Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

Abstract

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeastSchizosaccharomyces pombe. Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II–coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoebaDictyostelium, but in this case, by detaching actin filaments from myosin II–coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

Unraveling the mechanisms and evolution of a two-domain module in IQGAP proteins for controlling eukaryotic cytokinesis

“…The binding of the CHD to F-actin also inhibits myosin-II movement along actin filaments. 61 This antagonistic relationship between CHD and CykF can explain the observation that the node-to-ring transition was delayed in rng2-CykFΔ cells (presumably due to the dual role of the CHD in stabilizing F-actin and inhibiting myosin-II movement at the division site) but not in rng2-CHDΔ-CykFΔ cells. The functional analysis of the CHD-CykF module also provides experimental evidence for the hypothesis that F-actin dynamics and myosin-II movement along actin filaments are both required for efficient node-to-ring transition.…”

Deciphering the actin structure-dependent preferential cooperative binding of cofilin

“…Actin structure-dependent preferential binding of actin-binding proteins (ABPs) have been investigated at the molecular and cellular levels (Hayakawa et al, 2022;Jegou and Romet-Lemonne, 2019;Jégou and Romet-Lemonne, 2016;Ngo et al, 2016;Tokuraku et al, 2020Tokuraku et al, , 2009. Notably, Harris and colleagues reported biased localization of tandem calponin homology domains (CH1-CH2) that can be mutated to selectively bind different actin conformations at the leading or trailing edge of motile cells (Harris et al, 2020).…”

“…Accurately detecting actin pairs in this case was challenging due to cofilin decoration and the tip-sample dilation effect, as explained by (Ando, 2022). The HHPs and peak heights were determined using methods similar to our previous reports (Hayakawa et al, 2022;Ngo et al, 2015). (C) A comparison between MAD5 (5.7 ± 0.5 nm) observed in the shortened bare area and MAD6 (5.9 ± 0.3 nm) along with MAD7 (6.1 ± 0.3 nm) within the cofilactin region was presented.…”

“…To quantitatively analyze the HHPs and count the number of protomer pairs per HHP, we employed Umex Viewer for Drift Analysis, which enabled the semiautomatic determination and measurement of the distance between the highest "cross-over" points between two long-pitch strands. This involved creating a topographical line profile along the actin filaments, similar to previous studies conducted by (Amyot et al, 2022;Hayakawa et al, 2022). Specifically, one HHP was measured between the two highest cross-over points, while one helical pitch (HP) was measured between the three highest cross-over points.…”

“…The propagation of the supertwisted half helices induced by cofilin clusters results in the shortening of a bare half helix beyond the cofilactin/bare actin boundary. The HHPs and peak heights were determined using methods similar to our previous reports (Hayakawa et al, 2022; Ngo et al, 2015). Scale bars: 25 nm.…”