The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Jari Ylanne - One of the best experts on this subject based on the ideXlab platform.
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Evidence for the mechanosensor function of Filamin in tissue development
Scientific reports, 2016Co-Authors: Sven Huelsmann, Nina Rintanen, R. Sethi, Nicholas H. Brown, Jari YlanneAbstract:Cells 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 open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, our results show that an appropriate level of mechanical sensitivity is required for Filamins’ function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates Filamin binding to cellular components.
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model of a six immunoglobulin like domain fragment of Filamin a 16 21 built using residual dipolar couplings
Journal of the American Chemical Society, 2012Co-Authors: Helena Tossavainen, Outi Koskela, Pengju Jiang, Jari Ylanne, Iain D Campbell, Ilkka Kilpelainen, Perttu PermiAbstract: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 interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16–21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form ...
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atomic structures of two novel immunoglobulin like domain pairs in the actin cross linking protein Filamin
Journal of Biological Chemistry, 2009Co-Authors: Outi Heikkinen, Ilkka Kilpelainen, Perttu Permi, Salla Ruskamo, Peter V Konarev, D I Svergun, Tatu Iivanainen, Sami Heikkinen, Harri Koskela, Jari YlanneAbstract: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 NMR structures revealed that the domain packing in IgFLNa18-19 resembles the structure of IgFLNa20-21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16-17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem Filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of Filamin interactions through a mechanosensor mechanism.
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The Regulation Mechanism for the Auto-Inhibition of Binding of Human Filamin A to Integrin
Journal of molecular biology, 2009Co-Authors: Ulla Pentikäinen, Jari YlanneAbstract: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 β 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 found that mechanical force applied to Filamin can expose cryptic integrin binding sites. The forces required for this are considerably lower than those for Filamin immunoglobulin domain unfolding. The mechanical-force-induced unfolding of Filamin and exposure of integrin binding sites occur through stable intermediates where integrin binding is possible. Accordingly, our results support Filamin's role as a mechanotransducer, since force-induced conformational changes allow binding of integrin and other transmembrane and intracellular proteins. This observed force-induced conformational change can also be one of possible mechanisms involved in the regulation of integrin activation.
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structural basis of the migfilin Filamin interaction and competition with integrin β tails
Journal of Biological Chemistry, 2008Co-Authors: Yatish Lad, Pengju Jiang, Jari Ylanne, Iain D Campbell, Salla Ruskamo, David S Harburger, David A. CalderwoodAbstract: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. Human Filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The Filamin-binding site in migfilin is localized between Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21 interaction and migfilin and integrin β tails can compete with one another for binding to IgFLNa21. This suggests that competition between Filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating Filamin interactions and signaling. In this specific case, displacement of integrin tails from Filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.
David A. Calderwood - One of the best experts on this subject based on the ideXlab platform.
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asb2α an e3 ubiquitin ligase specificity subunit regulates cell spreading and triggers proteasomal degradation of Filamins by targeting the Filamin calponin homology 1 domain
Journal of Biological Chemistry, 2013Co-Authors: Ziba Razinia, Massimiliano Baldassarre, Gaia Cantelli, David A. CalderwoodAbstract:Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of Filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of Filamins. Here, we show that the calponin homology domain 1 (CH1), within the Filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant Filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of Filamins.
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Filamin A controls matrix metalloproteinase activity and regulates cell invasion in human fibrosarcoma cells
Journal of Cell Science, 2012Co-Authors: Massimiliano Baldassarre, Ziba Razinia, Nina N. Brahme, Roberto Buccione, David A. CalderwoodAbstract:Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of Filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of Filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within Filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in Filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of Filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered Filamin expression or Filamin mutations.
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structural basis of the migfilin Filamin interaction and competition with integrin β tails
Journal of Biological Chemistry, 2008Co-Authors: Yatish Lad, Pengju Jiang, Jari Ylanne, Iain D Campbell, Salla Ruskamo, David S Harburger, David A. CalderwoodAbstract: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. Human Filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The Filamin-binding site in migfilin is localized between Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21 interaction and migfilin and integrin β tails can compete with one another for binding to IgFLNa21. This suggests that competition between Filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating Filamin interactions and signaling. In this specific case, displacement of integrin tails from Filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.
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structure of three tandem Filamin domains reveals auto inhibition of ligand binding
The EMBO Journal, 2007Co-Authors: Yatish Lad, David A. Calderwood, Pengju Jiang, Iain D Campbell, Tiila-riikka Kiema, Olli T Pentikainen, C H Coles, Jari YlanneAbstract: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 β-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 Filamin binding to integrin β-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling Filamin–ligand interactions. This can explain the increased integrin binding of Filamin splice variants and provides a mechanism by which ligand binding might impact Filamin structure.
