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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 3 - ACTOMYOSIN RING (Understanding Cytokinetic Actomyosin Ring Assembly Through Genetic Code Expansion, Click Chemistry, DNA origami, and in vitro Reconstitution)

Reporting period: 2018-11-01 to 2020-04-30

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 in this 30 months we have achieved a number of the goals already.

Seven international journal publications have arisen from the work.

1. 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.

2. 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).

3. 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). Last Author TG Chew is a PDF in MKB group and MKB is the corresponding author.

4. 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).

5. 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.

6. Palani, S., Chew, TG, Srinivasan, R., Kamnev, A., Mishra, M., Sevugan, M., Chapa-y-Lazo, B., Gayathri, P., and Balasubramanian, M.K. (2017) Motor activity dependent and independent functions for Myosin II in Cytokinesis. Current Biology 12:751-757.

7. Zambon, P., Palani, S., Kamnev, A., Balasubramanian, M.K. (2017). Myo2p is the major motor involved in Actomyosin Ring Contraction in S.pombe. Current Biology 27:R99-100.

Through this work, we have advanced several new ideas and concepts that begin to deliver on 2 of the 4 aims (Aims 3 and 4). Aims 1 and 2 are much riskier and the key requirements are now in place and we should be able to deliver on these in a year or two.

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 unpublished 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.
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.