Roles of ADF-H domain proteins and their co-factors in promoting actin turnover
Creators: name: Aguilar Lopez, Rey Martin
Publisher: name: Brandeis University
Year: 2025
Subjects: actin
adf-h
cofilin
coronin
gmf
Biology
Cytoskeleton

description: Actin is one of the most abundant proteins in eukaryotic cells and polymerizes into helical ‘microfilaments’, which are further organized into dynamic networks and arrays. These actin structures play essential roles in cell and tissue morphogenesis, cell migration, cell adhesion, endocytosis, cytokinesis, and many other processes. Each of these actin structures is architecturally remodeled and disassembled (or ‘turned over’) at precise rates, and typically from one end of the network. This rapid turnover is achieved by a group of conserved actin binding proteins, each with highly specific activities on actin, all working in concert. Until now, most of the work in the field has focused on one actin-binding protein at a time, defining their individual effects on actin filaments. One of the goals of my thesis has been to better understand how the activities of multiple actin binding proteins influence each other and are coordinated, in some cases competing and in other cases synergizing, to regulate actin network turnover.My work has been focused on the actin depolymerizing factor-homology (ADF-H) family of proteins, which includes ADF/cofilin, Abp1, glia maturation factor (GMF), and twinfilin, each of which is highly conserved in the fungal and animal kingdoms. ADF/cofilin has three known effects on actin filaments: severing, depolymerization, and debranching; Abp1 promotes the nucleation and stabilization of branched actin filaments by binding to the Arp2/3 complex; GMF has opposite effects to Abp1, destabilizing and pruning branches; twinfilin has roles in promoting depolymerization at both the barbed and pointed ends of filaments, some of which depend on its interactions with another co-factor, Srv2/CAP.
description: In Chapter 2, I describe work that I contributed substantially to (Hilton, Aguilar et al., 2018), which defines the depolymerization activities of mouse twinfilin and Srv2/CAP, and reveals key differences from those of S. cerevisiae twinfilin and Srv2/CAP. Specifically, using direct visualization of effects by TIRF microscopy, we show that the barbed end depolymerization activity of mouse twinfilin isoforms is similar to yeast twinfilin, whereas the pointed end depolymerization effects of mouse twinfilin and Srv2/CAP are not nearly as robust as the yeast counterparts. In Chapter 3, I use an in vitro debranching assay to show that S. cerevisiae coronin (Crn1) synergizes with GMF in pruning branches, elevating the rate of debranching by ~10-fold. Further, I show that these activities depend on the ‘CA-like’ motif (which binds Arp2/3 complex) located in the ‘unique’ domain of coronin, and that the unique domain is sufficient for these effects. In Chapter 4, I explore the functional relationship between two ADF-H domain proteins, cofilin and Abp1, which have distinct activities but are both ubiquitous, abundant, and bind to the sides of actin filaments. My data suggest that cofilin and Abp1 compete for binding actin filaments, as expected, but surprisingly cytosolic concentrations of Abp1 (1 µM) do not interfere with the effects of cofilin in severing and depolymerizing actin filaments. I discuss a model for how both ADF-H domain proteins can interact with filamentous actin with high affinity, and perform their respective cellular functions, without interfering with each other due to key differences in their binding kinetics.
description: Overall, the results I present in this thesis add to the emerging view that groups of actin-binding proteins in cells work in coordination with one another. In some cases, these factors are cooperating or synergizing, in other cases directly competing, and in still other cases avoiding competition through differences in the kinetics of their interactions with actin.