Molecular dissection of the actin-binding ability of the fission yeast α-actinin, Ain1, in vitro and in vivo

Abstract

A contractile ring (CR) is involved in cytokinesis in animal and yeast cells. Although several types of actin-bundling proteins associate with F-actin in the CR, their individual roles in the CR have not yet been elucidated in detail. Ain1 is the sole α-actinin homologue in the fission yeast Schizosaccharomyces pombe and specifically localizes to the CR with a high turnover rate. S. pombe cells lacking the ain1+ gene show defects in cytokinesis under stress conditions. We herein investigated the biochemical activity and cellular localization mechanisms of Ain1. Ain1 showed weaker affinity to F-actin in vitro than other actin-bundling proteins in S. pombe. We identified a mutation that presumably loosened the interaction between two calponin-homology domains constituting the single actin-binding domain (ABD) of Ain1, which strengthened the actin-binding activity of Ain1. This mutant protein induced a deformation in the ring shape of the CR. Neither a truncated protein consisting only of an N-terminal ABD nor a truncated protein lacking a C-terminal region containing an EF-hand motif localized to the CR, whereas the latter was involved in the bundling of F-actin in vitro. We herein propose detailed mechanisms for how each part of the molecule is involved in the proper cellular localization and function of Ain1.

Molecular Mechanism for the Actin-Binding Domain of α-Actinin Ain1 Elucidated by Molecular Dynamics Simulations and Mutagenesis Experiments

“…For instance, the K255E mutation was located at the interface between the two CH domains, and it abnormally enhances actin-binding activity . Further, we and the other study demonstrated that the mutation (R216E) on Ain1 causes the deformation of the CR. , However, there is insufficient evidence about whether the closed–open conformational change exists. It is still unknown how the R216E mutation alters the actin-binding affinity.…”

“…When α-actinin forms an antiparallel dimer, the EF-hand motif of a pair α-actinin molecule is adjacent to ABD . Actually, the preceding study reported that the abnormal affinity of Ain1 R216E to F-actin is restored with the deletion of the EF-hand …”

“…39 Actually, the preceding study reported that the abnormal affinity of Ain1 R216E to F-actin is restored with the deletion of the EF-hand. 12 Finally, we would like to discuss how the surface interactions maintain the actin-binding affinity in Ain1 R216E (i.e., the reason why Ain1 R216E could bind to the actin without the closed−open transition). Until recently, it has been unclear how the R216E mutation increases the actin-binding affinity of Ain1.…”

“…11 CR. 12,13 However, there is insufficient evidence about whether the closed−open conformational change exists. It is still unknown how the R216E mutation alters the actin-binding affinity.…”

Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast

“…Compared to fimbrin Fim1, α-actinin Ain1 is a relatively weak F-actin bundling protein (Addario et al, 2016; Li et al, 2016; Morita et al, 2017). Remarkably, low-speed sedimentation (Figure 6A,B) and TIRFM assays (Figure 6C,D, Figure 6—video 1) revealed that tropomyosin Cdc8 significantly enhances the F-actin bundling ability of Ain1.…”