiianiia mitoticheskikh khromosom.

Function of condensin subunits XCAP-E and pEg7 (XCAP-D2) in the formation and maintaining of special organization of mitotic chromosomes has been studied in Xenopus laevis cells (XL-2). The experimental conditions involved blocking chromosomes being in the condensed state in cells treated by cytostatics, or during their reversible artificial decondensation. The latter was induced by incubation of living cells in hypotonic medium. In extensively mollen chromosomes, XCAP-E and pEg7, remained associated with axial regions of chromosomes. In contrast, upon adaptation of cells to hypotonic conditions and recondensation of chromosomes to nearly initial state, both proteins dissociated from chromosomes into the cytoplasm. In K-mitotic cells, after a 3-6 h treatment with nocodazole or taxol, considerable dissociation of XCAP-E and pEg7 from chromosomes was observed without significant changes in overall level of chromosome compactization. Taken together the data suggested that condensins play no important role in maintaining mitotic chromosomes being in condensed state. Rather, it seems probable that mitotic function of condensins may be associated either with the formation of the higher order chromosome structure, and/or segregation of sister chromatids, the processes being tightly linked with chromosome compactization. This paper is in memory of Professor Katherine Le Guellec of Rennes-1 University, who left us in June 2001. Professor Le Guellec initiated this work in Rennes and offered all the possible help that this work be continued in Moscow University. Let the memory of Katherine, a great scientist and sympathetic friend, live for ever in ours hearts.

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.

Kinetic analysis of cell population growth during cultivation in vitro.

XL-2 cells (Xenopus laevis) were used for kinetic analysis of cell population growth. The dependence of the time of cell duplication on the percentage of cells in the G0 phase of the cell cycle was studied and described by a mathematical expression. Possible causes of the changes in the ratio between the percentage of cells in the cell cycle and that in the G0 phase were analyzed. These are the decrease in the percentage of cells in the G0 phase due to the increase in the number of dividing cells, their position in the cell islets, the number of nuclei, the relative position of cells in the G0 phase. It was shown that the loss of the free edge by cells during their transition to the second layer of the cell islets without any changes in spreading led to a significant increase in the percentage of cells in the G0 phase. The percentage of cells in the G0 phase increased about five times for multinuclear cells. Analysis of the position of cells in the G0 phase showed that these cells were mostly in groups of two, three or four. Studies of a real cell culture in the logarithmic phase of growth (48-120 h of cultivation) showed that the percentage of cells in the G0 phase did not virtually change and all processes were equalized by one another. We propose a new method to determine the cell cycle duration under conditions from the time of cell culture duplication and the data on the percentage of cells in the G0 phase. This method can be used when traditional approaches using BrdU or [3H]]thymidine are difficult to implement or are unacceptable.

Postmitotic reconstruction of nucleoli in culture cells with UV-microbeam photoinactivated centrosome.

Ultraviolet microirradiation of one of the poles of the mitotic spindle of PK cells was performed 1 min after the onset of the anaphase. Formation of the nucleolus in the telophase and G1 period was studied by vital observation, electron microscopy and indirect immunofluorescence using antibodies against B23 protein. Sister cells with nonirradiated centrosomes and cells with partially irradiated cytoplasm were used as controls. During the first hour after the anaphase, the nuclei in both sister cells were identical and contained numerous small dense particles with granular ultrastructure. B23 protein detected in the mitotic poles and at the chromosome surface in the anaphase was dispersed in the cytoplasm in both cells in the early G1 period. Later, control cells did not display any difference from intact cells: nucleoli of a typical structure were formed, B23 protein appeared in the karyoplasm and was then accumulated in the nucleoli and disappeared from the cytoplasm and karyoplasm. Nucleoli in cells with irradiated centrosomes did not achieve the normal size and contained a significantly lower amount of granular component. B23 protein was dispersed in the karyoplasm and was not accumulated in the nucleoli. Nucleoli in cells with irradiated centrosomes contained small dense particles for at least 24 h. Telophase cells where microtubule formation had been inhibited by nocodazole formed normal nucleoli. It shows that the effects observed in cells with irradiated centrosomes are not due to the absence of the microtubule radial system. We conclude that UV microirradiation of the mitotic centrosome disturbs the postmitotic reconstruction of nucleoli probably because of the photodestruction of B23 protein accumulated in the mitotic pole.

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.

Neurotoxic glutamate treatment of cultured cerebellar granule cells induces Ca2+ -dependent collapse of mitochondrial membrane potential and ultrastructural alterations of mitochondria.

Rhodamine 123 staining and electron microscopy were used to reveal a correlation between the ultrastructural and functional state of cultured cerebellar granule cells after short glutamate treatment. Glutamate exposure (15 min, 100 microM) in Mg2+-free solution caused considerable ultrastructural alterations in a granule cell: clumping of the chromatin, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. After glutamate treatment, the mitochondria of the neurons lost their ability to sequester rhodamine 123. Both the N-methyl-D-aspartate receptor channel blocker MK-801 (30 microM) and cobalt chloride (2 mM) prevented the deteriorative effects of glutamate. These data suggest that glutamate-induced Ca2+ overload of the neurons can lead to non-specific permeability of the inner mitochondrial membrane, resulting in neuronal death.

