Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint.
In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation. Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries. We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2(+), is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells. Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A. Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation. Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1. The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN). The delay was also abolished in cells lacking Zfs1, a component of the previously described S. pombe cytokinesis checkpoint. Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner. Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation. We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.
Mulvihill
Daniel P
DP
Department of Biology, University College London, Gower Street, London WC1E 6BT, UK. d.mulvhill@ucl.ac.uk
Hyams
Jeremy S
JS
D000199:Actins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000502:physiology*; D002478:Cells, Cultured; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D018816:Genes, cdc / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D064246:Mad2 Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*
D000199:Actins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D008164:Luminescent Proteins; C577695:MAD2 protein, S cerevisiae; C090463:MID1 protein, S cerevisiae; D064246:Mad2 Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C097436:Zfs1 protein, S pombe; C577696:mad2 protein, S pombe; D049452:Green Fluorescent Proteins; C489404:CDC7 protein, S cerevisiae; C489406:cdc7 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D024462:Myosin Type II