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Biochemical, Cell Biological, and Molecular Dynamics Studies of the Fission Yeast α-Actinin Ain1

森田, 陸離 筑波大学

2020.07.21

概要

The actin cytoskeleton is involved in various cellular processes, such as crawling on substrates, intracellular transport of vesicles and organelles, cell morphogenesis, and cytokinesis. Actin-binding proteins modulate the structure and function of F-actin in these processes. In particular, actin-bundling proteins bundle F-actin and assemble the three-dimensional meshwork of actin cytoskeleton.

The fission yeast Schizosaccharomyces pombe forms a contractile ring (CR) during cytokinesis and utilizes its constriction to guide a septum. Fission yeast has several actin-bundling proteins, although the differences in their function have not yet been fully discovered. Ain1 is the sole homolog of α-actinin in fission yeast and specifically localizes to the CR. It has previously been shown that loss of the ain1+ gene induces cytokinetic defects under stressful conditions.

Here, I studied the biochemical interactions between Ain1 and F-actin and structural mechanism behind their interaction. It was found that Ain1 had a weaker affinity for F-actin than the other actin-bundling proteins. However, microscopy showed that Ain1 was able to bundle F-actin into thick filaments. A point-mutation R216E in the actin-binding domain (ABD), which corresponds to a pathogenic mutation in mammalian α-actinin, enhanced the actin-binding activity of Ain1 and bundled F-actin into a disorderly aggregation.

Next, to characterize the functional domain of Ain1, the cellular localization of truncated and mutated Ain1 was examined. I successfully demonstrated the role-sharing relationship of the two calponin homology domains in detail. In addition, the C-terminal EF-hand was found to be essential for actin-binding in vivo but unnecessary in vitro.

In addition, I studied the molecular mechanism of the interaction between Ain1 and F-actin. It had previously been proposed that the R216E mutation in α-actinin promoted the close-open conformational change of the ABD and enhanced its actin-binding activity. To investigate the transition mechanism, molecular dynamics simulations were performed. However, I did not observe the close-open conformational change. My study unexpectedly revealed a novel actin-binding region on Ain1 molecule and a mutation in this region abrogated the cellular localization of Ain1.

In this thesis, I examined the properties of the α-actinin Ain1 by biochemical, cell biological, and molecular dynamics studies.

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