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Understanding Cytokinetic Actomyosin Ring Assembly Through Genetic Code Expansion, Click Chemistry, DNA origami, and in vitro Reconstitution

Periodic Reporting for period 4 - ACTOMYOSIN RING (Understanding Cytokinetic Actomyosin Ring Assembly Through Genetic Code Expansion, Click Chemistry, DNA origami, and in vitro Reconstitution)

Reporting period: 2020-05-01 to 2021-04-30

Cell division requires a machine that generates force that can bisect a cell into two daughters.

In many organisms, including human, this process is achieved by a machine composed of proteins such as actin and myosin, both of which are key to force generation in the muscle. This actomyosin assembles into a ring that divides many cell types.

Understanding the mechanisms of actomyosin ring organization and assembly have a profound influence on basic sciences as well in disease, since actomyosin is altered in a number of diseases, such as cardiomyopathies, and some cancers.

The project aims to understand molecular mechanisms behind assembly of the cytokinetic actomyosin ring, a conserved structure that facilitates cytokinesis in a number of eukaryotes. In the project, we aim to understand 1. where actin filaments are nucleated from, using genetic code expansion and click chemistry 2. the polarity of actin filaments in the cytokinetic ring 3. the function of actin in ring assembly and 4. reconstitution of actomyosin rings using supported bilayers and in permeabilized protoplasts.
The project on actin filament and actomyosin ring assembly / organization mechanisms started in late 2015 and ended 30 April 2021.

15 papers have been already been published from the work and four more are in preparation.

1. Chin, S., Hatano, T., Sivashanmugam, L., Suchenko, A., Kashina, A., Balasubramanian, M.K. and Jansen, S. (2021). N-terminal modification of actin by acetylation and arginylation determines the architecture and assembly rate of linear and branched actin networks. Biorxiv. under revision eLife.

2. Palani, S., Ghosh, S., Clarke, S., Ivorra-Molla, E., Suchenko, A., Balasubramanian, M.K.,* and Koester, D (2021). Calponin-homology-domain mediated bending of membrane bound actin filaments. eLife doi: 10.7554/eLife.61078. MKB is a corresponding author. (S. Palani and D. Koester were PDFs in Balasubramanian laboratory).

3. Kamnev A, Palani S, Zambon P, Cheffings T, Burroughs N, Balasubramanian MK. (2021). Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe. Mol. Biol. Cell. doi: 10.1091/mbc. E20-09-0588.

4. Palani, S., Köster, D.V. and Balasubramanian, M.K. (2020). Phosphoregulation of actin-tropomyosin revealed through genetic code expansion. Wellcome Open Research. doi: 10.12688/wellcomeopenres.16082.1. eCollection 2020.

5. Lim, T, Chew, TG, Osaki, Y., Kamnev, A., Hatano., T., Osumi, M., Balasubramanian, M.K. (2020). Inhibition of cell membrane ingression at the division site by cell wall in fission yeast. Mol. Biol. Cell. doi: 10.1091/mbc.E20-04-0245.

6. Zambon, P., Palani, S., Jadhav, S., Gayathri, P., and Balasubramanian, M.K. (2020). Genetic Suppression of Defective Profilin by Attenuated Myosin II Reveals a Potential Role for Myosin II in Actin Dynamics in vivo in fission yeast. Mol. Biol. Cell. doi: 10.1091/mbc.E20-04-0224.

7. Chapa-y-Lazo, B., Hamanaka, M., Wray, A., Balasubramanian, M.K.* and Mishima, M. (2020) Polar relaxation by dynein-mediated removal of cortical myosin II. J Cell Biology: doi:10.1083/jcb.201903080. PMID: 32497213. MKB is a corresponding author and first author from MKB laboratory.

8. Hatano, T., Sivashanmugam, L., Suchenko, A., Hussain, H., and Balasubramanian, M.K. (2019). Pick-Ya actin: A method to purify actins with bespoke post-translational modifications. J Cell Science: doi: 10.1242/jcs.241406.

