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ACS Nano Apr 2017Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and...
Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.
Topics: Actins; Biomechanical Phenomena; Cell Adhesion; Cells, Cultured; Computer Simulation; Humans; Macrophages; Mechanotransduction, Cellular; Monocytes; Nanostructures; Particle Size; Paxillin; Podosomes; Surface Properties; Talin; Vinculin
PubMed: 28355484
DOI: 10.1021/acsnano.7b00622 -
FASEB Journal : Official Publication of... Aug 2016Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive...
Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.
Topics: Animals; Cell Movement; Down-Regulation; Endothelial Cells; Gene Expression Regulation; Humans; Male; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Podosomes; Stress, Physiological; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; p38 Mitogen-Activated Protein Kinases; src-Family Kinases
PubMed: 27103579
DOI: 10.1096/fj.201500091R -
Antioxidants & Redox Signaling May 2019Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2...
AIMS
Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2 either stimulatory or repressive influence on angiogenesis. Our findings unveil the mechanism of Nrf2-dependent vessel formation, which reaches beyond transactivation of gene expression and reconciles previous discrepancies.
RESULTS
We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system.
Topics: Actins; Animals; Cells, Cultured; Cellular Senescence; Chemokine CXCL12; Endothelial Cells; Endothelium; Growth Differentiation Factor 15; High-Throughput Nucleotide Sequencing; Kelch-Like ECH-Associated Protein 1; Mice; Mice, Knockout; MicroRNAs; Models, Biological; NF-E2-Related Factor 2; Neovascularization, Physiologic; Podosomes; Transcription, Genetic
PubMed: 30198307
DOI: 10.1089/ars.2018.7505 -
PloS One 2012Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been...
Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.
Topics: Actins; Animals; Antibodies, Monoclonal; Bone Marrow Cells; Cell Differentiation; Cofilin 1; Fluorescent Antibody Technique, Indirect; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Osteoclasts; Phosphoric Monoester Hydrolases; Phosphorylation; RANK Ligand; Retroviridae
PubMed: 23049890
DOI: 10.1371/journal.pone.0045909 -
Journal of Cell Science Jan 2018Tyrosine kinase substrate (Tks) adaptor proteins are considered important regulators of various physiological and/or pathological processes, particularly cell migration... (Review)
Review
Tyrosine kinase substrate (Tks) adaptor proteins are considered important regulators of various physiological and/or pathological processes, particularly cell migration and invasion, and cancer progression. These proteins contain PX and SH3 domains, and act as scaffolds, bringing membrane and cellular components in close proximity in structures known as invadopodia or podosomes. Tks proteins, analogous to the related proteins p47, p40 and NoxO1, also facilitate local generation of reactive oxygen species (ROS), which aid in signaling at invadopodia and/or podosomes to promote their activity. As their name suggests, Tks adaptor proteins are substrates for tyrosine kinases, especially Src. In this Cell Science at a Glance article and accompanying poster, we discuss the known structural and functional aspects of Tks adaptor proteins. As the science of Tks proteins is evolving, this article will point out where we stand and what still needs to be explored. We also underscore pathological conditions involving these proteins, providing a basis for future research to develop therapies for treatment of these diseases.
Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Gene Expression Regulation, Neoplastic; Humans; NADPH Oxidases; Neoplasms; Podosomes; Protein-Tyrosine Kinases; Reactive Oxygen Species; Signal Transduction
PubMed: 29311151
DOI: 10.1242/jcs.203661 -
Molecular Immunology Oct 2016Elucidating the molecular regulation of macrophage migration is essential for understanding the pathophysiology of multiple human diseases, including host responses to...
Elucidating the molecular regulation of macrophage migration is essential for understanding the pathophysiology of multiple human diseases, including host responses to infection and autoimmune disorders. Macrophage migration is supported by dynamic rearrangements of the actin cytoskeleton, with formation of actin-based structures such as podosomes and lamellipodia. Here we provide novel insights into the function of the actin-bundling protein l-plastin (LPL) in primary macrophages. We found that podosome stability is disrupted in primary resident peritoneal macrophages from LPL mice. Live-cell imaging of F-actin using resident peritoneal macrophages from LifeACT-RFP mice demonstrated that loss of LPL led to decreased longevity of podosomes, without reducing the number of podosomes initiated. Additionally, macrophages from LPL mice failed to elongate in response to chemotactic stimulation. These deficiencies in podosome stabilization and in macrophage elongation correlated with impaired macrophage transmigration in culture and decreased monocyte migration into murine peritoneum. Thus, we have identified a role for LPL in stabilizing long-lived podosomes and in enabling macrophage motility.
