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Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage.

Alsop GB, Zhang D - J. Cell Biol. (2003)

Bottom Line: We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present.Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress.We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331, USA.

ABSTRACT
Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced "cells" containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

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Microtubules alone are sufficient for cell cleavage. (A) Asters and chromosomes are removed in metaphase (a–c), which induces disassembly of the spindle (d). (B) After manipulation, the spindle collapses (0–37 min; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), inducing transient and repeated formations of bipolar (70 and 194) or monopolar (217) pseudospindles, and eventually gives rise to relatively disorganized arrays of bundled microtubules bearing pseudopoles (290–296, *). Furrow initiation (290–296, arrows) and accumulation of actin filaments (AF and Overlay) correspond to the midzone (MT, arrows) of bundled microtubule arrays. Due to microtubule reorganizations, furrow initiation is delayed by nearly 2 h in comparison with an initially synchronized cell (183, arrow). (C) Often, newly assembled microtubule bundles radiate randomly (0–108) and induce furrow initiation at multiple sites (118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1). However, only furrows initiated by persistent bundles sustain (118–137, arrows), others eventually regress (arrowheads). Furrow ingression forces the bundled microtubule arrays together (128–153), bearing a single midzone (MT, arrow) with an ingressed contractile ring (AF and Overlay). Furrow initiation is delayed by ∼1 h, judging by division in a neighboring cell (43, arrow). Bar, 10 μm.
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fig3: Microtubules alone are sufficient for cell cleavage. (A) Asters and chromosomes are removed in metaphase (a–c), which induces disassembly of the spindle (d). (B) After manipulation, the spindle collapses (0–37 min; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), inducing transient and repeated formations of bipolar (70 and 194) or monopolar (217) pseudospindles, and eventually gives rise to relatively disorganized arrays of bundled microtubules bearing pseudopoles (290–296, *). Furrow initiation (290–296, arrows) and accumulation of actin filaments (AF and Overlay) correspond to the midzone (MT, arrows) of bundled microtubule arrays. Due to microtubule reorganizations, furrow initiation is delayed by nearly 2 h in comparison with an initially synchronized cell (183, arrow). (C) Often, newly assembled microtubule bundles radiate randomly (0–108) and induce furrow initiation at multiple sites (118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1). However, only furrows initiated by persistent bundles sustain (118–137, arrows), others eventually regress (arrowheads). Furrow ingression forces the bundled microtubule arrays together (128–153), bearing a single midzone (MT, arrow) with an ingressed contractile ring (AF and Overlay). Furrow initiation is delayed by ∼1 h, judging by division in a neighboring cell (43, arrow). Bar, 10 μm.

Mentions: To produce cells containing microtubules as the only spindle structural constituent, we removed both asters and all chromosomes (Fig. 3 A, a–c; Fig. 3 B, 0–8 min) from cells in metaphase (12 of 20 cells had furrow initiation and ingression). These manipulations not only remove other confounding spindle constituents, but also induce disassembly of the spindle and assembly of radiating microtubules bundled together with mitochondria (Fig. 3 A, d; Fig. 3 B, 37). Obviously, disassembly destroys bipolarity of the metaphase spindle and normal distribution of furrow signaling molecules, potentially from asters (Rappaport, 1996) or chromosomes (Earnshaw and Mackay, 1994). Radiating microtubules exhibit dynamics similar to that observed in metaphase (Cassimeris et al., 1988), repeatedly changing their length and distribution to transiently organize mono- or bipolar pseudospindles (Fig. 3 B, 70–217; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), and ultimately give rise to relatively disorganized arrays of bundled microtubules (Fig. 3 B, 290–296). Induction of cell cleavage occurs at midzones of bundled microtubule arrays (Fig. 3 B, 290–296, arrows) organized by randomly formed pseudopoles (290–296, *). Cells fixed at initiation (296) show no chromosomes (DAPI) and exhibit central spindle–like bundled microtubule arrays with lightly stained midzones (Fig. 3 B, MT, arrows), where actin filaments are localized (AF and Overlay). Often, furrow induction occurs at multiple locations (Fig. 3 C, 118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1) due to random distribution of bundled microtubule arrays (43 onward). Many of these furrows, however, are transient and regress as microtubules reorganize (Fig. 3 C, 118–128, arrowheads). Successful furrows are usually induced independently at the midzones of persistent microtubule bundles (Fig. 3 C, 118–137, arrows), supporting earlier discoveries that furrow ingression does not require a complete contractile ring (Rieder et al., 1997). During ingression, these independent furrows can change directions of inward movement and force bundled microtubule arrays together (Fig. 3 C, 128–153, arrows). Cells fixed at completion of ingression (153) exhibit one central microtubule bundle with a distinct midzone (Fig. 3 C, MT, arrow) colocalized precisely with a contractile ring (AF and Overlay).


Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage.

