Kevin Carvalho - Academia.edu (original) (raw)
Papers by Kevin Carvalho
SSRN Electronic Journal, 2020
Highlights d IL-17-producing cells accumulate in the brain and the meninges of 3xTg-AD mice d The... more Highlights d IL-17-producing cells accumulate in the brain and the meninges of 3xTg-AD mice d The increase of IL-17 producers associates with short-term memory deficits d Neutralization of IL-17 prevents cognitive impairments and synaptic dysfunction d IL-17 triggers Alzheimer's disease onset independently of Ab and tau pathology
Soft Matter, 2016
Quantitative measurements of tension generated by an artificial actomyosin cortex reconstituted a... more Quantitative measurements of tension generated by an artificial actomyosin cortex reconstituted at the outer surface of liposome doublets, as sweetly schematized by halves of Italian cookies “baci di dama” that happen to stick.
Brain
Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer’... more Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer’s disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer’s disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by R...
La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squele... more La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squelette de la cellule (le cytosquelette), par l'intermédiaire de protéines d'ancrages et de lipides clefs qui jouent un rôle spécifique dans certains types d'interactions. Parmi les protéines intervenant dans l'ancrage direct de la membrane plasmique au cytosquelette, des protéines de la famille des ERM (Ezrine, Radixine, Moésine) peuvent interagir spécifiquement avec un lipide, le phosphatidylinositol (4,5) biphosphate (PIP2), d'une part et avec l'actine du cytosquelette d'autre part. Dans le but de comprendre les interactions entre membrane plasmique et cytosquelette, nous avons réalisé des expériences in vitro sur des systèmes comportant un nombre minimal de constituants : des vésicules géantes (GUV) contenant du PIP2, de l'ezrine recombinante et de l'actine purifiée. Nous avons mis en évidence que la liaison au PIP2 induit des changements conformationn...
This new volume of Methods in Cell Biology looks at building a cell from its component parts. Cha... more This new volume of Methods in Cell Biology looks at building a cell from its component parts. Chapters cover such topics as engineering motor scaffolds, artificial cytoskeletons, interconnected droplet networks and artificial cells; building cytoskeletal systems and artificial actin cortex on pillar arrays; reconstituting membrane fission, actin bundles at membranes, actin cortex in droplets, dynein-dynactin mediated cargo transport, MinD systems, protein-lipid machines that transform membranes, protein interactions and signaling on artificial lipid bilayers. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Methods in Cell Biology, 2015
Cells move and change shape by dynamically reorganizing their cytoskeleton next to the plasma mem... more Cells move and change shape by dynamically reorganizing their cytoskeleton next to the plasma membrane. In particular, actin assembly generates forces and stresses that deform the cell membrane. Cell-sized liposomes are designed to mimic this function. The activation of actin polymerization at their membrane is able to push the membrane forward, thus reproducing the mechanism of lamellipodium extension at the cell front. Moreover, the cell cortex, a submicrometer-thick actin shell right beneath the cell membrane can be reproduced; it contributes to cell tension with the action of molecular motors. We will describe experimental methods to prepare liposomes that mimic the inside geometry of a cell, and that reproduce actin-based propulsion of the liposome using an outside geometry. Such systems allow to study how actin-related proteins control and affect actin cortex assembly and can produce forces that drive cell shape changes.
Biophysical Journal, 2014
Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that part... more Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the "actin cloud" and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.
Proceedings of the National Academy of Sciences, 2012
Cells use their dynamic actin network to control their mechanics and motility. These networks are... more Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein.
Proceedings of the National Academy of Sciences, 2013
Animal cells actively generate contractile stress in the actin cortex, a thin actin network benea... more Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the cell membrane, to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex-membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Biophysical Journal, 2012
Biophysical Journal, 2008
Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying p... more Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP 2 ). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out z-potential measurements to prove the effective incorporation of PIP 2 as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP 2 -containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP 2 analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP 2 antibodies show PIP 2 clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca 21 and Mg 21 ) can induce aggregation of PIP 2 in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP 2 -containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP 2 and proteins are formed.
Biophysical Journal, 2008
The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of c... more The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP 2 ) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP 2 . For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ;5 mM for PIP 2 -LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP 2 -LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ;30 nm 2 ) binding to a single PIP 2 can block access to neighboring PIP 2 molecules and thus contributes to lower the accessible PIP 2 concentration. In addition, no evidence exists for a clustering of PIP 2 induced by ezrin addition.
Biochimie, 2013
Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a lar... more Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Recent reviews extensively discuss their biological functions [1e4]. In this review, we will first remind the main features of this family of proteins, which are known as linkers and regulators of plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments.
