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The International Journal of... May 2014Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM)...
Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.
Topics: Animals; Cell Membrane Structures; Cell Movement; Dendritic Cells; Extracellular Matrix; Humans; Male; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Transfection; Wiskott-Aldrich Syndrome Protein
PubMed: 24508783
DOI: 10.1016/j.biocel.2014.01.021 -
Journal of Immunology (Baltimore, Md. :... Nov 2015Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium....
Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.
Topics: Animals; COS Cells; Cell Movement; Chlorocebus aethiops; Humans; Imatinib Mesylate; Macrophages; Mice; Neoplasm Invasiveness; Neoplasms; Phosphorylation; Podosomes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-abl; SOS1 Protein; rac GTP-Binding Proteins; src-Family Kinases
PubMed: 26447228
DOI: 10.4049/jimmunol.1500579 -
Molecular Biology of the Cell Sep 2011Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on...
Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on bead-seeded soft substrates that podosome assemblies, such as rings, are involved in tension forces. During the expansion of a podosome ring, substrate displacement is oriented outward, suggesting that podosomal structures push the substrate away. To further elucidate the function of forces generated by podosomes, we analyze osteoclast migration. Determining the centers of mass of the whole cell (G) and of actin (P), we demonstrate that osteoclasts migrate by "jumps" and that the trajectories of G and P are strongly correlated. The velocity of the center of mass as a function of time reveals that osteoclasts rapidly catch up with podosomal structures in a periodic pattern. We conclude that actin dynamics inside the cell are not only correlated with cell migration, but drive it.
Topics: Actins; Algorithms; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Line; Cell Movement; Cell-Matrix Junctions; Green Fluorescent Proteins; Mice; Microscopy, Fluorescence; Osteoclasts; Recombinant Fusion Proteins; Single-Cell Analysis; Stress, Mechanical; Time-Lapse Imaging
PubMed: 21737683
DOI: 10.1091/mbc.E11-01-0086 -
Molecular Biology of the Cell Aug 2019Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for...
Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.
Topics: Actin-Related Protein 2-3 Complex; Actins; Animals; Cell Communication; Cell Movement; Cytochalasin B; Female; Macrophages; Male; Membrane Fusion; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Podosomes; Wiskott-Aldrich Syndrome Protein; cdc42 GTP-Binding Protein
PubMed: 31242090
DOI: 10.1091/mbc.E19-01-0009 -
Journal of Cell Science Jan 2020Cysteinyl-leukotrienes (cys-LTs) have well-characterized physiopathological roles in the development of inflammatory diseases. We have previously found that protein...
Cysteinyl-leukotrienes (cys-LTs) have well-characterized physiopathological roles in the development of inflammatory diseases. We have previously found that protein tyrosine phosphatase ε (PTPε) is a signaling partner of CysLTR, a high affinity receptor for leukotriene D4 (LTD). There are two major isoforms of PTPε, receptor-like (RPTPε) and cytoplasmic (cyt-)PTPε, both of which are encoded by the gene but from different promoters. In most cells, their expression is mutually exclusive, except in human primary monocytes, which express both isoforms. Here, we show differential PTPε isoform expression patterns between monocytes, M1 and M2 human monocyte-derived macrophages (hMDMs), with the expression of glycosylated forms of RPTPε predominantly in M2-polarized hMDMs. Using PTPε-specific siRNAs and expression of RPTPε and cyt-PTPε, we found that RPTPε is involved in monocyte adhesion and migration of M2-polarized hMDMs in response to LTD Altered organization of podosomes and higher phosphorylation of the inhibitory Y-722 residue of ROCK2 was also found in PTPε-siRNA-transfected cells. In conclusion, we show that differentiation and polarization of monocytes into M2-polarized hMDMs modulates the expression of PTPε isoforms and RPTPε is involved in podosome distribution, ROCK2 activation and migration in response to LTD.
Topics: Humans; Macrophages; Phosphorylation; Podosomes; Protein Tyrosine Phosphatases; Signal Transduction; rho-Associated Kinases
PubMed: 31722979
DOI: 10.1242/jcs.234641 -
European Journal of Cell Biology Sep 2008Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and...
Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.
Topics: Actin Cytoskeleton; Actins; Animals; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Endothelial Cells; Humans; Microscopy, Fluorescence; Protein Kinase C-alpha; Protein Kinase C-delta; Signal Transduction; Smad Proteins; Swine; cdc42 GTP-Binding Protein
PubMed: 18397815
DOI: 10.1016/j.ejcb.2008.02.006 -
Journal of Thrombosis and Haemostasis :... Nov 2020Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the...
BACKGROUND
Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production.
OBJECTIVE
The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids.
METHODS
We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells.
RESULTS
We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis.
CONCLUSION
This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.
Topics: Animals; Blood Platelets; Bone Marrow; Capillaries; Endothelial Cells; Megakaryocytes; Mice; Podosomes; Thrombopoiesis
PubMed: 32702204
DOI: 10.1111/jth.15024 -
The Journal of Biological Chemistry 2021Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast,...
Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.
Topics: Animals; Bone Resorption; Cell Differentiation; HEK293 Cells; Humans; Mice; Osteoclasts; Podosomes; src Homology Domains; src-Family Kinases
PubMed: 34019873
DOI: 10.1016/j.jbc.2021.100790 -
Nature Communications Oct 2016Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In...
Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area.
Topics: Actin Cytoskeleton; Actins; Actomyosin; Cell Surface Extensions; Cytoskeleton; Dendritic Cells; Humans; Models, Biological; Nonmuscle Myosin Type IIA; Podosomes; Polymerization; Rheology; Talin; Time Factors; Vinculin
PubMed: 27721497
DOI: 10.1038/ncomms13127 -
Experimental Cell Research Apr 2006Cortactin, a multi-domain scaffolding protein involved in actin polymerization, is enriched in podosomes induced by phorbol ester in vascular smooth muscle cells. We...
Cortactin, a multi-domain scaffolding protein involved in actin polymerization, is enriched in podosomes induced by phorbol ester in vascular smooth muscle cells. We generated several functional and truncation mutants of cortactin to probe the roles of various protein interaction domains in the regulation of the dynamics of podosome formation. At the onset of podosome genesis, cortactin clustered near the ends of stress fibers that appeared to act as nucleation platforms onto which the actin polymerization machinery assembled. Translocation of cortactin to these pre-podosome clusters required the intact N-WASp-binding SH3 domain. Overexpression of the C-terminal third of cortactin containing the intact SH3 domain inhibited podosome formation presumably by sequestering of N-WASp and prevented cortactin clustering. Subsequent assembly of the actin-rich core of podosomes required translocation of additional cortactin to the actin core, a process that required the actin-binding repeats, but not the Arp2/3-binding N-terminal acidic region nor the SH3 domain. These results suggest that the SH3 domain and the actin-binding repeat region are involved, respectively, in the early and late stages of podosome formation process.
Topics: Actins; Animals; Binding Sites; Cell Membrane Structures; Cells, Cultured; Cortactin; Liver; Muscle, Smooth, Vascular; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Tertiary; Rats
PubMed: 16434035
DOI: 10.1016/j.yexcr.2005.11.032