Abstract
Cytokinesis in many eukaryotes involves the contraction of an actomyosin-based contractile ring1,2. However, the detailed mechanism of contractile ring contraction is not fully understood. Here, we establish an experimental system to study contraction of the ring to completion in vitro. We show that the contractile ring of permeabilized fission yeast cells undergoes rapid contraction in an ATP- and myosin-II-dependent manner in the absence of other cytoplasmic constituents. Surprisingly, neither actin polymerization nor its disassembly is required for contraction of the contractile ring, although addition of exogenous actin-crosslinking proteins blocks ring contraction. Using contractile rings generated from fission yeast cytokinesis mutants, we show that not all proteins required for assembly of the ring are required for its contraction in vitro. Our work provides the beginnings of the definition of a minimal contraction-competent cytokinetic ring apparatus.
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Acknowledgements
We thank D. McCollum (University of Massachusetts Medical School, USA), K. Gould (Vanderbilt University, USA), J-Q. Wu (Ohio State University, USA), V. Simanis (ISREC, Switzerland), J. Bähler (University College London, UK), T. D. Pollard (Yale University, USA), T. Toda (Cancer Research, UK), I. Hagan (Paterson Institute, UK), F. Chang (Columbia University, USA), D. Mulvihill (University of Kent, UK), P. Perez (CSIC, Spain), Y. Hiraoka (Osaka University, Japan), K. Nakano (University of Tsukuba, Japan), S. Oliferenko (TLL, Singapore), and M. Sato (Waseda University, Japan) and the Yeast Genetic Resource Center Japan for providing plasmids and strains, Y. Oba and M. Ojika (Nagoya University, Japan) for their kind gift of DXQ, Y. Toyoshima (University of Tokyo, Japan) for her kind gift of kinesin, R. Amikura for help in electron microscopy, and S. Oliferenko, S. Bulchand and D. Subramanian for critical reading of this manuscript. We thank K. Gull (Oxford University, UK) for antibodies. We thank M. Sevugan for technical assistance. This work was supported by a Japan Society for Promotion of Science (JSPS) grant-in-aid for scientific research (I.M., #22247031); JSPS research fellowships for young scientists (J.K.); NUS JSPS collaborative grant (I.M. and M.B.), Temasek Life Sciences Laboratory and Singapore Millennium Foundation (M.B. and M.M.); visiting scientist fellowship from the Gakushuin University (M.M.) and Mechanobiology Institute (M.B., R.S. and Y.H.).
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I.M. (in vitro activation and biochemistry) and M.K.B. (establishment of ring assembly in protoplasts and mutant studies) conceived the study, I.M., M.M., M.K.B. and J.K. designed the experiments. I.M., J.K., M.M., T.T., R.S. and Y.H. conducted the experiments. J.K., I.M. and M.M. analysed the data. M.K.B., M.M., J.K. and I.M. wrote the manuscript.
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ATP drives ring contraction in vitro.
A contracting ring in the presence of 0.5 mM ATP at 25 °C corresponds to the frames shown in Fig. 2a. X-Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 199 kb)
ATP drives ring contraction.
A contracting ring in the presence of 0.01 mM ATP at 25 °C corresponds to the frames shown in Fig. 2c. X–Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 118 kb)
AMP-PNP does not induce ring contraction in vitro.
Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 2d. XY and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 1038 kb)
The plasma membrane of Rlc1-3xGFP expressing cell ghosts stained with FM4-64 during contraction in vitro.
Experiment was done at 25 °C. The membrane did not ingress as the ring contracts. The video corresponds to the frames shown in Fig. 2g. (MOV 450 kb)
Incomplete actomyosin arcs do not show ATP-dependent contraction in vitro.
The GFP fluorescence decays the presence of 0.5 mM ATP but the actomyosin arc do not show any contraction at 25 °C. The video corresponds to the frames shown in Fig. 2j. X–Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 329 kb)
Contractile ring of myo2-E1ts undergoes slow in vitro contraction even at the permissive temperature of 25 °C.
The video corresponds to the frames shown in Fig. 3b. (MOV 105 kb)
Contractile ring of myp2 null mutant (myp2Δ) in vitro.
Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 3b. (MOV 287 kb)
Contractile ring contraction of the double mutant of myo2 and myp2 (myo2-E1ts myp2Δ) in vitro.
These rings did not show any contraction and instead fragmented and disassembled in presence of ATP at 25 °C. The video corresponds to the frames shown in Fig. 3b. (MOV 912 kb)
Addition of Jasplakinolide does not affect ring contraction in vitro.
Experiment was done at 25 °C. (MOV 548 kb)
ADF/cofilin (Adf1p) is not required for ring contraction in vitro.
Cell ghosts from adf1-1ts were held at 36 °C for 15 min before ATP addition. (MOV 534 kb)
LatA has minimal effect on ring contraction in vitro.
Experiment was done at 25 °C. (MOV 515 kb)
Formin (Cdc12p) is not required for ring contraction in vitro.
Experiment was done at 36 °C. Cell ghosts from cdc12-112ts were held at 36 °C for 15 min before ATP addition. (MOV 204 kb)
F-BAR protein (Cdc15p) is not required for ring contraction in vitro.
Experiment was done at 36 °C. Cell ghosts from cdc15-140ts were held at 36 °C for 15 minutes before ATP addition. (MOV 571 kb)
Tropomyosin (Cdc8p) is required for integrity and contraction of ring in vitro.
Cell ghosts from cdc8-110ts were held at 36 °C for 15 min before ATP addition. The video corresponds to the frames shown in Fig. 5a. (MOV 105 kb)
Addition of 1 μM purified N-terminal region of IQGAP (Rng2Ns) completely blocks ring contraction in vitro.
Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 5b. (MOV 253 kb)
Addition of 0.9 μM purified fimbrin (Fim1p) completely blocks ring contraction in vitro.
Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 5c. (MOV 212 kb)
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Mishra, M., Kashiwazaki, J., Takagi, T. et al. In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics. Nat Cell Biol 15, 853–859 (2013). https://doi.org/10.1038/ncb2781
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DOI: https://doi.org/10.1038/ncb2781
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