The force-dependent filamin A-G3BP1 interaction regulates phase-separated stress granule formation.

Filamin A (FLNA) is an actin crosslinking protein that mediates mechanotransduction. External and internal mechanical forces, through the actin cytoskeleton, can induce conformational changes of the FLNA molecule to expose cryptic binding sites for its binding partners. Here, we identified Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) as a new FLNA mechanobinding partner. Unlike other FLNA binding partners to the mechanosensing domain repeat 21 (R21), G3BP1 requires an additional neighboring repeat R22 to interact. We demonstrated that their interaction occurs in the cytosol of living cells in an actin polymerization-dependent manner. We also mapped the FLNA-binding site on G3BP1 and found that a F360A point mutation in the RNA recognition motif disrupts the interaction. RNA interfered with the FLNA-G3BP1 interaction, and FLNA did not localize in RNA-rich stress granules (SGs). Disruption of the interaction was sufficient to promote phase-separated SG formation, and arsenite treatment further stimulated the formation of SGs. Taken together, these data identify G3BP1 as a new mechanobinding protein that interacts with the FLNA mechanosensing domain R21 and suggest that SG formation is partially regulated by mechanical force.

Identification of Filamin A Mechanobinding Partner III: SAV1 Specifically Interacts with Filamin A Mechanosensitive Domain 21.

Filamin A (FLNA) cross-links actin filaments and mediates mechanotransduction by force-induced conformational changes of its domains. FLNA's mechanosensitive immunoglobulin-like repeats (R) interact with each other to create cryptic binding sites, which can be exposed by physiologically relevant mechanical forces. Using the FLNA mechanosensing domains as an affinity ligand followed by stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, we recently identified smoothelin and fimbacin as FLNA mechanobinding proteins. Here, using the mechanosensing domain as an affinity ligand and two labeled amino acids, we identify salvador homologue 1 (SAV1), a component of the Hippo pathway kinase cascade, as a new FLNA mechanobinding partner. We demonstrate that SAV1 specifically interacts with the cryptic C-D cleft of FLNA R21 and map the FLNA-binding site on SAV1. We show that point mutations on the R21 C strand block the SAV1 interaction and find that SAV1 contains a FLNA-binding motif in the central region (116Phe-124Val). Point mutations F116A and T118A (FT/AA) disrupt the interaction. A proximity ligation assay reveals that their interaction occurs in the cytosol in an actin polymerization-dependent manner. Although SAV1 is typically found in the cytosol, disrupting the interaction between SAV1 and FLNA causes SAV1 to diffuse to the nucleus and YAP1 to diffuse to the cytosol in an inverse relationship. These results suggest that FLNA mediates regulation of the Hippo pathway through actin polymerization-dependent interaction with SAV1.

Structure and Function of Filamin C in the Muscle Z-Disc.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

Interaction of LARP4 to filamin A mechanosensing domain regulates cell migrations.

Filamin A (FLNA) is an actin cross-linking protein that mediates mechanotransduction. Force-dependent conformational changes of FLNA molecule expose cryptic binding site of FLNA, allowing interaction with partners such as integrin, smoothelin, and fimbacin. Here, we identified La-related protein 4 (LARP4) as a new FLNA mechanobinding partner. LARP4 specifically interacts with the cleft formed by C and D strands of immunoglobulin-like repeat 21 (R21) which is blocked by A strand of R20 without force. We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro. We also determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 (F277A in human: F273A in mouse Larp4) that disrupts the interaction. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled LARP4 in living cells demonstrated that mutant LARP4 diffuses faster than WT LARP4. Proximity ligation assay (PLA) also confirmed their interaction and disruption of actin polymerization diminishes the interaction. Data mining of RNAseq analysis of LARP4 knockdown (KD) HEK293T cells suggested that LARP4 is involved in morphogenesis and cell motility. Consistent with this prediction, we found that KD of LARP4 increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells also leads to a higher cell migration speed compared to WT LARP4. These results demonstrated that the LARP4 interaction with FLNA regulates cell migration.