Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the checkpoint, chromosome movement. We hypothesized that, inliving cells, suppression of microtubule is responsible for ability taxol to inhibit progression cell proliferation. Using quantitative fluorescence video microscopy, we examined effects (30–100 nM) on individual microtubules two living human tumor lines: Caov-3 ovarian adenocarcinoma cells A-498 kidney carcinoma...

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted the hypothesis this was due to a nocodazole-induced stabilization microtubules. We tested by examining effects on dynamic instability interphase vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized video-enhanced differential interference contrast microscopy perfused solutions ranging concentration from 4...

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and cell line permanently expressing GFP-α was established (LLCPK-1α). The mitotic index doubling time for LLCPK-1α not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the in GFP-tubulin; level unlabeled reduced to 82% parameters microtubule dynamic instability compared interphase injected rhodamine-labeled tubulin. Dynamic very similar two cases,...

Microtubule (MT) turnover within the four principal MT arrays, cortical array, preprophase band, mitotic spindle and phragmoplast, has been measured in living stamen hair cells of Tradescantia that have injected with fluorescent neurotubulin. Using combined techniques confocal laser scanning microscopy fluorescence redistribution after photobleaching (FRAP), we report half-time MTs is t 1/2 = 31 +/- 6 seconds, which excellent agreement previous measurements animal cell spindles. interphase...

Recent experiments have demonstrated that the behavior of interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters instability responsible for this difference, we examined individual both cell types after injection with rhodamine-labeled tubulin subunits. Individual were observed to grow, shorten, pause, as expected. The average amount time...

Carboxyfluorescein-labeled brain tubulin has been microinjected into stamen hair cells of Tradescantia, and its distribution during mitosis cytokinesis was examined using confocal laser scanning fluorescence microscopy. The results show that incorporates plant microtubules is utilized throughout cytokinesis. Microtubule structures incorporate include the preprophase band, perinuclear sheath at late prophase, kinetochore fibers prometaphase, metaphase, anaphase, interzone spindle finally...

We have characterized the effects of vinblastine on dynamic instability behavior individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and found that at low concentrations (3-64 nM), potently suppresses without causing net microtubule depolymerization. Vinblastine suppressed rates growth shortening, decreased frequency transitions from or pause to also called catastrophe. In vinblastine-treated cells, both average duration a (a state attenuated dynamics...

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state assembly was abruptly blocked with either high concentrations nocodazole (10 micrograms/ml) or exposure cold temperature (3 degrees C). At various times after inhibition assembly, cells processed for anti-tubulin immunofluorescence microscopy. Stained observed an intensified video camera attached fluorescence microscope. A tracing entire...

One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" microtubules occurs within half-spindle between chromosomes and poles (Margolis, R. L., L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling using fluorescent analog cytochemistry measurements fluorescence redistribution after photobleaching to examine microtubule behavior metaphase mitosis. Mitotic BSC 1 mammalian tissue...

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, Kinesin-5 motor Eg5 interacts with factor TPX2, but how this interaction contributes to formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking domain. Spindles in these were highly disorganized multiple poles. The TPX2–Eg5 was required for kinetochore fiber contributed localization microtubules...

The temporal regulation of protein abundance and post-translational modifications is a key feature cell division. Recently, we analysed gene expression changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, show that by using specific intracellular immunolabelling protocols, FACS separation mitotic cells, including subphases, can be combined with proteomic analysis mass spectrometry. Using this PRIMMUS (PRoteomic Intracellular iMMUnolabelled Subsets)...

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by loss extensive interphase array and formation bipolar mitotic spindle. Microtubules in stably expressing GFP–α-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) early prometaphase. Our results demonstrate transient stimulation individual dynamic turnover inward motion microtubule bundles these cells. Motion was inhibited...

Abstract The development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells Tradescantia. Microtubules actin microfilaments identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with confocal laser scanning microscope has permitted sequential imaging fluorescent cytoskeletal elements single progressing through division. Phragmoplast microtubules initially emerge lateral coalescence...

ABSTRACT Heat-stable brain microtubule associated proteins (MAPs) and purified protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual micro-tubules was then examined using low-light-level fluorescence microscopy quantitative tracking methods. Both MAP preparations suppressed dynamics in vivo, by reducing the average rate extent both growing shortening events. duration...

The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report AtKRP125c, one four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle appears to function both interphase mitosis. In temperature-sensitive mutant, cortical are disorganized restrictive temperature spindles massively disrupted, consistent with defect stabilization anti-parallel midzone, as previously...

Microtubules stimulate contractile-ring formation in the equatorial cortex and simultaneously suppress contractility polar cortex; how they accomplish these differing activities is incompletely understood. We measured behavior of GFP-actin mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly resulted functional contractile rings were wider than controls had altered dynamic...