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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01007

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Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes

Department of Zoology/Center for Gene Research and Biotechnology (CGRB), Oregon State University, Corvallis, OR 97331, USA

View larger version (141K): [in a new window]   Fig. 1. Cytokinesis in grasshopper spermatocytes. (A) A polarization microscope sequence (Movie 1, http://jcs.biologists.org/supplemental/) showing behavior of mitochondria during cell cleavage in Melanoplus femurrubrum spermatocytes. Time is given in minutes. In metaphase (0 minutes), mitochondria (m) are scattered around the spindle apparatus (microtubules appear as black fibers). During early to mid anaphase (10-14 minutes), mitochondria accumulate around the spindle midzone (arrowheads, 32 minutes), and later bundle with and move poleward along peripheral microtubules of the central spindle (32-54 minutes). The cleavage furrow initiates (arrows, 54 minutes) and ingresses (72-93 minutes) at the midzone, which is symmetric to the bipolar spindle. (B) Localization of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at various stages during cell cleavage. Actin filaments, distributed randomly through the cell in early anaphase (e, red), relocate to the microtubule overlapping zone where mitochondria accumulate (b,f, arrows) in late anaphase, corresponding to the 32-minute stage shown in A. During furrow initiation, actin filaments (g, red) further accumulate at the light-stained midzone (c, arrows) of the newly assembled central spindle (c,g, green). During furrow ingression, actin filaments (h, red) become extremely condensed on both sides of the narrowed midzone (d, arrows). Scale bars: 10 µm.

View larger version (56K): [in a new window]   Fig. 2. Schematic illustration of the generation of microtubule asymmetry in grasshopper spermatocytes. (A) Bipolar spindles with an asymmetric microtubule distribution were produced by removing all chromosomes, except the X, in prometaphase of meiosis I cells (a,b). The X chromosome subsequently causes spindle asymmetry by promoting microtubule assembly at its proximal pole (c). (B) Greater microtubule asymmetry was produced by further removal of the aster from the achromosomal spindle pole (a,b) following extraction of all but the X chromosome, yielding a cell with a monopolar spindle bearing only the X chromosome (c). (C) The greatest spindle asymmetry was created by cutting cells between segregating chromosomes in anaphase (a,b), producing two cells, each containing a half-spindle with all segregated chromosomes (c).

View larger version (139K): [in a new window]   Fig. 3. Microtubule distribution and furrow induction in cells containing a bipolar spindle and the X chromosome. Following removal of all bivalents, microtubule density increases at the pole bearing the X chromosome, which is shown in both polarization microscope images (A, 0-57 minutes; see also Movie 2, http://jcs.biologists.org/supplemental/) and a cell fixed at the corresponding stage and stained (B) for microtubules (a, green), actin filaments (e, red), and the chromosome (a, blue). The asymmetric microtubule distribution shifts mitochondria (m) away from the chromosome bearing pole (0-57 minutes). Anaphase onset is recognized as a spilt of the X chromosome (82 minutes, circle in A) arms (b, blue) and disassembly of spindle microtubules, which appears as a reduction in spindle birefringence (82-104 minutes) and fluorescence (a,b, green). During anaphase (87 minutes) mitochondria (arrowheads) and actin filaments (f, red) accumulate at the midzone (82-87 minutes; b, arrows), which is shifted away from the pole with greater microtubule density. Following anaphase, mitochondria extend along microtubules (87-122 minutes) as actin filaments bundle into the contractile ring (f,g, red; see also Movie 3, http://jcs.biologists.org/supplemental/) around the shifted midzone (c, arrows), asymmetrically inducing cell cleavage 16±2% from the genuine central position of the spindle (104 minutes, arrows; n=7). Despite spindle reorganization (c,d, arrows) that symmetrically shifts the midzone and furrow (104 minutes onward; g,h, red) along the central spindle, the initial asymmetry to the cell remains (152 minutes; d,h). Scale bars: 10 µm.