[The effect of ultraviolet microbeam irradiation of the centrosome on cellular behavior. IV. Synthetic activity, cell spreading and growth with an inactivated centrosome]. / Vliianie ul'trafioletovogo mikrooblucheniia tsentrosomy na povedenie kletok. IV. Sinteticheskaia aktivnost', rasplastyvanie i rost kletok s inaktivirovannoi tsentrosomoi.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam at anaphase. After irradiation, cell division completed with a minor delay and two daughter cells were spreading synchronously. Later on, cells with irradiated centrosomes slightly shrunk, while their sister cells enlarged normally. Sister cells entered S-phase, some of them undergoing mitosis. In the cells with irradiated centrosomes the formation of nucleoli was disturbed and numerous primary nucleoli remained for 50 h (the maximum time of observation). RNA synthesis in the cells with irradiated centrosomes was twice less than in the sister cells, with ribosomal RNA synthesis being suppressed predominantly. Cells with irradiated centrosomes did not enter S-phase for as long as 24 h. The same irradiation of a portion of cytoplasm outside the spindle performed during anaphase did not change the pattern behaviour in daughter cell. In is concluded that the centrosome regulates progression throughout the cell cycle, and that centrosome irradiation induces specific and irreversible damage of interphase cells.

[Behavior of the mitotic apparatus after ultraviolet microirradiation of the spindle pole]. / Povedenie mitoticheskogo apparata posle ul'travioletovogo mikrooblucheniia poliusa veretena.

UV-microirradiation of the centrosome (spindle pole) alters behaviour of mitotic cell. Cell reaction strongly depends on the stage of mitosis when irradiation has been made. Irradiation of one pole at metaphase blocked cell in c-mitosis for several hours. The same or even more strong irradiation of the pole at anaphase slightly postponed cytokinesis, but didn't prevent chromosome separation and normal exit of cell into interphase. Irradiation at metaphase resulted in immediate shift of chromosomes towards the nonirradiated pole. Afterwards spindle disassembled. Irradiation at anaphase slowed down chromosome motion towards irradiated pole without prominent effect on the opposite halfspindle. Electron microscopy demonstrated that immediately after irradiation centrosome lost association with kinetochore microtubules. At metaphase irradiated pole moved towards chromosomes, while at anaphase--away from them. Same irradiation of other besides spindle poles regions had no effect on continuation of mitosis.

Role of the centrosome in mitosis: UV micro-irradiation study.

Ultraviolet micro-irradiation (UV-MI) of the PK (pig kidney embryo) cell centrosome (lambda max = 280 nm, spot diameter 1.6 mm, exposure time 5-15 s) at metaphase and anaphase resulted in functional damage of the centrosome. After UV-MI of the centrosome at early metaphase, chromosomes quickly (in 1-3 min) moved away from the irradiated pole and then encircled the non-irradiated pole. Within 10 min after UV-MI the spindle disassembled and chromosomes remained unseparated. The minimal dose inducing this effect in 90% of cells was accumulated in 5 s. After the same UV-MI at late metaphase, chromosomes shifted towards the non-irradiated pole; however, anaphase started and chromosome motion towards the non-irradiated pole continued normally. UV-MI of the centrosome at early anaphase for 5-15 s slowed down and then stopped chromosome motion towards the irradiated pole. This was a result of rapid (within 2-3 min) disorganization of the half-spindle. Chromosomes continued to move towards the opposite pole normally, while cytokinesis was significantly retarded. No visible lesion was revealed by electron microscopy after 5 s UV-MI, while 15 s irradiation resulted in the truncation of the microtubule bundles 1.5-2 microns from the centrosome. We concluded that UV-MI inactivates the centrosome and induces disaggregation of microtubule initiation sites. The critical point (checkpoint) in mitosis up to which this damage induces mitotic arrest is mid-metaphase.

[The effect of the UV microirradiation of the centrosome on cell behavior. III. The ultrastructure of the centrosome after irradiation]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. III. Ul'trastruktura tsentrosomy posle oblucheniia.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.

[The effect of UV microirradiation of the centrosome on cell behavior. I. The destruction of the mitotic spindle and disruption of cell division during irradiation in the metaphase]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. I. Raspad mitoticheskogo veretena i narushenie deleniia kletki pri obluchenii v metafaze.

A 5 second UV microirradiation of the centrosome during the early metaphase leads to a rapid (within 5 minutes) chromosome shift towards the normal pole and disorganizes the spindle. The later metaphase plate is also disorganized, chromosomes being situated chaotically in the central part of the cell. Numerous (up to 10 or more) microtubule convergence centers are observed instead of the spindle. 2-4 hours after the microirradiation some cells may enter cytotomy. The microirradiation of chromosomes and cytoplasm in similar and greater doses (exposure up to 15 seconds) did not lead to disorganization of the spindle and did not effect the normal completion of mitosis. Sometimes the 5 second microirradiation in the middle metaphase also blocked anaphase, but the microirradiation within the last 5 minutes of the metaphase always failed to block anaphase and normal completion of division.

[The effect of centrosome UV microbeam irradiation on cell behavior. II. The radiation sequelae in the anaphase: the completion of division and the fate of the interphase cell]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. II. Posledstviia oblucheniia v anafaze: zavershenie deleniia i sud'ba interfaznoi kletki.

Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.