Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes

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.

Chromosomes selectively detach at one pole and quickly move towards the opposite pole when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes

“…Several lines of evidence indicate that the cells do not die after NOC treatment. For one, in the continuing presence of NOC the furrow ingresses and moves toward the pole with fewer chromosomes (Fegaras and Forer, 2017), sometimes moving off the end of the cell but often forming two different sized cells, still attached, resembling somewhat the appearance of a "shmoo" (Capp, 1948), seen in supplemetal video 3. Further, univalents left behind at a pole sometimes move across the equator to the opposite pole, up to 15 minutes after addition of NOC.…”

Evidence of Non-microtubule Spindle Forces in Mesostoma ehrenbergii Spermatocytes

“…When cells are treated with 10 μM NOC the bivalent chromosomes immediately stop oscillating and each kinetochore moves toward its respective pole as the bivalent stretches out. The chromosomes pause (i.e., remain stretched out) and after a few minutes all three kinetochores at one pole detach and move toward the opposite pole, after which the cleavage furrow moves toward the vacated half spindle ( Fegaras and Forer, 2018a , b ).…”

“…The procedures have been described in detail elsewhere ( Fegaras and Forer, 2018a , b ). In brief, Mesostoma ehrenbergii were reared in lab ( Hoang et al, 2013 ).…”

“…Mesostoma spermatocytes have three bivalent chromosomes and two sets of univalent chromosomes positioned at either pole, as first described in Husted and Ruebush, 1940 ( Figure 1 ). A precocious pre-anaphase cleavage furrow develops and ingresses slightly during early prometaphase giving the cell its characteristic dumbbell-like shape, then recommences ingression during anaphase at which time it cleaves the cell in the usual manner ( Forer and Pickett-Heaps, 2010 ; Fegaras and Forer, 2018b ). The univalent chromosomes are never paired: there are only 3 synaptonemal complexes in pre-division nuclei, corresponding to each of the 3 bivalents ( Oakley and Jones, 1982 ).…”

“…This causes the bivalents to stretch, possibly due to non-microtubules associated force production via the spindle matrix and/or actin-myosin. After a few minutes being stretched, all the kinetochores oriented to one pole detach and move rapidly toward the opposite pole, at speeds averaging 38 μm/min and with instantaneous speeds up to 100 μm/min ( Fegaras and Forer, 2018a , b ). One reason that all kinetochores move to the same pole might be because this is related to non-random segregation, because all male partners segregate to one pole and all females to the other.…”