Cytokinesis is the mechanism by which the genetic complement and the cellular cytoplasm are accurately and permanently segregated at the end of mitosis. Its proper completion is fundamental to the maintenance of the euploid genome and thus involves a coordinated series of changes whose temporal and spatial accuracy cannot be left to chance.
Cytokinesis occurs through the interaction of actin and myosin II on the cell cortex at the position of the spindle equator during late anaphase, creating the contractile event that ultimately separates the two daughter cells (Mabuchi, 1986
). Evidence obtained from various systems, including invertebrate embryos and mammalian culture cells, demonstrates that a microtubule-dependent process transmits the signal for cytokinesis (Rappaport, 1986
; Cao and Wang, 1996
). Signaling communicates the spindle position to the cell cortex, to ensure that effective furrowing occurs at the precise midpoint of the spindle after the chromatid sets are well separated.
The anaphase spindle has two distinct components, astral arrays of microtubules that emanate from the two spindle poles and that do not interact, and a central spindle that is comprised of an array of tightly bundled antiparallel microtubules that arise from the two spindle poles and interdigitate in the equatorial region of the cell. The relative importance of the two microtubule arrays to furrowing seems to be divergent in different systems. Sea urchin embryos and Caenorhabditis elegans
cells seem to require astral arrays for furrowing, whereas Drosophila melanogaster
cells require the central spindle (for review, see Glotzer, 2004
). This divergence may be due to differences in embryonic and nonembryonic cell cleavage mechanisms.
Mechanisms of cell cleavage are highly variable. Therefore, results in C. elegans
or D. melanogaster
do not necessarily predict cleavage requirements in mammalian cells. In mammalian cells the bulk of evidence, based on micromanipulation studies, supports an important role for the central spindle in furrow formation (Cao and Wang, 1996
). On the other hand, it has been demonstrated that astral microtubules also can play a role in the positioning of cleavage apparatus (Rieder et al., 1997
) and of the passenger proteins in mammalian cells (Murata-Hori and Wang, 2002
). Interestingly, the absence of overlapping microtubules does not inhibit passenger protein localization or furrowing in cells with monopolar spindles (Canman et al., 2003
), suggesting the central spindle is not required for furrowing.
The central spindle, created by PRC1 (Jiang et al., 1998
; Mollinari et al., 2002
; Kurasawa et al., 2004
) and the central-spindlin complex, consisting of a kinesin-like protein ZEN-4/MKLP1 and a Rho-family GAP, CYK4/MgcRacGAP (Mishima et al., 2002
), serves as a platform that enables the proper positioning at the anaphase spindle equator of proteins required for furrow progression. Such proteins include the passenger proteins, a group of proteins (INCENP, Survivin, Aurora B, Borealin, and TD-60) that seem to play a key role in the proper completion of cytokinesis in mammalian cells (Martineau et al., 1995
; Mackay et al., 1998
; Skoufias et al., 2001
; Terada, 2001
; Gassmann et al., 2004
; Sampath et al., 2004
Using a combination of small interfering RNA (siRNA) ablation of PRC1 and both time-lapse and immunofluorescence microscopy, we have here addressed the role of the central spindle bundle in furrow completion and in final cell abscission. We have found that furrowing proceeds to completion in the absence of the central spindle bundle, followed by failure of abscission, and reversion of the furrow an hour later to create a binucleate cell. In the absence of the anaphase spindle bundle, the passenger proteins organize into a subcortical ring instead of forming a telophase disc. This ring coincides with the deposition of actin and anillin at the cell cortex and thus coincides with the position of the cleavage event. The passenger proteins seem to be most prominent near the tips of astral microtubules. Our observations lead us to conclude that the central spindle bundle is not obligatory for completion of furrowing in mammalian cells but is required for the final abscission event.