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When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.
Figure 1. Mt remodeling in prophase/metaphase cells. (A) GFP-tubulin fluorescence in a prophase cell; time is relative to the onset of inward collapse of the Mt array. Inset in the first panel shows the nucleus in phase contrast; the focal plane was changed for subsequent panels. (B) Prophase cells imaged starting at NE perforation complete mitosis and cytokinesis normally; time as in A. (C) Higher magnification of the boxed regions in B show the rapid loss of individual Mts (top, single Mts) and the formation and motion of Mt bundles (bottom, bundles). (D) Immunofluorescence staining for Mts (top) and the inner NE protein LAP2 (middle, two different focal planes) and overlay (bottom). Inward collapse of the Mt array occurs at NEBD. Bars: (A, B and D) 10 μm; (C) 5 μm.
Figure 2. Formation and motion of Mt bundles in prophase/prometaphase cells. (A and B) Quantification of fluorescence intensity; boxed areas in A are enlarged below; (B) Histograms of normalized fluorescence intensity values. (C) Prophase Mt bundles, visualized using immunofluorescence, in LLCPK1 parental, BSC-1, and MDCK cells; boxed areas are enlarged below. (D) Motile behavior of Mts in prophase cells; times are the interval between successive images in min:s. Top four rows of panels are oriented so that the NE is to the bottom of each series; bottom row is a metaphase cell; arrow shows a small focus of Mts; the dark sphere is a vacuole. Bars: (A and C, top) 10 μm; (A and C, bottom, and D) 5 μm.
Figure 3. Localization of Mt bundle components. Immunolocalization of tubulin (left), Eg5 (top), dynactin (p150; middle), and XMAP215 (bottom) in fixed LLCPK1α cells; overlays (tubulin in green; Eg5, p150 and XMAP215 in red) are shown at right. Eg5 and p150, but not XMAP215, localize to the Mt bundles.
Figure 4. Dynein/dynactin-dependent motion of Mt bundles. (A) Images of prophase LLCPK1α cells incubated with ML-7 (top) or monastrol (bottom). Time (min:s) after treatment is shown in the top right, elapsed time is shown in the bottom right. (B) Cells injected with anti-70.1 antibodies. Time after injection is shown in the top right. (Top) 33 min after injection bundles have formed, but inward motion is inhibited; elapsed time in the bottom right. (Middle) Inward collapse of bundles is inhibited in 70.1 injected cells. (Bottom) Spindle formation is abnormal in injected cells; asterisk indicates a different focal plane. (CâE) Models for Mt behavior at NEBD; cytoplasmic dynein moves clustered (C) and bundled (D and E) Mts toward the minus ends of centrosomal Mts. Bars, 10 μm.
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