Alsop GB, Zhang D - J. Cell Biol. (2003)

Microtubules alone are sufficient for cell cleavage. (A) Asters and chromosomes are removed in metaphase (a–c), which induces disassembly of the spindle (d). (B) After manipulation, the spindle collapses (0–37 min; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), inducing transient and repeated formations of bipolar (70 and 194) or monopolar (217) pseudospindles, and eventually gives rise to relatively disorganized arrays of bundled microtubules bearing pseudopoles (290–296, *). Furrow initiation (290–296, arrows) and accumulation of actin filaments (AF and Overlay) correspond to the midzone (MT, arrows) of bundled microtubule arrays. Due to microtubule reorganizations, furrow initiation is delayed by nearly 2 h in comparison with an initially synchronized cell (183, arrow). (C) Often, newly assembled microtubule bundles radiate randomly (0–108) and induce furrow initiation at multiple sites (118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1). However, only furrows initiated by persistent bundles sustain (118–137, arrows), others eventually regress (arrowheads). Furrow ingression forces the bundled microtubule arrays together (128–153), bearing a single midzone (MT, arrow) with an ingressed contractile ring (AF and Overlay). Furrow initiation is delayed by ∼1 h, judging by division in a neighboring cell (43, arrow). Bar, 10 μm.
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Related In: Results  -  Collection

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fig3: Microtubules alone are sufficient for cell cleavage. (A) Asters and chromosomes are removed in metaphase (a–c), which induces disassembly of the spindle (d). (B) After manipulation, the spindle collapses (0–37 min; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), inducing transient and repeated formations of bipolar (70 and 194) or monopolar (217) pseudospindles, and eventually gives rise to relatively disorganized arrays of bundled microtubules bearing pseudopoles (290–296, *). Furrow initiation (290–296, arrows) and accumulation of actin filaments (AF and Overlay) correspond to the midzone (MT, arrows) of bundled microtubule arrays. Due to microtubule reorganizations, furrow initiation is delayed by nearly 2 h in comparison with an initially synchronized cell (183, arrow). (C) Often, newly assembled microtubule bundles radiate randomly (0–108) and induce furrow initiation at multiple sites (118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1). However, only furrows initiated by persistent bundles sustain (118–137, arrows), others eventually regress (arrowheads). Furrow ingression forces the bundled microtubule arrays together (128–153), bearing a single midzone (MT, arrow) with an ingressed contractile ring (AF and Overlay). Furrow initiation is delayed by ∼1 h, judging by division in a neighboring cell (43, arrow). Bar, 10 μm.
Mentions: To produce cells containing microtubules as the only spindle structural constituent, we removed both asters and all chromosomes (Fig. 3 A, a–c; Fig. 3 B, 0–8 min) from cells in metaphase (12 of 20 cells had furrow initiation and ingression). These manipulations not only remove other confounding spindle constituents, but also induce disassembly of the spindle and assembly of radiating microtubules bundled together with mitochondria (Fig. 3 A, d; Fig. 3 B, 37). Obviously, disassembly destroys bipolarity of the metaphase spindle and normal distribution of furrow signaling molecules, potentially from asters (Rappaport, 1996) or chromosomes (Earnshaw and Mackay, 1994). Radiating microtubules exhibit dynamics similar to that observed in metaphase (Cassimeris et al., 1988), repeatedly changing their length and distribution to transiently organize mono- or bipolar pseudospindles (Fig. 3 B, 70–217; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), and ultimately give rise to relatively disorganized arrays of bundled microtubules (Fig. 3 B, 290–296). Induction of cell cleavage occurs at midzones of bundled microtubule arrays (Fig. 3 B, 290–296, arrows) organized by randomly formed pseudopoles (290–296, *). Cells fixed at initiation (296) show no chromosomes (DAPI) and exhibit central spindle–like bundled microtubule arrays with lightly stained midzones (Fig. 3 B, MT, arrows), where actin filaments are localized (AF and Overlay). Often, furrow induction occurs at multiple locations (Fig. 3 C, 118–128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1) due to random distribution of bundled microtubule arrays (43 onward). Many of these furrows, however, are transient and regress as microtubules reorganize (Fig. 3 C, 118–128, arrowheads). Successful furrows are usually induced independently at the midzones of persistent microtubule bundles (Fig. 3 C, 118–137, arrows), supporting earlier discoveries that furrow ingression does not require a complete contractile ring (Rieder et al., 1997). During ingression, these independent furrows can change directions of inward movement and force bundled microtubule arrays together (Fig. 3 C, 128–153, arrows). Cells fixed at completion of ingression (153) exhibit one central microtubule bundle with a distinct midzone (Fig. 3 C, MT, arrow) colocalized precisely with a contractile ring (AF and Overlay).

Bottom Line: We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present.Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress.We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331, USA.

ABSTRACT
Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced "cells" containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

Show MeSH