Cells use the actin cytoskeleton for many of their functions, including their division, adhesion, mechanosensing, endo- and phagocytosis, migration, and invasion. Actin bundles are the main constituent of actin-rich structures involved in these processes. An ever-increasing number of proteins that crosslink actin into bundles or regulate their morphology is being identified in cells. With recent advances in high-resolution microscopy and imaging techniques, the complex process of bundles formation and the multiple forms of physiological bundles are beginning to be better understood. Here, we review the physiochemical and biological properties of four families of highly conserved and abundant actin-bundling proteins, namely, α-actinin, fimbrin/plastin, fascin, and espin. We describe the similarities and differences between these proteins, their role in the formation of physiological actin bundles, and their properties—both related and unrelated to their bundling abilities. We also review some aspects of the general mechanism of actin bundles formation, which are known from the available information on the activity of the key actin partners involved in this process.

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures—but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined—including that it is driven by actin’s polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical’s disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical’s F-actin disassembly in vitro and in vivo—and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin -binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the fulllength protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy. (c) 2023 The Author(s). Published by Elsevier Ltd.

The spatiotemporal regulation of an actin mesh during Drosophila oogenesis is essential for proper localization of cell polarity determinants that establish the future patterning of the embryo. Here, we reveal an unexpected role for Semaphorin-2a (Sema2a) in actin mesh regulation and oogenesis. Sema2a classically functions as a secreted guidance cue that binds its cognate Plexin-B (PlexB) receptor to establish neural circuits. In contrast, we find that Sema2a is expressed inside the germarium, germline, and follicle cells of the developing ovary. Sema2a mutants possess small ovaries that fail to develop past mid-oogenesis. We demonstrate that Sema2a interacts with Cappuccino (Capu), a key actin nucleator crucial for building the actin mesh in Drosophila oocytes. Sema2a inhibits the actin assembly activity of Capu in vitro. Furthermore, genetic interaction between Sema2a and Capu influences mesh density and disrupts oskar mRNA localization. PlexB mutants, however, exhibit wild-type size ovaries with oskar mRNA localization distinct from Sema2a mutants, confirming the non-canonical role of Sema2a in oogenesis.

Actin’s transition from monomers (G-actin) to polymers (F-actin) and then into bundled and branched networks underlies many cellular and system functions. Yet, how these networks are dynamically assembled and disassembled is incompletely understood—including why F-actin is often simultaneously and redundantly bundled by different proteins. Here, we focus on fascin and espin, two bundlers that often coexist and robustly bundle F-actin. We find that they synergistically bundle F-actin compared to equal amounts of each one alone. However, we show that bundles containing these two proteins are robustly destabilized by a synergism between Mical and cofilin, indicating mechanisms of how complex bundles are disassembled and remodeled. Yet, our results also reveal that together fascin and espin protect F-actin from this disassembly more effectively than each one alone—including to regulate F-actin disassembly and cellular remodeling in vivo. These findings reveal mechanisms for assembling and disassembling complex networks of bundled F-actin, including a synergism between different bundlers and disassemblers in these processes.