View larger version (135K): [in a new window]   Fig. 4. Microtubule distribution and furrow induction in cells containing a monopolar spindle and the X chromosome. Following removal of all bivalents and one aster, the spindle becomes monopolar as microtubules are stabilized around the X chromosome and the remaining pole (A, 0 minutes; see also Movie 4, http://jcs.biologists.org/supplemental/; Ba, green). Mitochondria, scattered at the spindle periphery initially, accumulate at the plus-ends of radiating spindle microtubules (0-32 minutes, arrowheads; a, green). Following anaphase, as recognized by the separation of the X chromosome (50 minutes, arrow), mitochondria move back toward the original pole along central spindle microtubules (50-87 minutes). Accumulation of actin filaments (f, red) and furrow initiation are shifted 42±2.1% (87 minutes, arrows; n=5) from the central position to the `midzone' (b, arrows), asymmetrically defined by microtubules from the original and newly established half-spindles. The furrow ingresses (87 minutes onward) as actin filaments accumulate and constrict (g,h, red) the midzone (c,d, arrows). Owing to microtubule elongation at the new half-spindle (c,d, green), the furrow eventually becomes more symmetric with respect to the central spindle (142 minutes) but retains its asymmetry with respect to the cell (97 minutes onwards). Scale bars: 10 µm.

View larger version (151K): [in a new window]   Fig. 5. Microtubule distribution and furrow induction in cut cells containing a half-spindle and segregated chromosomes. Spermatocytes (A, 0 minutes) were cut in two (8 minutes) between segregating chromosomes in early anaphase, exposing plus-ends of overlapping microtubules (Ba, green). As chromosomes continue to move poleward (8-40 minutes; see also Movie 5, http://jcs.biologists.org/supplemental/), both mitochondria (0-8 minutes, m) and actin filaments (f, red), slightly disorganized by cutting, are excluded toward microtubule plus-ends at the cell periphery (15-25 minutes; f,g, red). When chromosomes arrive at the pole (40 minutes), a new half-spindle emerges and overlaps with cut plus-ends of original half-spindle microtubules (c, green), forming an extremely asymmetric `midzone' (c, arrows). Actin filaments (h, red) continue to follow microtubule plus-ends, now overlapping at the midzone, whereas mitochondria begin to extend along microtubules (40 minutes onwards). Furrow initiation (68-83 minutes, arrows) and contractile ring formation (i, red) occur at the asymmetric midzone, displaced 48.6±3.8% (n=8) away from the central position. As the furrow ingresses, the contractile ring (i,j, red) and furrow (83 minutes onward) follow the midzone position (c-e, arrows), becoming progressively more symmetric with respect to the central spindle because of elongation of the new half-spindle (c-e, green). But the furrow remains asymmetric to the cell because of the initial asymmetry at furrow initiation (140 minutes). Scale bars: 10 µm.

View larger version (77K): [in a new window]   Fig. 6. Re-establishment of spindle bipolarity relative to changes in half-spindle length in cut cells. (A) Polarization microscope images illustrating length change measurements (n=8) of the new (d1) and original (d2) half-spindles with respect to the furrow position (line). Time is given in minutes. (B) Bar chart showing that during furrow ingression, the length of the new half-spindle (d1) has more than doubled, from 5.48±1.75 to 12.45±1.76 µm, whereas that of the original half-spindle remains relatively unchanged (d2). Scale bars: 10 µm.

View larger version (20K): [in a new window]   Fig. 7. Model for asymmetric furrow induction through microtubule-mediated distribution of actin filaments in cut cells. Following cutting in early anaphase (a), chromosomes (blue) continue to move poleward (b,c), whereas actin filaments (red) and mitochondria (gray) accumulate toward exposed plus-ends of spindle microtubules (green). Spindle bipolarity begins to re-establish when short antiparallel microtubules organize a new half-spindle and overlap with cut plus-ends of the original half-spindle to form a new `midzone' (c). While mitochondria become aligned with spindle microtubules, actin filaments continue to follow microtubule plus-ends to the cell cortex where they bundle into a contractile ring and initiate an asymmetric furrow (d). The furrow ingresses as the new half-spindle elongates, shifting along with the midzone toward the middle of the central spindle (d,e).

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