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Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends
Published on: March 15, 2014
Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in
1Department of Biology, York University, 4700 Keele St, Toronto, ON, M3J 1P3, Canada.
Abstract:
A "precocious" cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2-3 μm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~ 1 μm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.
Insights
In Mesostoma ehrenbergii spermatocytes, a precocious cleavage furrow ingressed even without spindle microtubules. This indicates cleavage furrows can resume ingression independently of anaphase and microtubule presence.
Area of Science:
- Cell Biology
- Cytokinesis
- Mitosis
Background:
- A "precocious" cleavage furrow forms and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes.
- This furrow dynamically shifts position in response to chromosome movements during prometaphase, aiming for proper segregation before anaphase.
- Spindle microtubules (MTs) are traditionally implicated in regulating cleavage furrow positioning and ingression.
Purpose of the Study:
- To investigate the role of spindle microtubules in the positioning and ingression of the precocious cleavage furrow.
- To determine if cleavage furrows can ingress in the absence of spindle MTs during prometaphase.
Main Methods:
- Depolymerization of spindle microtubules during prometaphase using nocodazole (NOC) and colcemid in Mesostoma ehrenbergii spermatocytes.
- Observation of cleavage furrow behavior and chromosome movement following microtubule depolymerization.
- Immunofluorescence staining to confirm complete microtubule depolymerization.
Main Results:
- Contrary to expectations, precocious cleavage furrows did not regress but instead commenced ingression in the absence of spindle MTs.
- In 33/61 treated cells, furrows ingressed at rates similar to normal anaphase, often moving towards one pole.
- Chromosome segregation was aberrant, with bivalent chromosomes moving to one pole and univalents showing varied distribution.
Conclusions:
- Cleavage furrows can resume ingression independently of spindle microtubules and anaphase.
- The results challenge existing models that heavily rely on spindle MTs for furrow positioning and ingression control.
- This suggests an intrinsic mechanism within the cell cortex may drive furrow ingression even when MTs are absent.
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