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 g/ml and 0.2 g/ml all c-metaphase cells divide normally into two daughter cells. During recovery after a short (1–4 h) treatment with 0.6 g/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 g/ml kinetochores are not associated with microtubules. At a nocodazole concentration of 0.6 g/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.by M. Trendelenburg     

Nocodazole, vinblastine and taxol at low concentrations affect fibroblast locomotion and saltatory movements of organelles

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatory movements of particles in their lamella Fibroblasts extended their leading edges into the experimental wound at a rate of 20+/-11 microm/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 microM) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 microM) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.     

Centrosome-dependent anisotropic random walk of cytoplasmic vesicles

We approach the problem of an apparently random movement of small cytoplasmic vesicles and its relationship to centrosome functioning. Motion of small vesicles in the cytoplasm of BSC-1 cells was quantified using computer-assisted microscopy. The vesicles move across the cytoplasm frequently changing their directions with negligible net displacement. The autocorrelation function for consecutive velocities of individual vesicles becomes indistinguishable from zero in 10s. Variance in the displacement is proportional to time. The motion of vesicles is anisotropic: It has diffusivity along the radii drawn from the centrosome several times higher than the tangential diffusivity. This anisotropy is abolished by ultraviolet microbeam irradiation of the centrosome when the microtubule array loses radial structure. We conclude that the motion of the vesicles in the cytoplasm can be described as diffusion-like random walk with centrosome-dependent anisotropy. The present analysis quantitatively corroborates the 'trial and error' model of vesicular transport.     

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

The Dynamics of Microtubules in Cultured Cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording with a digital camcorder. In the cell lamella, the positive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode close to 0. The maximum rate of lengthening and shortening reaches 30 and 50 m/min, respectively. The positive ends of microtubules in PtK cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were usually displaced to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK cells appeared mostly due to spontaneous assembly in the cytoplasm (not in the relationship with the preexisting microtubules) and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1–2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place mostly at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.     

Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.     

Blue Light Inhibits Mitosis in Tissue Culture Cells

Irradiation of the mitotic (prophase and prometaphase) tissue culture PK (pig kidney embryo) cells using mercury arc lamp and band-pass filters postponed or inhibited anaphase onset. The biological responses observed after irradiation were: (i) normal cell division, (ii) delay in metaphase and then normal anaphase and incomplete cytokinesis, (iii) exit into interphase without separation of chromosomes, (iv) complete mitotic blockage. Cell sensitivity to the light at wavelengths from 423 and 488 nm was nearly the same; to the near UV light (wavelength 360 nm) it was 5–10 times more; to the green light (wavelength >500 nm) it was at least 10 times less. To elucidate the possible mechanism of the action of blue light we measured cell adsorption and examined cell autofluorescence. Autofluorescence of cytoplasmic granules was exited at wavelengths of 450–490 nm, but not at >500 nm. In mitotic cells fluorescent granules accumulated around the spindle. We suppose blue light irradiation induces formation of the free radicals and/or peroxide, and thus perturb the checkpoint system responsible for anaphase onset.     

Imaging flow cytometry: coping with heterogeneity in biological systems

Imaging flow cytometry (IFC) platforms combine features of flow cytometry and fluorescent microscopy with advances in data-processing algorithms. IFC allows multiparametric fluorescent and morphological analysis of thousands of cellular events and has the unique capability of identifying collected events by their real images. IFC allows the analysis of heterogeneous cell populations, where one of the cellular components has low expression (<0.03%) and can be described by Poisson distribution. With the help of IFC, one can address a critical question of statistical analysis of subcellular distribution of proteins in a cell. Here the authors review advantages of IFC in comparison with more traditional technologies, such as Western blotting and flow cytometry (FC), as well as new high-throughput fluorescent microscopy (HTFM), and discuss further developments of this novel analytical technique.