Mechanical Strain in Actin Networks Regulates FilGAP and Integrin Binding to Filamin A

Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 09/2011; 478(7368):260-3. DOI: 10.1038/nature10430
Source: PubMed


Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.

Download full-text


Available from: Thomas P Stossel
  • Source
    • "pmCherry-RhoGDI2 was constructed by ligating PCR product of RhoGDI2 cDNA digested with BamHI/EcoRI into pmCherry-C1 digested with BglII/EcoRI. peGFP-FLNA wt and del41 were previously described ([14] [27]). Mutagenesis were performed using Quickchange site directed mutagenesis kit (Agilent). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylated at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona-fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA.
    Full-text · Article · Dec 2015 · Biochemical and Biophysical Research Communications
  • Source
    • "Recent literature emphasizes the microscopic, bottomup , granular approach in the context of specific molecules, their roles and their interdependencies [1] [2] [3]. This can range from identifying mechanically sensitive actin-linkers [4], to characterizing signaling pathways, [5] to finding downstream effectors of mechanotransduction such as YAP/TAZ [6]. The older literature, by contrast, emphasizes the macroscopic, coarse-grained, topdown approach in the context of mechanical forces, fields, and integrative physiological function. "
    [Show abstract] [Hide abstract]
    ABSTRACT: As do all things in biology, cell mechanosensation, adhesion and migration begin at the scale of the molecule. Collections of molecules assemble to comprise microscale objects such as adhesions, organelles and cells. And collections of cells in turn assemble to comprise macroscale tissues. From the points of view of mechanism and causality, events at the molecular scale are seen most often as being the most upstream and, therefore, the most fundamental and the most important. In certain collective systems, by contrast, events at many scales of length conspire to make contributions of equal importance, and even interact directly and strongly across disparate scales. Here we highlight recent examples in cellular mechanosensing and collective cellular migration, where physics at some scale bigger than the cell but smaller than the tissue -the mesoscale- becomes the missing link that is required to tie together findings that might otherwise seem counterintuitive or even unpredictable. These examples, taken together, establish that the phenotypes and the underlying physics of collective cellular migration are far richer than previously anticipated.
    Full-text · Article · Nov 2015 · Experimental Cell Research
    • "The internal tension of the fibers not only changes the basic mechanical properties of the filaments [12] [46], but is also crucial in maintaining the mechanical shape and integrity of the cell [47]. Furthermore, microfilament tension alters the affinities of binding proteins [48] [49], and can directly serve as a mechanism of mechanotransduction by exposing previously inaccessible cryptic binding sites [50]. Because of the dominant role of active processes in the cell, the incorporation of motors into reconstituted in vitro systems has received considerable attention over the past decade. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The intracellular cytoskeleton is an active dynamic network of filaments and associated binding proteins that control key cellular properties, such as cell shape and mechanics. Due to the inherent complexity of the cell, reconstituted model systems have been successfully employed to gain an understanding of the fundamental physics governing cytoskeletal processes. Here, we review recent advances and key aspects of these reconstituted systems. We focus on the importance of assembly kinetics and dynamic arrest in determining network mechanics, and highlight novel emergent behavior occurring through interactions between cytoskeletal components in more complex networks incorporating multiple biopolymers and molecular motors. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jun 2015 · Biochimica et Biophysica Acta
Show more