Gelsolin-Like Domain 3 Plays Vital Roles in Regulating the Activities of the Lily Villin/Gelsolin/Fragmin Superfamily

RESEARCH ARTICLE Gelsolin-Like Domain 3 Plays Vital Roles in Regulating the Activities of the Lily Villin/ Gelsolin/Fragmin Superfamily Dong Qian1☯, Qiong Nan1☯, Yueming Yang1, Hui Li1, Yuelong Zhou1, Jingen Zhu1, Qifeng Bai2, Pan Zhang1, Lizhe An1, Yun Xiang1* 1 MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, 730000, China, 2 College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Qian D, Nan Q, Yang Y, Li H, Zhou Y, Zhu J, et al. (2015) Gelsolin-Like Domain 3 Plays Vital Roles in Regulating the Activities of the Lily Villin/ Gelsolin/Fragmin Superfamily. PLoS ONE 10(11): e0143174. doi:10.1371/journal.pone.0143174 Editor: Haitao Shi, Hainan University, CHINA Received: July 25, 2014 Accepted: November 2, 2015 Published: November 20, 2015 Copyright: © 2015 Qian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The villin/gelsolin/fragmin superfamily is a major group of Ca2+-dependent actin-binding proteins (ABPs) involved in various cellular processes. Members of this superfamily typically possess three or six tandem gelsolin-like (G) domains, and each domain plays a distinct role in actin filament dynamics. Although the activities of most G domains have been characterized, the biochemical function of the G3 domain remains poorly understood. In this study, we carefully compared the detailed biochemical activities of ABP29 (a new member of this family that contains the G1-G2 domains of lily ABP135) and ABP135G1-G3 (which contains the G1-G3 domains of lily ABP135). In the presence of high Ca2+ levels in vitro (200 and 10 μM), ABP135G1-G3 exhibited greater actin severing and/or depolymerization and nucleating activities than ABP29, and these proteins had similar actin capping activities. However, in the presence of low levels of Ca2+ (41 nM), ABP135G1-G3 had a weaker capping activity than ABP29. In addition, ABP29 inhibited F-actin depolymerization, as shown by dilution-mediated depolymerization assay, differing from the typical superfamily proteins. In contrast, ABP135G1-G3 accelerated F-actin depolymerization. All of these results demonstrate that the G3 domain plays specific roles in regulating the activities of the lily villin/gelsolin/fragmin superfamily proteins. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Basic Research Program (2014CB954203), National Natural Science Foundation of China (31270326 and 30800079), the Natural Science Foundation for Distinguished Yong Scholars of Gansu Province (2013GS10064), the Program for New Century Excellent Talents in University (NCET-13-0264) and the Fundamental Research Funds for the Central Universities (lzujbky-2013-243). Competing Interests: The authors have declared that no competing interests exist. Introduction The villin/gelsolin/fragmin superfamily is an important class of multifunctional actin-binding proteins (ABPs) that regulate dynamic remodeling of the actin cytoskeleton via actin nucleating, severing, capping and bundling activities in eukaryotes [1–3]. These superfamily proteins are typified by the possession of three or six G domains [1,4,5]. In mammals and bacteria, each member of this superfamily is encoded by distinct genes, such as villin, which contains six G domains and one head piece domain [6,7]; gelsolin, which contains six G domains [1,4]; PLOS ONE | DOI:10.1371/journal.pone.0143174 November 20, 2015 1 / 21 The Gelsolin-Like Domain 3 in Lily fragmin/severin, which has three G domains [8]; and GSNL-1 in Caenorhabditis elegans, which contains four G domains [9]. However, although the genes enconding villin have been reported in detail[10–12], gelsolin-like proteins in plants have only been mentioned in a few reports [12,13]. The first two plant villins to be identified were ABP135 [14,15] and ABP115 [16,17], which were isolated from lily pollen. In addition, there are six villin homologs in Oryza sativa and five in Arabidopsis thaliana [10,18]. Recently, two new members were identified: PrABP80 [13], which was isolated from poppy pollen and contains six G domains, and LdABP41 [19], which was isolated from Lilium davidii pollen and contains the G1-G3 domains. Interestingly, the smallest identified member of the superfamily is ABP29 [20], which contains only the G1 and G2 domains and part of the G2-G3 linker from Lilium pollen and is an alternative splicing product of plant ABP135 [20,21]. Despite sharing conserved protein sequences and three-dimensional structures, each G domain of the villin/gelsolin/fragmin superfamily plays a distinct role in actin dynamics, endowing the proteins with multifunctional and distinct activities [1,12]. Through several decades of research, the biochemical functions of most G domains in this superfamily have been well characterized. For example, the N-terminal 17-kD chymotryptic segment (CT14N) that retains weak actin-binding functions was described as the G1 domain, which can bind to monomeric actin in a Ca2+-insensitive manner [22]. The truncation of gelsolin with G1 domain and 10 extra amino acids from the start of the G2 domain has weak F-actin severing activity, but this severing activity of the truncation is only one percent of the full gelsolin protein [23]. The G2 domain contains an F-actin binding site, which also has actin capping activity [24]. In addition, the G2 domain has a phosphatidylinositol 4,5-bisphosphate (PIP2) binding site and binds to tropomyosin in a Ca2+- and pH-sensitive manner [24,25]. Further, the G2 domain of GSNL-1 functions as a regulatory domain for Ca2+-dependent conformational changes [26]. Similar to the G1 domain, the G4 domain also binds to monomeric actin [5,27] In addition, it is generally accepted that the C-terminus of gelsolin acts as a helix latch and binds the G2 domain to maintain gelsolin in a Ca2+-free compact state [5,28,29]. However, the biochemical function of the G3 domain is still poorly understood beyond its role as a spacer. We have previously demonstrated that both ABP29 and LdABP41 accelerate actin nucleation, severing and capping of actin filaments in a Ca2+- and/or PIP2-regulated manner in vitro [19,20,30]. Surprisingly, significant differences between ABP29 and LdABP41 and other gelsolin-like proteins have been shown in dilution-mediated F-actin depolymerization assays [20,30]. Specifically, ABP29 inhibits F-actin depolymerization, whereas LdABP41 accelerates F-actin depolymerization similar to other typical members of this superfamily. Why is this? Does the loss of the G3 domain cause this difference? Unfortunately, the gene (...truncated)