Evidence for the mechanosensor function of filamin in tissue development

AbstractCells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. Here we tested, how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The ope…

Characterization of the interaction between Actinin-Associated LIM Protein (ALP) and the rod domain of α-actinin

Abstract Background The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown. Results In this study, using circular dichroism and nuclear magnetic resonance (NMR), we showed that the ALP internal region (residues 107–273) was largely unfolded in solution, but was able to interact with the α-actinin rod domain in vitro, and to co-localize with α-actinin on stress fibres in vivo. NMR analysis revealed that the ti…

Negative regulators of integrin activity

Integrins are heterodimeric transmembrane adhesion receptors composed of α- and β-subunits. They are ubiquitously expressed and have key roles in a number of important biological processes, such as development, maintenance of tissue homeostasis and immunological responses. The activity of integrins, which indicates their affinity towards their ligands, is tightly regulated such that signals inside the cell cruicially regulate the switching between active and inactive states. An impaired ability to activate integrins is associated with many human diseases, including bleeding disorders and immune deficiencies, whereas inappropriate integrin activation has been linked to inflammatory disorders…

Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20-21.

Filamins (FLNs) are large, multidomain actin cross-linking proteins with diverse functions. Besides regulating the actin cytoskeleton, they serve as important links between the extracellular matrix and the cytoskeleton by binding cell surface receptors, functioning as scaffolds for signaling proteins, and binding several other cytoskeletal proteins that regulate cell adhesion dynamics. Structurally, FLNs are formed of an amino terminal actin-binding domain followed by 24 immunoglobulin-like domains (IgFLNs). Recent studies have demonstrated that myosin-mediated contractile forces can reveal hidden protein binding sites in the domain pairs IgFLNa18-19 and 20-21, enabling FLNs to transduce me…

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances fi…

The regulation mechanism for the auto-inhibition of binding of human filamin A to integrin.

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Human filamins are large actin cross-linking proteins that connect integrins to the cytoskeleton. Filamin binding to the cytoplasmic tail of beta integrins has been shown to prevent integrin activation in cells, which is important for controlling cell adhesion and migration. The molecular-level mechanism for filamin binding to integrin has been unclear, however, as it was recently demonstrated that filamin undergoes intramolecular auto-inhibition of integrin binding. In this study, using steered molecular dynamics simulations, we f…

Structure of the human filamin A actin-binding domain.

Filamin A (FLNa) is a large dimeric protein that binds to actin filaments via its actin-binding domain (ABD). The crystal structure of this domain was solved at 3.2 A resolution. The domain adopts a closed conformation typical of other ABDs, but also forms a dimer both in crystallization conditions and in solution. The structure shows the localization of the residues mutated in patients with periventricular nodular heterotopia or otopalatodigital syndrome. Structural analysis predicts that mutations in both types of disorder may affect actin binding.

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with α-actinin—Analysis of patient mutations

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either …

Crystal Structure of Human Filamin C Domain 23 and Small Angle Scattering Model for Filamin C 23–24 Dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation …

Binding properties and stability of the Ras-association domain of Rap1-GTP interacting adapter molecule (RIAM).

The Rap1-GTP interacting adapter protein (RIAM) is an important protein in Rap1-mediated integrin activation. By binding to both Rap1 GTPase and talin, RIAM recruits talin to the cell membrane, thus facilitating talin-dependent integrin activation. In this article, we studied the role of the RIAM Ras-association (RA) and pleckstrin-homology (PH) domains in the interaction with Rap1. We found that the RA domain was sufficient for GTP-dependent interaction with Rap1B, and the addition of the PH domain did not change the binding affinity. We also detected GTP-independent interaction of Rap1B with the N-terminus of RIAM. In addition, we found that the PH domain stabilized the RA domain both in …

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16–21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagen…

Molecular basis of filamin a-filGAP interaction and its impairment in congenital disorders associated with filamin a mutations

Background Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/Principal Findings We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D β-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the b…

Dynamic force sensing of filamin revealed in single-molecule experiments

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20–21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that fi…

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin …

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Phosphorylated immunoreceptor tyrosine-based activation motifs and integrin cytoplasmic domains activate spleen tyrosine kinase via distinct mechanisms

Spleen tyrosine kinase (Syk) is involved in cellular adhesion and also in the activation and development of hematopoietic cells. Syk activation induced by genomic rearrangement has been linked to certain T-cell lymphomas, and Syk inhibitors have been shown to prolong survival of patients with B-cell lineage malignancies. Syk is activated either by its interaction with a double-phosphorylated immunoreceptor tyrosine-based activation motif (pITAM), which induces rearrangements in the Syk structure, or by the phosphorylation of specific tyrosine residues. In addition to its immunoreceptor function, Syk is activated downstream of integrin pathways, and integrins bind to the same region in Syk a…

Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLN…

Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.

Platelet activation causes conformational changes of integrin GPIIb/IIIa (α IIb β 3 ), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which ar…

Small-angle X-ray scattering reveals compact domain-domain interactions in the N-terminal region of filamin C

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the Nterminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain in…

Novel structural insights into F-actin-binding and novel functions of calponin homology domains.

Tandem calponin homology (CH) domains are well-known actin filaments (F-actin) binding motifs. There has been a continuous debate about the details of CH domain-actin interaction, mainly because atomic level structures of F-actin are not available. A recent electron microscopy study has considerably advanced our structural understanding of CH domain:F-actin complex. On the contrary, it has recently also been shown that CH domains can bind other macromolecular systems: two CH domains from separate polypeptides Ncd80, Nuf2 can form a microtubule-binding site, as well as tandem CH domains in the EB1 dimer, while the single C-terminal CH domain of alpha-parvin has been observed to bind to a alp…

Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells

Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear a…

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NM…

A novel structural unit in the N-terminal region of filamins.

Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces. Here, we investigated if there are similar inter-domain interactions around domain 4 in the N-terminal rod 1 region of FLN. Protein crystal structures revealed a new type of dom…

The molecular basis of filamin binding to integrins and competition with talin.

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notabl…

Structural basis of the migfilin-filamin interaction and competition with integrin beta tails.

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. H…

Integrin cytoplasmic domain and pITAM compete for spleen tyrosine kinase binding

ABSTRACTIn hematopoietic tissues cell-cell communication involves immunoreceptors and specialized cell adhesion receptors that both mediate intracellular signals. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in the downstream signaling of both immunoreceptors tyrosine activation motif (ITAM) receptors and integrin family cell adhesion receptors. Both phosphorylated ITAM (pITAM) and integrins bind to the regulatory domain of Syk composed of two Src homology 2 (SH2) domains. The interaction with pITAM is mediated by binding of a specific phosphotyrosine to each of the SH2 domains, leading to conformational changes and Syk kinase activation. Integrins bind to the int…

Skeletal Dysplasia Mutations Effect on Human Filamins’ Structure and Mechanosensing

AbstractCells’ ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered mo…

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

AbstractLeukocyte integrins of the β2 family are essential for immune cell-cell adhesion. In activated cells, β2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the β2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of β2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated β2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (Kd, 261…

Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/Principal Findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D b-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the…