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The Molecular Basis of Filamin Binding to Integrins and Competition with Talin.
Molecular cell, 2006Co-Authors: Tiila-riikka Kiema, Massimiliano Baldassarre, Pengju Jiang, Jari Ylanne, Iain D Campbell, Yatish Lad, Camilla L. Oxley, Kate L. Wegener, David A. CalderwoodAbstract: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. Notably, the structurally defined Filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-Filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit Filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.
Massimiliano Baldassarre - One of the best experts on this subject based on the ideXlab platform.
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asb2α an e3 ubiquitin ligase specificity subunit regulates cell spreading and triggers proteasomal degradation of Filamins by targeting the Filamin calponin homology 1 domain
Journal of Biological Chemistry, 2013Co-Authors: Ziba Razinia, Massimiliano Baldassarre, Gaia Cantelli, David A. CalderwoodAbstract:Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of Filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of Filamins. Here, we show that the calponin homology domain 1 (CH1), within the Filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant Filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of Filamins.
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Filamin A controls matrix metalloproteinase activity and regulates cell invasion in human fibrosarcoma cells
Journal of Cell Science, 2012Co-Authors: Massimiliano Baldassarre, Ziba Razinia, Nina N. Brahme, Roberto Buccione, David A. CalderwoodAbstract:Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of Filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of Filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within Filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in Filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of Filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered Filamin expression or Filamin mutations.
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Functional and Structural Insights into ASB2α, a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells
Journal of Biological Chemistry, 2011Co-Authors: Isabelle Lamsoul, Massimiliano Baldassarre, Ziba Razinia, David Calderwood, Christel Moog-lutz, Clara Burande, Thibault Houles, Delphine Menoret, Monique Erard, Pierre LutzAbstract:By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2β) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets Filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through Filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of β1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with Filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the Filamin-binding motifs of migfilin and β integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to Filamin.
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The E3 ubiquitin ligase specificity subunit ASB2 targets Filamins for proteasomal degradation by interacting with the Filamin actin-binding domain
Journal of Cell Science, 2011Co-Authors: Ziba Razinia, Massimiliano Baldassarre, Mohamed Bouaouina, Isabelle Lamsoul, Pierre Lutz, David CalderwoodAbstract:Filamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that Filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASBα and that acute degradation of Filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2α triggers Filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with Filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on Filamin and that the actin-binding domain (ABD) of Filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated Filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting Filamins for degradation. These studies provide the molecular basis for ASB2α-mediated Filamin degradation and unravel an important mechanism by which Filamin levels can be acutely regulated.
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ASB2 targets Filamins A and B to proteasomal degradation
Blood, 2008Co-Authors: Mélina Heuzé, Massimiliano Baldassarre, Ziba Razinia, Isabelle Lamsoul, David Calderwood, Yatish Lad, Sophie Lévêque, Christel Moog-lutz, Pierre LutzAbstract:The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins Filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and Filamin degradation; conversely, ASB2 expression in leukemia cells induces Filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both Filamin A and Filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of Filamins for degradation.
Robert J. Bryson-richardson - One of the best experts on this subject based on the ideXlab platform.
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G.P.59 Characterization and investigation of zebrafish models of Filamin related myofibrillar myopathy
Neuromuscular Disorders, 2012Co-Authors: Avnika A. Ruparelia, M. Zhao, Peter D. Currie, Robert J. Bryson-richardsonAbstract:Abstract Myofibrillar myopathies are characterized by the disintegration of skeletal muscle fibers and formation of ectopic sarcomeric protein aggregates. All the proteins known to be involved in myofibrillar myopathies localize to a region of the sarcomere known as the Z-disk, the site at which defects are first observed. Given the common cellular phenotype observed in this group of disorders it is thought that there is a common mechanism of pathology. Mutations in Filamin C, which has several proposed roles in the development and function of skeletal muscle, can result in Filamin related myofibrillar myopathy. The lack of a suitable animal model system has limited investigation into the mechanism of pathology in this disease and the role of Filamin C in muscle development. Here we characterize stretched out (sot), a zebrafish Filamin Cb mutant, together with targeted knockdown of zebrafish Filamin Ca, revealing fiber dissolution and formation of protein aggregates strikingly similar to those seen in Filamin related myofibrillar myopathies. Through knockdown of both zebrafish Filamin C homologues we demonstrate that Filamin C is not required for fiber specification and that fiber damage is a consequence of muscle activity. The remarkable similarities in the myopathology between our models and Filamin related myofibrillar myopathy makes them suitable for the study of these diseases and provides unique opportunities for the investigation of the function of Filamin C in muscle and development of therapies.
