Mechanics of the F-actin cytoskeleton Stricker, Jonathan; Falzone, Tobias; Gardel, Margaret L
Journal of biomechanics,
01/2010, Volume:
43, Issue:
1
Journal Article
Peer reviewed
Open access
Abstract Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is critical to numerous physical cellular processes, including cell adhesion, migration and division. Each of these ...processes require precise regulation of cell shape and mechanical force generation which, to a large degree, is regulated by the dynamic mechanical behaviors of a diverse assortment of F-actin networks and bundles. In this review, we review the current understanding of the mechanics of F-actin networks and identify areas of further research needed to establish physical models. We first review our understanding of the mechanical behaviors of F-actin networks reconstituted in vitro, with a focus on the nonlinear mechanical response and behavior of “active” F-actin networks. We then explore the types of mechanical response measured of cytoskeletal F-actin networks and bundles formed in living cells and identify how these measurements correspond to those performed on reconstituted F-actin networks formed in vitro. Together, these approaches identify the challenges and opportunities in the study of living cytoskeletal matter.
Microtubules (MTs) govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal cross-talk have remained obscure. We used single-molecule ...fluorescence microscopy to show that the MT plus-end–associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers cotracking growing filament ends for several minutes. CLIP-170–mDia1 complexes promoted actin polymerization ~8 times faster than free–barbed-end growth while simultaneously enhancing protection from capping proteins. We used a MT-actin dynamics co-reconstitution system to observe CLIP-170–mDia1 complexes being recruited to growing MT ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the MT surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing MT plus ends direct rapid actin assembly.
Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During
oogenesis, intercellular bridges (referred to as ring ...canals; RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10 μm. Although multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Thus, here, we optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence prey were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins and uncharacterized proteins. A proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective prey. Furthermore, an RNA interference screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks ...respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
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•Lamellipodial actin density co-fluctuates with the size of the projected area•Lamellipodial actin density adapts to changes in membrane tension•Actin branch geometry prescribes adaptations in lamellipodial actin
How do mechanical perturbations influence the density and the geometry of actin networks at the leading edge of migrating cells?
The actin cytoskeleton plays a central role in many essential cellular processes. Its involvement requires actin filaments to form multiple populations with different structural and therefore ...functional properties in specific subcellular locations. This diversity is facilitated through the interaction between actin and a number of actin binding proteins. One family of proteins, the tropomyosins, are absolutely essential in regulating actin’s ability to form such diverse structures. In this review we integrate studies from different organisms and cell types in an attempt to provide a unifying view of tropomyosin dependent regulation of the actin cytoskeleton.
Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR ...screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B–Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.
The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the ...nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.
An increasing number of studies have shown that circular RNAs (circRNAs) play important roles in malignant tumor initiation and progression; however, many circRNAs are yet unidentified, and the role ...of circRNAs in nasopharyngeal carcinoma (NPC) is unclear. Using RNA sequencing, we discovered a novel circRNA, termed circARHGAP12, that was processed from the pre-mRNA of the ARHGAP12 gene. CircARHGAP12 was significantly upregulated in NPC tissues and cell lines and promoted NPC cell migration and invasion. Overexpression or knockdown experiments revealed that circARHGAP12 regulates the expression of cytoskeletal remodeling-related proteins EZR, TPM3, and RhoA. CircARHGAP12 was found to bind directly to the 3′ UTR of EZR mRNA and promote its stability; moreover, EZR protein interacted with TPM3 and RhoA and formed a complex to promote NPC cell invasion and metastasis. This study identified the novel circRNA circARHGAP12, characterized its biological function and mechanism, and increased our understanding of circRNAs in NPC pathogenesis. In particular, circARHGAP12 was found to promote the malignant biological phenotype of NPC via cytoskeletal remodeling, thus providing a clue for targeted therapy of NPC.
•We found a novel circRNA which is back-spliced by exons 2–3 of ARHGAP12 gene.•CircARHGAP12 is significantly upregulated in NPC and promotes NPC cell migration and invasion in vitro and in vivo.•CircARHGAP12 binds directly to the 3′UTR of EZR mRNA and promotes its stability.•EZR, TPM3, and RhoA form a complex and a feedback loop.•CircARHGAP12 can promote the aggregation of microfilaments, and further promote NPC cell invasion and metastasis via EZR.
Collective cell migration has a key role during morphogenesis and during wound healing and tissue renewal in the adult, and it is involved in cancer spreading. In addition to displaying a coordinated ...migratory behaviour, collectively migrating cells move more efficiently than if they migrated separately, which indicates that a cellular interplay occurs during collective cell migration. In recent years, evidence has accumulated confirming the importance of such intercellular communication and exploring the molecular mechanisms involved. These mechanisms are based both on direct physical interactions, which coordinate the cellular responses, and on the collective cell behaviour that generates an optimal environment for efficient directed migration. The recent studies have described how leader cells at the front of cell groups drive migration and have highlighted the importance of follower cells and cell-cell communication, both between followers and between follower and leader cells, to improve the efficiency of collective movement.
Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We ...here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.
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