SSRN Electronic Journal, 2020
Highlights d IL-17-producing cells accumulate in the brain and the meninges of 3xTg-AD mice d The... more Highlights d IL-17-producing cells accumulate in the brain and the meninges of 3xTg-AD mice d The increase of IL-17 producers associates with short-term memory deficits d Neutralization of IL-17 prevents cognitive impairments and synaptic dysfunction d IL-17 triggers Alzheimer's disease onset independently of Ab and tau pathology
Soft Matter, 2016
Quantitative measurements of tension generated by an artificial actomyosin cortex reconstituted a... more Quantitative measurements of tension generated by an artificial actomyosin cortex reconstituted at the outer surface of liposome doublets, as sweetly schematized by halves of Italian cookies “baci di dama” that happen to stick.
Brain
Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer’... more Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer’s disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer’s disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by R...
La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squele... more La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squelette de la cellule (le cytosquelette), par l'intermédiaire de protéines d'ancrages et de lipides clefs qui jouent un rôle spécifique dans certains types d'interactions. Parmi les protéines intervenant dans l'ancrage direct de la membrane plasmique au cytosquelette, des protéines de la famille des ERM (Ezrine, Radixine, Moésine) peuvent interagir spécifiquement avec un lipide, le phosphatidylinositol (4,5) biphosphate (PIP2), d'une part et avec l'actine du cytosquelette d'autre part. Dans le but de comprendre les interactions entre membrane plasmique et cytosquelette, nous avons réalisé des expériences in vitro sur des systèmes comportant un nombre minimal de constituants : des vésicules géantes (GUV) contenant du PIP2, de l'ezrine recombinante et de l'actine purifiée. Nous avons mis en évidence que la liaison au PIP2 induit des changements conformationn...
This new volume of Methods in Cell Biology looks at building a cell from its component parts. Cha... more This new volume of Methods in Cell Biology looks at building a cell from its component parts. Chapters cover such topics as engineering motor scaffolds, artificial cytoskeletons, interconnected droplet networks and artificial cells; building cytoskeletal systems and artificial actin cortex on pillar arrays; reconstituting membrane fission, actin bundles at membranes, actin cortex in droplets, dynein-dynactin mediated cargo transport, MinD systems, protein-lipid machines that transform membranes, protein interactions and signaling on artificial lipid bilayers. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Methods in Cell Biology, 2015
Cells move and change shape by dynamically reorganizing their cytoskeleton next to the plasma mem... more Cells move and change shape by dynamically reorganizing their cytoskeleton next to the plasma membrane. In particular, actin assembly generates forces and stresses that deform the cell membrane. Cell-sized liposomes are designed to mimic this function. The activation of actin polymerization at their membrane is able to push the membrane forward, thus reproducing the mechanism of lamellipodium extension at the cell front. Moreover, the cell cortex, a submicrometer-thick actin shell right beneath the cell membrane can be reproduced; it contributes to cell tension with the action of molecular motors. We will describe experimental methods to prepare liposomes that mimic the inside geometry of a cell, and that reproduce actin-based propulsion of the liposome using an outside geometry. Such systems allow to study how actin-related proteins control and affect actin cortex assembly and can produce forces that drive cell shape changes.
Biophysical Journal, 2014
Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that part... more Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the "actin cloud" and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.
Proceedings of the National Academy of Sciences, 2012
Cells use their dynamic actin network to control their mechanics and motility. These networks are... more Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein.
Proceedings of the National Academy of Sciences, 2013
Animal cells actively generate contractile stress in the actin cortex, a thin actin network benea... more Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the cell membrane, to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex-membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Biophysical Journal, 2012
Biophysical Journal, 2008
Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying p... more Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP 2 ). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out z-potential measurements to prove the effective incorporation of PIP 2 as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP 2 -containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP 2 analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP 2 antibodies show PIP 2 clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca 21 and Mg 21 ) can induce aggregation of PIP 2 in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP 2 -containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP 2 and proteins are formed.
Biophysical Journal, 2008
The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of c... more The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP 2 ) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP 2 . For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ;5 mM for PIP 2 -LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP 2 -LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ;30 nm 2 ) binding to a single PIP 2 can block access to neighboring PIP 2 molecules and thus contributes to lower the accessible PIP 2 concentration. In addition, no evidence exists for a clustering of PIP 2 induced by ezrin addition.
Biochimie, 2013
Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a lar... more Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Recent reviews extensively discuss their biological functions [1e4]. In this review, we will first remind the main features of this family of proteins, which are known as linkers and regulators of plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments.