ABSTRACT
Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.
Subject(s)
Cytoskeleton , Proteomics , Signal Transduction , src-Family Kinases , Animals , Cells, Cultured , Molecular Dynamics Simulation , Phosphorylation , src Homology Domains , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a ß1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to ß1 integrin. The non-ubiquitylatable form of ICAP-1 modifies ß1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a ß1-integrin-independent manner. ICAP-1 monoubiquitylation regulates rigidity sensing by increasing MRCKα (also known as CDC42BPA)-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitylation helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitylation serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a crucial modulator of cell migration and mechanosensing.
Subject(s)
Cell Movement , Extracellular Matrix/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Myotonin-Protein Kinase/metabolism , Ubiquitination , rho-Associated Kinases/metabolism , Adaptor Proteins, Signal Transducing , Animals , Binding Sites , Biomechanical Phenomena , Cell Adhesion , Cell Line , Fibronectins/metabolism , Focal Adhesions/metabolism , Humans , Integrin beta1/chemistry , Integrin beta1/metabolism , Mice , Models, Biological , Signal Transduction , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND INFORMATION: Integrins are key receptors that allow cells to sense and respond to their mechanical environment. Although they bind the same ligand, ß1 and ß3 integrins have distinct and cooperative roles in mechanotransduction. RESULTS: Using traction force microscopy on unconstrained cells, we show that deleting ß3 causes traction forces to increase, whereas the deletion of ß1 integrin results in a strong decrease of contractile forces. Consistently, loss of ß3 integrin also induces an increase in ß1 integrin activation. Using a genetic approach, we identified the phosphorylation of the distal NPXY domain as an essential process for ß3 integrin to be able to modulate traction forces. Loss of ß3 integrins also impacted cell shape and the spatial distribution of traction forces, by causing forces to be generated closer to the cell edge, and the cell shape. CONCLUSIONS: Our results emphasize the role of ß3 integrin in spatial distribution of cellular forces. We speculate that, by modulating its affinity with kindlin, ß3 integrins may be able to locate near the cell edge where it can control ß1 integrin activation and clustering. SIGNIFICANCE: Tensional homeostasis at the single cell level is performed by the ability of ß3 adhesions to negatively regulate the activation degree and spatial localization of ß1 integrins. By combining genetic approaches and new tools to analyze traction distribution and cell morphology on a population of cells we were able to identify the molecular partners involved in cellular forces regulation.
Subject(s)
Carrier Proteins/genetics , Fibroblasts/metabolism , Integrin alphaVbeta3/genetics , Integrin beta1/genetics , Integrin beta3/genetics , Mechanotransduction, Cellular , Amino Acid Sequence , Animals , Biomechanical Phenomena , Carrier Proteins/metabolism , Cell Adhesion , Cell Line , Fibroblasts/ultrastructure , Gene Deletion , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Integrin alphaVbeta3/metabolism , Integrin beta1/metabolism , Integrin beta3/metabolism , Mice , Phosphorylation , Protein Binding , Protein DomainsABSTRACT
Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.
Subject(s)
CARD Signaling Adaptor Proteins , Inflammasomes , Optogenetics , Pyroptosis , Inflammasomes/metabolism , Optogenetics/methods , Animals , Humans , CARD Signaling Adaptor Proteins/metabolism , CARD Signaling Adaptor Proteins/genetics , Mice , Caspase 1/metabolism , Caspase 1/genetics , Interleukin-1beta/metabolism , Interleukin-1beta/geneticsABSTRACT
Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation. We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.
Subject(s)
Adherens Junctions/metabolism , Cadherins/metabolism , Cell Differentiation , Enterocytes/cytology , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Animals , Apoptosis , Blotting, Western , Cell Proliferation , Fluorescent Antibody Technique , HT29 Cells , Humans , Immunoenzyme Techniques , Immunoprecipitation , Rats , p120 GTPase Activating Protein/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into ß3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires ß3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling.
Subject(s)
Bone Morphogenetic Protein Receptors, Type II , Bone Morphogenetic Protein Receptors, Type I , Integrin beta3 , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Protein Receptors, Type II/metabolism , Cell Adhesion , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/metabolism , Integrin beta3/metabolism , Protein Serine-Threonine Kinases/metabolismABSTRACT
Invadosomes support cell invasion by coupling both acto-adhesive and extracellular matrix degradative functions, which are apparently antagonistic. ß1-integrin dynamics regulate this coupling, but the actual sensing mechanism and effectors involved have not yet been elucidated. Using genetic and reverse genetic approaches combined with biochemical and imaging techniques, we now show that the calcium channel TRPV4 colocalizes with ß1-integrins at the invadosome periphery and regulates its activation and the coupling of acto-adhesive and degradative functions. TRPV4-mediated regulation of podosome function depends on its ability to sense reactive oxygen species (ROS) in invadosomes' microenvironment and involves activation of the ROS/calcium-sensitive kinase Ask1 and binding of the motor MYO1C. Furthermore, disease-associated TRPV4 gain-of-function mutations that modulate ECM degradation are also implicated in the ROS response, which provides new perspectives in our understanding of the pathophysiology of TRPV4 channelopathies.
Subject(s)
Podosomes/metabolism , Reactive Oxygen Species/metabolism , TRPV Cation Channels/metabolism , Actins/metabolism , Animals , Calcium/metabolism , Cell Adhesion , Cysteine/metabolism , Dithionitrobenzoic Acid , Extracellular Matrix/metabolism , HEK293 Cells , Humans , Hydrogen Peroxide/metabolism , Integrin beta1/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Mice , Models, Biological , Myosin Type I/metabolism , Protein Transport , RAW 264.7 CellsABSTRACT
Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instruments or limited by the complexity of the required analysis. With the recent development and expanding utilization of FRET-based biosensors, it becomes essential to allow biologists to produce quantitative results that can directly be compared. Here, we present a new calibration and analysis method allowing for quantitative FRET imaging in living cells with a simple fluorescence microscope. Aside from the spectral crosstalk corrections, two additional correction factors were defined from photophysical equations, describing the relative differences in excitation and detection efficiencies. The calibration is achieved in a single step, which renders the Quantitative Three-Image FRET (QuanTI-FRET) method extremely robust. The only requirement is a sample of known stoichiometry donor:acceptor, which is naturally the case for intramolecular FRET constructs. We show that QuanTI-FRET gives absolute FRET values, independent of the instrument or the expression level. Through the calculation of the stoichiometry, we assess the quality of the data thus making QuanTI-FRET usable confidently by non-specialists.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer/methods , Evaluation Studies as Topic , FluorescenceABSTRACT
Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy.
Subject(s)
Cell Adhesion Molecules/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Phosphoproteins/metabolism , Catenins , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Cell Cycle/physiology , Cell Growth Processes/physiology , Centrosome/metabolism , Colonic Neoplasms/genetics , Cytoplasm/metabolism , Disease Progression , Gene Amplification , Genomic Instability , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HT29 Cells , Humans , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Phosphorylation , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Up-Regulation , Delta CateninABSTRACT
Integrins are transmembrane receptors that have a pivotal role in mechanotransduction processes by connecting the extracellular matrix to the cytoskeleton. Although it is well established that integrin activation/inhibition cycles are due to highly dynamic interactions, whether integrin mobility depends on local tension and cytoskeletal organization remains surprisingly unclear. Using an original approach combining micropatterning on glass substrates to induce standardized local mechanical constraints within a single cell with temporal image correlation spectroscopy, we measured the mechanosensitive response of integrin mobility at the whole cell level and in adhesion sites under different mechanical constraints. Contrary to ß1 integrins, high tension increases ß3 integrin residence time in adhesive regions. Chimeric integrins and structure-function studies revealed that the ability of ß3 integrins to specifically sense local tensional organization is mostly encoded by its cytoplasmic domain and is regulated by tuning the affinity of its NPXY domains through phosphorylation by Src family kinases.
Subject(s)
Integrin beta1/metabolism , Integrin beta3/metabolism , src-Family Kinases/metabolism , Animals , Biomechanical Phenomena , Cell Adhesion , Fibroblasts/cytology , Fibroblasts/metabolism , Green Fluorescent Proteins/metabolism , Integrin beta3/chemistry , Mechanotransduction, Cellular , Mice , Models, Biological , Phosphorylation , Protein Domains , Protein Transport , Spectrum Analysis , src-Family Kinases/antagonists & inhibitorsABSTRACT
Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes.
Subject(s)
Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Extracellular Matrix/metabolism , LIM Domain Proteins/metabolism , Paxillin/metabolism , Podosomes/metabolism , Amino Acid Motifs , Animals , Cell Adhesion , Janus Kinase 1/metabolism , Mice , Models, Biological , Paxillin/chemistry , Protein Binding , Protein Domains , Structure-Activity Relationship , ras GTPase-Activating Proteins/metabolismABSTRACT
The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.
Subject(s)
Actins/metabolism , Dynamins/metabolism , Animals , Cells, Cultured , Fibroblasts/metabolism , Mice , Oncogene Protein pp60(v-src)ABSTRACT
Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization-depolymerization associated with ß1 and ß3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using ß1â»/â» or ß3â»/â» cells, it seemed that ß1A but not ß3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the ß1A tail. Moreover, our study clearly showed that ß1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the ß1A cytoplasmic domain allowed specific and immediate loss of function of ß1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.
Subject(s)
Actins/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion , Cell Membrane Structures/physiology , Integrin beta1/metabolism , Animals , Cell Membrane Structures/ultrastructure , Cell Movement , Cells, Cultured , Extracellular Matrix/metabolism , Focal Adhesions/metabolism , Gene Knockout Techniques , Genes, src , Integrin beta1/chemistry , Integrin beta3/metabolism , Mesoderm/metabolism , Mice , Phosphorylation , Polymerization , Protein Kinase C/metabolism , Signal TransductionABSTRACT
In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.