Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Single-cell electroendocytosis on a micro chip using in situ fluorescence microscopy.

Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent intensity from a large number (>2,000) of single cells. The EED efficiency was determined as a function of three electric parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the time-dependent mechanism of EED at the single-cell level.

Comparison of mitostatic effect, cell uptake and tubulin-binding activity on colchicine and colcemid.

Mitostatic action, cellular uptake and the binding of colchicine and colcemid to tubulin were compared. It was shown that mitostatic action of low doses of colchicine developed only after 24 h incubation of the drug with mouse L fibroblasts, while the colcemid-induced block of mitosis was evident after 2 h incubation. The initial rate of uptake was about 10 times greater for colcemid than for colchicine. Cellular uptake of the drugs reached an equilibrium after 2 and 15-18 h incubation for colcemid and colchicine, respectively, and the plateau values were identical. The kinetics of colchicine and colcemid binding to bovine brain tubulin was studied by the DEAE-filter binding assay. Colcemid binds to tubulin much faster than does colchicine. The rate of colcemid efflux from L cells is much higher than that of colchicine. According to the efflux data, colcemid dissociates readily from a complex with tubulin (t1/2 = 10 min), while the colchicine-tubulin complex is stable for at least 1 h. These results are consistent with previously published data (Frankel, F.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 72, 2798-2802), which showed that colcemid action on cells is more reversible than that of colchicine. We suggest that differences between colchicine and colcemid in the rate of mitostatic action and its reversibility are determined by the differences in parameters of tubulin binding.

Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

[Mouse cell accumulation of Colcemid and colchicine in tissue culture]. / Nakoplenie koltsemida i kolkhitsina myshinymi kletkami v kul'ture tkani.

Kinetics of the uptake of 3H-colchicine and 3H-colcemid by mouse transformed L cells has been studied. The amounts of colchicine and of colcemid, accumulated by cells during a long-term incubation appeared to be equal; however, the initial uptake rates being higher for colcemid. The rates of the efflux of 3H-colchicine and 3H-colcemid were also different. After a 3 hours incubation of cells in antitubulin-free medium, the cells retain as much as 70% of colchicine, and only 10% of colcemid. It is known that colchicine does not bind tubulin at 0 degrees C; a colchicine analog lumicolchicine does not bind tubulin at any temperature. We have shown that the rate of 3H-lumicolchicine efflux at 37 degrees C is very rapid. The same was true for colchicine, if the incubation with the drug was performed at 0 degrees. We suggest that the difference in the rates of influx and efflux between colchicine and colcemid may be due to differences in their capacities of binding tubulin and dissociating from it.

The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.

The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs) are unknown. By studying the effect of transient microtubule depolymerization and centrosome mutant conditions, we have found that such orientation memory requires both the centrosome-organized interphase aster and centrosome-independent functions. We have also found that the span of such memory is limited to the last mitosis. Furthermore, the orientation of the NB axis of polarity can be reset to any angle with respect to the surrounding tissue and is, therefore, cell autonomous.

Nucleolin functions in nucleolus formation and chromosome congression.

A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of high-resolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation.

Identification of tubulin from the yeast Saccharomyces cerevisiae.

A tubulin-like protein was identified in the lower eukaryote Saccharomyces cerevisiae. The following criteria were used: (i) copolymerization of the 35S-labeled yeast protein with porcine brain tubulin; (ii) immunoprecipitation of the 35S-labeled yeast protein with antiflagellar tubulin antibody; (iii) the presence of the yeast protein as a constituent of isolated yeast nuclei; and (iv) splitting of the yeast protein in a gel electrophoretic system containing sodium dodecyl sulfate that resolved the alpha- and beta-tubulin chains from other sources. This protein did not appear to have significant affinity for the plant alkaloid, Colcemid.

Changes in colchicine and demecolcine content during vegetation period of colchicum autumnale L.

Colchicine and demecolcine were determined in raw and dried leaves, stems, mother and daughter corms of Colchicum autumnale L. in four stages of its ontogenesis. The colchicine content in raw material varies during plant growth. The content of both alkaloids decreases with drying. The HPLC method used is suitable both for the phytochemical analysis of Colchicum and for the toxicological evaluation in cases of intoxication by these plants.

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)colcemid, a probe for different classes of colchicine-binding site on tubulin.

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of a colchicine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the colchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 x 10(5) M-1 s-1 for the association and 0.79 s-1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 444.4 M-1 s-1 and 11.67 M-1 s-1 [corrected], respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

Modifications of the Ca2+ release mechanisms of mouse oocytes by fertilization and by sperm factor.

A cytosolic factor from sperm (SF) is thought to be responsible for the generation of intracellular calcium oscillations ([Ca2+]i) associated with fertilization in mammalian oocytes. Whether or not mouse oocytes injected with SF exhibit modifications of their Ca2+ release mechanisms similar to those observed in fertilized oocytes is not known and this was investigated here by injecting porcine SF (pSF). First, pSF-activated oocytes injected with CaCl2 showed persistent sensitization of the Ca2+-induced Ca2+ release mechanism, but this sensitization was absent in SrCl2-activated oocytes. Second, pSF-injected oocytes re-initiated oscillations when fused with untreated oocytes, although the Ca2+ responses were short-lived compared to those initiated by fertilization. Likewise, in the presence of colcemid, pSF-initiated oscillations were prolonged but ceased in advance of those in fertilized zygotes. Also, pronuclear envelope breakdown induced by okadaic acid was not associated with Ca2+ release in pSF-generated zygotes, whereas it was observed in fertilized zygotes. Finally, roscovitine, an inhibitor of maturation promoting factor, blocked pSF-induced [Ca2+]i oscillations. Together, these results show that pSF-induced [Ca2+]i responses exhibit properties similar to those triggered by the sperm, although the SF's Ca2+ active component(s) may be less stable or more susceptible to degradation, resulting in shorter modification of the oocyte's Ca2+ release mechanisms.

The Invs gene encodes a microtubule-associated protein.

Microtubule networks are important for many vital processes such as mitosis, cell polarity, and differentiation. Ciliary architecture and function closely depend on the microtubule cytoskeleton, and recent studies suggest a role of apical cilia of renal epithelia in the pathogenesis of polycystic kidney disease. This study evaluates the localization, potential interacting partners, and functional aspects of the Invs gene product inversin. Only recently, INVS has been identified as the gene that is mutated in nephronophthisis type 2, an autosomal recessive polycystic kidney disease. Using immunoprecipitation and co-pelleting assays, we show that the Invs gene product inversin forms a stable complex with tubulin in cultured renal epithelial cells. Inversin localizes to several components of the cytoskeleton including ciliary, random, and polarized microtubule pools. During cell divison, inversin is recruited to mitotic spindle fibers. After microtubule depolymerization using colcemid inversin and tubulin staining is no longer characterized by a network pattern but by homogeneous, diffuse distribution. Inversin does not coprecipitate with tubulin after addition of colcemid. After removal of colcemid, inversin immunofluorescence reappears together with tubulin in centrioles. Treatment with the microtubule stabilizing agent paclitaxel leads to severe alteration of the microtubule cytoskeleton with bundling and formation of long spindles of tubulin and inversin. In conclusion, inversin is closely associated with the microtubule cytoskeleton, and its spatial distribution is dependent on tubulin polymerization. Hence, altered inversin-tubulin interaction may impair ciliary function and thereby contribute to cyst development in nephronophthisis.

Role of B-ring of colchicine in its binding to tubulin.

The chemical specificity of the colchicine-binding site of tubulin is less stringent for the presence of the B-ring than the A- and C-rings of colchicine, Colchicine analogues with modifications in the B-ring bind to tubulin at the same site as colchicine. Analogues with smaller or no substituents in the B-ring bind tubulin remarkably faster than colchicine. Thus, a compound without the B-ring [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] binds tubulin even at 4 degrees C and the binding is almost instantaneous at 37 degrees C. Colcemid and 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone bind reversibly to tubulin, whereas colchicine and desacetamidocolchicine bind almost irreversibly, suggesting that the size of the B-ring moiety of colchicine is not related to the reversibility of binding. We conclude that although the presence of the B-ring of colchicine does not appear to be an essential prerequisite for the drug-tubulin interaction, the B-ring substituents play an important role in determining the binding properties of colchicine to tubulin.

A fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid, as a probe for the colcemid-binding sites of tubulin and microtubules.

The synthesis and biological testing of the fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid (NBD-colcemid), are here described. NBD-colcemid exhibited a visible absorption maximum at 465 nm and fluoresced in the range of 520-540 nm, highly in environments of low polarity, whereas only slightly in aqueous solution. The addition of NBD-colcemid to bovine brain tubulin was accompanied by a striking enhancement of fluorescence. The fluorescent titration study suggested a stoichiometric binding of NBD-colcemid to tubulin. Assembled microtubules were directly visualized after mixing with NBD-colcemid using a fluorescence microscope. NBD-colcemid reversibly disrupted the metaphase spindles of sea urchin eggs as well as unlabeled colcemid. However, even when the birefringence of spindles was mostly lost, self-quenching properties of the NBD fluorescence allowed tubulin and its oligomers aggregated in higher concentrations in eggs to be visualized under a fluorescence microscope. The results suggest a wide applicability of NBD-colcemid as a fluorescent probe for studying the interactions of colcemid with tubulin and microtubules, as well as for localizing other colcemid-binding structures within cells.

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.

Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro.

The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal tubulin.

Anion-induced increases in the affinity of colcemid binding to tubulin.

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.

Mutants of Chinese hamster ovary (CHO) cells with altered colcemid-binding affinity.

Clones of CHO cells stably resistant to colcemid have been isolated in the presence of the nonionic detergent Tween 80 after mutagen treatment. Successive single-step selections for increasing resistance were performed resulting in lines after three selection steps about 10 fold more resistant to colcemid than the parental cells. Three observations indicate that these colcemid-resistant (CMR) mutants are different from the colchicine-resistant permeability mutants isolated previously. First, their relative resistance to colcemid was not diminished in the presence of detergent which promoted increased drug permeability. Second, the CMR clones displayed limited cross-resistances only to tubulin-binding compounds. Third, the binding affinity of labeled colcemid by cytoplasmic extracts from CMR clones was reduced, and the reduction was greater in the more resistant clones. No reduction in binding of labeled colcemid was observed in the membrane-altered colchicine-resistant mutants. All these observations are consistent with the CMR clones being tubulin-altered mutants. In further support of this conclusion, we observed that tubulin purified from a CMR mutant still possessed reduced colcemid-binding affinity compared with that from parental cells.

Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine.

Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5-50 mug) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.

The synthesis of the brain specific S100 protein in colcemid resistant mutants of rat glial cells.

We have isolated two colcemid-resistant mutant sublines, CMR (7A) and CMR (7B), from rat glial cells, C6, using multiple consecutive selections with increasing concentrations of colcemid. The mutant sublines show a decreased uptake of [3H]colchicine but have no apparent defect in the cytoplasmic binding of the drug. The synthesis of the brain-specific S100 protein is less sensitive to colcemid inhibition in the mutant cell lines than in parental C6 cells, suggesting that colcemid must enter the cell to inhibit S100 protein synthesis.