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Characterization and investigation of zebrafish models of Filamin-related myofibrillar myopathy
Human molecular genetics, 2012Co-Authors: Avnika A. Ruparelia, M. Zhao, Peter D. Currie, Robert J. Bryson-richardsonAbstract:Myofibrillar myopathies are a group of muscle disorders characterized by the disintegration of skeletal muscle fibers and formation of sarcomeric protein aggregates. All the proteins known to be involved in myofibrillar myopathies localize to a region of the sarcomere known as the Z-disk, the site at which defects are first observed. Given the common cellular phenotype observed in this group of disorders, it is thought that there is a common mechanism of pathology. Mutations in Filamin C, which has several proposed roles in the development and function of skeletal muscle, can result in Filamin-related myofibrillar myopathy. The lack of a suitable animal model system has limited investigation into the mechanism of pathology in this disease and the role of Filamin C in muscle development. Here, we characterize stretched out (sot), a zebrafish Filamin Cb mutant, together with targeted knockdown of zebrafish Filamin Ca, revealing fiber dissolution and formation of protein aggregates strikingly similar to those seen in Filamin-related myofibrillar myopathies. Through knockdown of both zebrafish Filamin C homologues, we demonstrate that Filamin C is not required for fiber specification and that fiber damage is a consequence of muscle activity. The remarkable similarities in the myopathology between our models and Filamin-related myofibrillar myopathy makes them suitable for the study of these diseases and provides unique opportunities for the investigation of the function of Filamin C in muscle and development of therapies.
Ziba Razinia - One of the best experts on this subject based on the ideXlab platform.
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asb2α an e3 ubiquitin ligase specificity subunit regulates cell spreading and triggers proteasomal degradation of Filamins by targeting the Filamin calponin homology 1 domain
Journal of Biological Chemistry, 2013Co-Authors: Ziba Razinia, Massimiliano Baldassarre, Gaia Cantelli, David A. CalderwoodAbstract:Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of Filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of Filamins. Here, we show that the calponin homology domain 1 (CH1), within the Filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant Filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of Filamins.
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Filamin A controls matrix metalloproteinase activity and regulates cell invasion in human fibrosarcoma cells
Journal of Cell Science, 2012Co-Authors: Massimiliano Baldassarre, Ziba Razinia, Nina N. Brahme, Roberto Buccione, David A. CalderwoodAbstract:Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of Filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of Filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within Filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in Filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of Filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered Filamin expression or Filamin mutations.
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Functional and Structural Insights into ASB2α, a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells
Journal of Biological Chemistry, 2011Co-Authors: Isabelle Lamsoul, Massimiliano Baldassarre, Ziba Razinia, David Calderwood, Christel Moog-lutz, Clara Burande, Thibault Houles, Delphine Menoret, Monique Erard, Pierre LutzAbstract:By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2β) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets Filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through Filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of β1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with Filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the Filamin-binding motifs of migfilin and β integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to Filamin.
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The E3 ubiquitin ligase specificity subunit ASB2 targets Filamins for proteasomal degradation by interacting with the Filamin actin-binding domain
Journal of Cell Science, 2011Co-Authors: Ziba Razinia, Massimiliano Baldassarre, Mohamed Bouaouina, Isabelle Lamsoul, Pierre Lutz, David CalderwoodAbstract:Filamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that Filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASBα and that acute degradation of Filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2α triggers Filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with Filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on Filamin and that the actin-binding domain (ABD) of Filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated Filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting Filamins for degradation. These studies provide the molecular basis for ASB2α-mediated Filamin degradation and unravel an important mechanism by which Filamin levels can be acutely regulated.
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ASB2 targets Filamins A and B to proteasomal degradation
Blood, 2008Co-Authors: Mélina Heuzé, Massimiliano Baldassarre, Ziba Razinia, Isabelle Lamsoul, David Calderwood, Yatish Lad, Sophie Lévêque, Christel Moog-lutz, Pierre LutzAbstract:The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins Filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and Filamin degradation; conversely, ASB2 expression in leukemia cells induces Filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both Filamin A and Filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of Filamins for degradation.