9. Palani, S, Köster, D.V. Hatano, T., Kamnev, T., Kanamaru, T., Brooker, H., Hernandez-Fernaud, J.R. Jones, A.M.E. Millar, J.B.A. Mulvihill, D.P. and Balasubramanian, M.K. (2019). Phospho-regulation of tropomyosin is crucial for actin cable turnover and division site placement. J Cell Biology (DOI: 10.1083/jcb.201809089).

10. Syed, J., Palani, S., Clarke, S.T. Asad, Z., Bottrill, A., Jones, A.M.E. Sampath, K., Balasubramanian, M.K. (2019). Expanding the zebrafish genetic code through site-specific incorporation of azido-lysine, bicyclononyne-lysine, and diazirine-lysine. Int. J. Mol. Sci. (doi: 10.3390/ijms20102577).

11. Meadows, J.C.,Messin L.J. Kamnev, A., Lancaster, T.C. Balasubramanian, M.K. Cross, R.C. and Millar, J.B.A (2018). Stabilizing and destabilizing kinesin complexes queue at the +TIP to ensure microtubule disassembly at the cell cortex. EMBO Reports (doi: 10.15252/embr.201846196).

12. Hatano, T., Alioto, S., Roscioli, E., Palani, S., Clarke, S.T. Kamnev, A., Hernandez-Fernaud, J.R. Sivashanmugam, L., Chapa-Y-Lazo, B., Jones, A., Robinson, R.C. Sampath, K., Mishima, M., McAinsh., A., Goode, B.l. Balasubramanian, M.K. (2018). Rapid production of pure recombinant actin in Pichia pastoris. J Cell Science (doi: 10.1242/jcs.213827).

13. Lim, T., Hatano, T., Kamnev, A., Balasubramanian, M.K.,* and Chew, T.G. (2018). Equatorial Assembly of the Cell Division Actomyosin Ring in the Absence of Cytokinetic Spatial Cues. Current Biology (doi: 10.1242/jcs.213827). MKB is the corresponding author and all authors from MKB laboratory.

14. Palani, S., Srinivasan, R., Zambon, P., Kamnev, A., Gayathri, P., and Balasubramanian, M.K. (2017). Evidence that a steric clash in the upper 50KDa domain of the motor Myo2p leads to cytokinesis defects in fission yeast. J Cell Science. (doi: 10.1242/jcs.205625).

15. Chew, TG, Huang J, Palani, S., Kamnev, A, Hatano, T., Somesse, R., Gu, Y., Oliferenko, S., Sivaramakrishnan, S., and Balasubramanian, M.K. (2017) Actin turnover ensures actin filament homeostasis during cytokinetic ring contraction. J Cell Biology. 216: 2657-2667.

We have established the role of myosin II in cytokinetic actomyosin ring assembly and contraction. This is key to the synthetic reconstitution of cytokinetic actomyosin rings. We have also established the length regime of actin filaments that can support proper actomyosin ring assembly. In recently published work, we have also established supported bilayer based assays and permeabilized spheroplast assays for cytokinetic ring assembly.
We have provided insight into why cytokinetic rings need to turnover continuously, which is also important to understand mechanism of actomyosin ring assembly.

We have developed a method to purify fission yeast actin, which is helping us advance ideas on the role of actin dynamics in actomyosin ring assembly.

We have reconstituted curved actin filaments and have developed the first steps in synthetic cell generation.
1. As part of the work, we have devised a method to purify the key cytoskeletal protein actin without any contaminants. This protein has been purified in mixtures, in the past, but not by itself. We plan to develop this method further to enable purification of actin mutants that cause human disease. Such a method to purify human actins should aid in drug discovery.
2. We have succeeded in assembling curved actin filaments on supported lipid bilayers.
3. We have made significant progress towards synthetic cell division apparatuses.
4. We have developed a variety of cell biological and synthetic biological tools.