Alexandre Deshiere | Université Laval (original) (raw)

Papers by Alexandre Deshiere

Research paper thumbnail of Regulation of Mechanical Equilibrium in Multicellular Arrangements

Biophysical Journal, 2010

The organization of cells into epithelium depends on cell interaction with both the extracellular... more The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

Research paper thumbnail of Protein kinase CK2 and cell polarity

Molecular and Cellular Biochemistry, 2008

There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellula... more There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellular processes, in the maintenance of mammalian cell morphology and cell polarity. Here, we show that in epithelial cells, a fraction of CK2 is associated to the plasma membrane and that this localization is controlled by cell–matrix interactions. In addition, inhibition of CK2 activity in mammary epithelial cells (MCF10A), using either the specific CK2 inhibitor TBB or siRNA-mediated CK2β knockdown, induced differential phenotypes revealing an important role of this enzyme in epithelial cell morphology.

Research paper thumbnail of Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

Biochemical Journal, 2007

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged... more X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr 188 and Phe 190 , which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr 188 -Phe 190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

Research paper thumbnail of RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged... more X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr 188 and Phe 190 , which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr 188 -Phe 190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

Research paper thumbnail of PNAS-2011-Gay-11464-9

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of Unbalanced expression of CK2 kinase subunits is sufficient to drive epithelial-to-mesenchymal transition by Snail1 induction

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours... more Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2a in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2b regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2b-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3b through synergistic hierarchal phosphorylation. This process depends strongly on CK2b, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2b underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2b in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

Research paper thumbnail of Spatial organization of the extracellular matrix regulates cell–cell junction positioning

The organization of cells into epithelium depends on cell interaction with both the extracellular... more The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

Research paper thumbnail of Regulation of Mechanical Equilibrium in Multicellular Arrangements

Biophysical Journal, 2010

The organization of cells into epithelium depends on cell interaction with both the extracellular... more The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

Research paper thumbnail of Protein kinase CK2 and cell polarity

Molecular and Cellular Biochemistry, 2008

There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellula... more There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellular processes, in the maintenance of mammalian cell morphology and cell polarity. Here, we show that in epithelial cells, a fraction of CK2 is associated to the plasma membrane and that this localization is controlled by cell–matrix interactions. In addition, inhibition of CK2 activity in mammary epithelial cells (MCF10A), using either the specific CK2 inhibitor TBB or siRNA-mediated CK2β knockdown, induced differential phenotypes revealing an important role of this enzyme in epithelial cell morphology.

Research paper thumbnail of Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

Biochemical Journal, 2007

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged... more X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr 188 and Phe 190 , which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr 188 -Phe 190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

Research paper thumbnail of RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged... more X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr 188 and Phe 190 , which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr 188 -Phe 190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

Research paper thumbnail of PNAS-2011-Gay-11464-9

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

The intracellular localization and shape of the nucleus plays a central role in cellular and deve... more The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actinbinding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actinbased network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. cfm1 | cfm2 | neural progenitors P hysical connections between perinuclear actin bundles and the nuclear envelope are essential for nuclear movement that controls cell migration and mammalian developmental processes. Defects in perinuclear actin organization are associated with many disease states (1). In fibroblasts, cell shape, nuclear shape, and movement are controlled by specific perinuclear actin networks of actin cables anchored to the nuclear membrane, called transmembrane actin-associated nuclear (TAN) lines (2) or actin cap (3). Despite extensive work on cytoskeleton anchorage at the nuclear envelope, it remains to be determined how actin bundle dynamics are regulated at the nuclear surface. Here we identify a unique family of F-actin bundling proteins that we call refilin (RefilinA and RefilinB) (for REgulator of FILamin proteIN), which function to organize perinuclear actin networks in fibroblasts and in epithelial cells during epithelial-mesenchymal transition (EMT). Refilins bind to the actin-binding filamins. Filamins (FLNA, FLNB, and FLNC) are a family of actin binding and scaffolding proteins that integrate cellular architecture and signaling and are essential for normal fetal development (4-6). Mammalian FLNA is composed of an amino-terminal actin binding domain followed by 24 repeats of which the last repeat mediates dimerization. Two flexible hinge regions, H1 and H2, separate repeats 15 and 16 and 23 and 24, respectively (7). Dimerization of FLNA forms V-shaped molecules that cross-link actin filaments into orthogonal networks (7). FLNA-null murine embryos die with severe vascular, cardiac, and brain morphogenic defects . In humans, pathogenic mutations in FLNA cause a wide range of developmental malformations in the heart, skeleton, and brain (10-12). A comprehensive model of FLNA functions is still difficult to formulate and it remains unclear how different mutations in the same protein can cause such a broad spectrum of diseases. Here we provide evidence that refilin converts FLNA from an F-actin branching protein into an Factin bundler and that the refilin/FLNA complex functions to organize an actin cap in fibroblasts and a unique perinuclear actin network in epithelial cells during EMT mediated by TGFbeta (TGF-β). EMT is a biological process that plays crucial roles in the differentiation of multiple tissues and organs . These findings open unique perspectives for understanding FLNA function during embryonic development and in human syndromes associated with FLNA mutations.

Research paper thumbnail of Unbalanced expression of CK2 kinase subunits is sufficient to drive epithelial-to-mesenchymal transition by Snail1 induction

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours... more Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2a in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2b regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2b-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3b through synergistic hierarchal phosphorylation. This process depends strongly on CK2b, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2b underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2b in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

Research paper thumbnail of Spatial organization of the extracellular matrix regulates cell–cell junction positioning

The organization of cells into epithelium depends on cell interaction with both the extracellular... more The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.