Precocious (pre-anaphase) cleavage furrows in Mesostoma spermatocytes

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Abstract

It generally is assumed that cleavage furrows start ingression at anaphase, but this is not always true. Cleavage furrows are initiated during prometaphase in spermatocytes of the flatworm Mesostoma, becoming detectable soon after the spindles achieve bipolarity. The furrows deepen during prometaphase, but ingression soon arrests. After anaphase the pre-existing furrow recommences its ingression and rapidly cleaves the cell. Such “precocious” furrowing also commonly occurs in diatoms and other algae.

The position of the “precocious” cleavage furrow changes when there are changes in the distribution of chromosomes. Each of the 4 unipolarly-oriented univalent chromosomes moves to a pole at the start of prometaphase but later in prometaphase may move to the opposite pole. The furrow position adjusts during prometaphase according to the numbers of univalents at the two poles: when there are two univalent chromosomes at each pole the furrow is symmetrical at the spindle equator, but when there are unequal numbers at the poles the furrow shifts 2–3 μm toward the half-spindle with fewer univalents. Nocodazole causes spindle microtubules to disappear. After addition of nocodazole, bivalents become detached from one pole and move toward the other, which causes the furrow to shift 2–3 μm toward the pole with fewer chromosomes. Furrow positioning thus is sensitive to the positioning of chromosomes in the spindle and furrow positions change in the absence of spindle microtubules.

Introduction

Cell cleavage (cytokinesis) in animals cells generally begins during mid-anaphase or late-anaphase: the cleavage furrow indents the cell cortex in the plane that was occupied by the former metaphase plate of chromosomes and it continuously constricts the cytoplasm until the cell is cleaved into two cells (Burgess and Chang, 2005, Rappaport, 1996). The contractile ring that cleaves the cell generates the force using actin and myosin, and contains as well a variety of other proteins [e.g., anillin, septins, formins, Arp2/3, profilin, MLCK, etc. (Eggert et al., 2006, Guertin et al., 2002, Oegama and Hyman, 2006)], including regulatory proteins such as Rho (Bement et al., 2005, von Dassow, 2009). The work we report here deals with two large scale attributes of the cytokinetic machinery: when does the furrow begin to constrict the cell? And how is the furrow position specified?

There is general agreement in the literature that in animal cells furrowing does not begin until after chromosomes separate in anaphase (e.g., reviews in Barr and Gruneberg, 2007, Burgess and Chang, 2005), but specification of the furrow position occurs earlier (e.g., Rappaport, 1981, Rappaport and Rappaport, 1993). As summarised by Rappaport, 1996 (page 138): “The event that establishes the division [furrow position] can be relatively brief, and it normally occurs (in the small number of cell types that have actually been tested) at metaphase–anaphase.”

There is no solid consensus on how the site of the furrow is determined. Most consider that spindle microtubules interact with the cell cortex to specify furrow position (e.g., Albertson et al., 2008, Alsop and Zhang, 2003, Barr and Gruneberg, 2007, Burgess and Chang, 2005, Canman et al., 2003, Chen et al., 2008, D’Avino et al., 2005, Murthy and Wadsworth, 2008, Rappaport and Rappaport, 1993, Rappaport, 1996, Wadsworth, 2005). The spindle-cortex interaction may be generated by spindle microtubules in the spindle equator (e.g., Rappaport, 1996), or associated with the midzone of the anaphase spindle (e.g., Alsop and Zhang, 2003, Wheatley and Wang, 1996), or associated with spindle asters, e.g., transporting components to the cortex or causing relaxation at the poles and thence contraction at the equator (e.g., Oegama and Mitchison, 1997, Rappaport, 1996, Sisson et al., 1999), though recent experiments show that cleavage can take place even when astral microtubules do not extend to the cortex (von Dassow et al., 2009). Or cells may specify furrow positions in more than one way (e.g., Bringmann and Hyman, 2005, Chen et al., 2008).

The work on Mesostoma spermatocytes that we present here raises questions about both these generalisations, that is, that furrow constriction does not begin until after the start of anaphase, and that spindle microtubules specify the furrow position.

The cleavage furrow in the spermatocyte of the flatworm Mesostoma appears in prometaphase, more than one hour before the start of anaphase. Once the furrow appears, its ingression is arrested until anaphase, or continues very slowly until anaphase, after which it cleaves the cell in the usual manner. Precocious cleavage furrows also are seen in diatoms (Pickett-Heaps et al., 1980) and various other algae such as Spirogyra (e.g., McIntosh et al., 1995), in which precocious furrows may appear as early as prophase (e.g., Lokhorst et al., 1988, Mattox and Stewart, 1974, McIntosh et al., 1995, Sawitzky and Grolig, 1995). We know of no animal cells except Mesostoma spermatocytes in which a precocious furrow persists throughout division and eventually cleaves the cell after anaphase.

The furrow's position in a Mesostoma spermatocyte changes when the distribution of chromosomes changes. In early prometaphase each of the 4 univalent chromosomes moves to a pole as the bivalents become bipolarly oriented, but univalent chromosomes often move between the poles during prometaphase in order to achieve their proper segregation (Oakley, 1985). As we illustrate in this article, the prometaphase cleavage furrow is sensitive to these redistributions; its position changes when there are unequal numbers of univalents at the two poles, though the spindle remains in its one, fixed position.

Section snippets

Materials and methods

Mesostoma were reared from diapausing (overwintering) eggs originally provided by Dr. Paul Hebert, University of Guelph (e.g., Hebert and Beaton, 1990) and kept in pond water at 4 °C for several months before being placed in a jar that contained algae and Daphnia; the algae were food for the Daphnia which in turn were food for the Mesostoma that hatched from the eggs. Mesostoma are hermaphrodites, with both testes and ovaries; adults can be viviparous, or can bear diapausing eggs (Ferguson and

Results

Mesostoma spermatocytes contain three bivalent and four univalent chromosomes (Fig. 1). The three bivalents (arrows in Fig. 1) are bipolarly oriented from early prometaphase onwards; they are monochiasmatic, with prominent free arms at each end (the grey arrowheads in Fig. 1 point to kinetochores from which the free arms extend). The four univalents are at the poles from early prometaphase onwards (Oakley, 1985) and thus would seem to be unipolarly oriented. The univalents (open arrows in Fig. 1

Precocious formation of the cleavage furrow

Precocious cleavage furrows are initiated in early prometaphase Mesostoma spermatocytes, nearly two hours before anaphase; furrow ingrowth proceeds for a short time but is arrested until telophase when ingression recommences and cleaves the cell (e.g., Fig. 3, Fig. 5, Fig. 10). Initiation of furrows in prometaphase is an apparent exception to the general consensus that cleavage begins as cells transit through anaphase into telophase. From viewing cleavage in several types of entirely unrelated

Acknowledgements

This work was supported by grants from the Natural Sciences and Engineering Council of Canada to A.F. We thank Karen Rethoret, York University, for sectioning the cells and taking the electron micrographs.

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