Topics: Animals; Cell Movement; Cytoskeletal Proteins; Macrophages, Peritoneal; Mice; Mice, Knockout; Microfilament Proteins; Microscopy, Confocal; Phosphoproteins; Podosomes
PubMed: 27614263
DOI: 10.1016/j.molimm.2016.08.012 -
The Journal of Investigative Dermatology Aug 2023Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph...
Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Met). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.
Topics: Mice; Animals; Podosomes; Cell Movement; Skin; Langerhans Cells; Signal Transduction; Dendritic Cells; Lymph Nodes
PubMed: 36813160
DOI: 10.1016/j.jid.2022.12.025 -
Journal of Cell Science Jul 2016Podosomes are dynamic cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based...
Podosomes are dynamic cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based cytoskeletal structures have been mostly studied in monocytic cells, but much less is known about those formed in other lineages. In this study, we characterise podosomes in capillary-derived microvascular endothelial cells. We identify two types of podosomes: constitutive podosomes that form in the absence of specific stimulation and induced podosomes that arise in response to the angiogenic factor VEGF-A. Constitutive and VEGF-A-induced podosomes share similar components but exhibit marked differences in terms of gelatinolytic activity. We also show that the extracellular matrix proteins laminin and collagen-IV are key determinants of the VEGF-A response, but neither collagen-I nor fibronectin are conducive for podosome induction. Moreover, only collagen-IV elicits the formation of proteolytically active podosomes through a mechanism involving increased Src phosphorylation, p190RhoGAP-B (also known as ARHGAP5) relocalisation and MT1-MMP (also known as MMP14) cell surface exposure at podosome sites. We hypothesise that by promoting podosome formation, VEGF-A enables endothelial cells to overcome the basement membrane barrier to allow sprouting outwards from the existing vasculature.
Topics: Actins; Collagen Type IV; Cytoskeleton; Endothelial Cells; GTPase-Activating Proteins; Gene Expression Regulation; Humans; Matrix Metalloproteinase 14; Phosphorylation; Podosomes; Proteolysis; Vascular Endothelial Growth Factor A
PubMed: 27231093
DOI: 10.1242/jcs.186585 -
European Journal of Cell Biology Apr 2006Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal...
Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.
Topics: Animals; Cell Adhesion Molecules; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Extracellular Matrix; Fluorescent Antibody Technique; Mutation; Swine; cdc42 GTP-Binding Protein
PubMed: 16546575
DOI: 10.1016/j.ejcb.2005.09.009 -
Frontiers in Immunology 2018The immune system serves as a crucial line of defense from infection and cancer, while also contributing to tissue homeostasis. Communication between immune cells is... (Review)
Review
The immune system serves as a crucial line of defense from infection and cancer, while also contributing to tissue homeostasis. Communication between immune cells is mediated by small soluble factors called cytokines, and also by direct cellular interactions. Cell-cell interactions are particularly important for T cell activation. T cells direct the adaptive immune response and therefore need to distinguish between self and foreign antigens. Even though decades have passed since the discovery of T cells, exactly why and how they are able to recognize and discriminate between antigens is still not fully understood. Early imaging of T cells was very successful in capturing the early stages of conjugate formation of T cells with antigen-presenting cells upon recognition of peptide-loaded major histocompatibility complexes by the T cell receptor (TCR). These studies lead to the discovery of a "supramolecular activation cluster" now known as the immunological synapse, followed by the identification of microclusters of TCRs formed upon receptor triggering, that eventually coalesce at the center of the synapse. New developments in light microscopy have since allowed attention to turn to the very earliest stages of T cell activation, and to resting cells, at high resolution. This includes single-molecule localization microscopy, which has been applied to the question of whether TCRs are pre-clustered on resting T cells, and lattice light-sheet microscopy that has enabled imaging of whole cells interacting with antigen-presenting cells. The utilization of lattice light-sheet microscopy has yielded important insights into structures called microvilli, which are small membrane protrusions on T cells that seem likely to have a large impact on T cell recognition and activation. Here we consider how imaging has shaped our thinking about T cell activation. We summarize recent findings obtained by applying more advanced microscopy techniques and discuss some of the limitations of these methods.
Topics: Cell Communication; Humans; Immunological Synapses; Lymphocyte Activation; Microscopy, Confocal; Microscopy, Fluorescence; Microvilli; Podosomes; Receptors, Antigen, T-Cell; Single Molecule Imaging; T-Lymphocytes
PubMed: 30319617
DOI: 10.3389/fimmu.2018.02152