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Nature Communications May 2023Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for...
Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.
Topics: Podosomes; Actins; Macrophages
PubMed: 37217555
DOI: 10.1038/s41467-023-38598-z -
Journal of Cell Science Jan 2013Podosomes are actin-enriched membrane protrusions that play important roles in extracellular matrix degradation and invasive cell motility. Podosomes undergo...
Podosomes are actin-enriched membrane protrusions that play important roles in extracellular matrix degradation and invasive cell motility. Podosomes undergo self-assembly into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and certain highly invasive cancer cells. Several protein tyrosine kinases have been shown to be important for the formation of podosome rosettes, but little is known regarding the role of protein tyrosine phosphatases in this process. We found that knockdown of the Src homolog domain-containing phosphatase 2 (SHP2) significantly increased podosome rosette formation in Src-transformed fibroblasts. By contrast, SHP2 overexpression suppressed podosome rosette formation in these cells. The phosphatase activity of SHP2 was essential for the suppression of podosome rosette formation. SHP2 selectively suppressed the tyrosine phosphorylation of Tks5, a scaffolding protein required for podosome formation. The inhibitory effect of SHP2 on podosome rosette formation was associated with the increased activation of Rho-associated kinase (ROCK) and the enhanced polymerization of vimentin filaments. A higher content of polymerized vimentin filaments was correlated with a lower content of podosome rosettes. Taken together, our findings indicate that SHP2 serves as a negative regulator of podosome rosette formation through the dephosphorylation of Tks5 and the activation of ROCK-mediated polymerization of vimentin in Src-transformed fibroblasts.
Topics: Animals; Fibroblasts; Gene Knockdown Techniques; Humans; Mice; NIH 3T3 Cells; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; src-Family Kinases
PubMed: 23178938
DOI: 10.1242/jcs.116624 -
Experimental Cell Research Apr 2016Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formation and aggressiveness. Adhesion-based signaling driven by a rigid... (Review)
Review
Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formation and aggressiveness. Adhesion-based signaling driven by a rigid microenvironment is thought to facilitate invasion and migration of cancer cells away from primary tumors. Proteolytic degradation of extracellular matrix (ECM) is a key component of this process and is mediated by subcellular actin-rich structures known as invadopodia. Both ECM rigidity and cellular traction stresses promote invadopodia formation and activity, suggesting a role for these structures in mechanosensing. The presence and activity of mechanosensitive adhesive and signaling components at invadopodia further indicates the potential for these structures to utilize myosin-dependent forces to probe and remodel their ECM environments. Here, we provide a brief review of the role of adhesion-based mechanical signaling in controlling invadopodia and invasive cancer behavior.
Topics: Extracellular Matrix; Humans; Mechanotransduction, Cellular; Models, Biological; Podosomes; Tumor Microenvironment
PubMed: 26546985
DOI: 10.1016/j.yexcr.2015.10.038 -
Cell Reports Mar 2021Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETR) drives...
Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETR) drives invadopodia by a not fully explored coordinated function of β-arrestin1 (β-arr1). Here, we report that β-arr1 links the integrin-linked kinase (ILK)/βPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/β-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.
Topics: Actin Depolymerizing Factors; Animals; Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Cell Movement; Coculture Techniques; Databases, Genetic; Endothelin A Receptor Antagonists; Endothelin-1; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Ovarian Neoplasms; Peritoneum; Phenylpropionates; Phosphorylation; Podosomes; Protein Serine-Threonine Kinases; Pyridazines; Receptor, Endothelin A; Rho Guanine Nucleotide Exchange Factors; Signal Transduction; Tumor Microenvironment; Xenograft Model Antitumor Assays; beta-Arrestin 1; p21-Activated Kinases; rac GTP-Binding Proteins; Mice
PubMed: 33657382
DOI: 10.1016/j.celrep.2021.108800 -
Cell Reports Dec 2019p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to...
p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover.
Topics: Cells, Cultured; Disulfides; Extracellular Matrix; Focal Adhesions; HEK293 Cells; Humans; Myeloid Cells; Naphthols; Phosphorylation; Podosomes; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; THP-1 Cells; p21-Activated Kinases
PubMed: 31825823
DOI: 10.1016/j.celrep.2019.11.016 -
Cells Aug 2021Non-receptor tyrosine kinases (NRTKs) are crucial mediators of intracellular signaling and control a wide variety of processes such as cell division, morphogenesis, and... (Review)
Review
Non-receptor tyrosine kinases (NRTKs) are crucial mediators of intracellular signaling and control a wide variety of processes such as cell division, morphogenesis, and motility. Aberrant NRTK-mediated tyrosine phosphorylation has been linked to various human disorders and diseases, among them cancer metastasis, to which no treatment presently exists. Invasive cancer cells leaving the primary tumor use invadopodia, feet-like structures which facilitate extracellular matrix (ECM) degradation and intravasation, to escape the primary tumor and disseminate into distant tissues and organs during metastasis. A major challenge in metastasis research is to elucidate the molecular mechanisms and signaling pathways underlying invadopodia regulation, as the general belief is that targeting these structures can potentially lead to the eradication of cancer metastasis. Non-receptor tyrosine kinases (NRTKs) play a central role in regulating invadopodia formation and function, but how they coordinate the signaling leading to these processes was not clear until recently. Here, we describe the major NRTKs that rule invadopodia and how they work in concert while keeping an accurate hierarchy to control tumor cell invasiveness and dissemination.
Topics: Cell Movement; Extracellular Matrix; Humans; Morphogenesis; Neoplasm Invasiveness; Neoplasm Metastasis; Podosomes; Protein-Tyrosine Kinases; Signal Transduction; src-Family Kinases
PubMed: 34440806
DOI: 10.3390/cells10082037 -
Nature Communications Jul 2022Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize...
Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin are characterized.
Topics: Actins; Microscopy; Myosin Type II; Podosomes; Talin
PubMed: 35896550
DOI: 10.1038/s41467-022-32038-0 -
Cancer Research Jan 2016Tumor-associated macrophages (TAM) play complex and pivotal roles during cancer progression. A subset of metastasis-associated macrophages accumulates within metastatic...
Tumor-associated macrophages (TAM) play complex and pivotal roles during cancer progression. A subset of metastasis-associated macrophages accumulates within metastatic sites to promote the invasion and growth of tumor cells. Src kinase-associated phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages from various tumors, but its contribution to the tumor-promoting behavior of TAMs is unknown. Here, we report that SKAP2 regulates podosome formation in macrophages to promote tumor invasion and metastasis. SKAP2 physically interacted with Wiskott-Aldrich syndrome protein (WASP) and localized to podosomes, which were rarely observed in SKAP2-null macrophages. The invasion of peritoneal macrophages derived from SKAP2-null mice was significantly reduced compared with wild-type macrophages, but could be rescued by the restoration of functional SKAP2 containing an intact tyrosine phosphorylation site and the ability to interact with WASP. Furthermore, SKAP2-null mice inoculated with lung cancer cells exhibited markedly decreased lung metastases characterized by reduced macrophage infiltration compared with wild-type mice. Moreover, intravenously injected SKAP2-null macrophages failed to efficiently infiltrate established tumors and promote their growth. Taken together, these findings reveal a novel mechanism by which macrophages assemble the appropriate motile machinery to infiltrate tumors and promote disease progression, and implicate SKAP2 as an attractive candidate for therapeutically targeting TAMs.
Topics: Animals; Breast Neoplasms; Carcinoma, Lewis Lung; Cell Line, Tumor; Disease Progression; Female; Humans; Intracellular Signaling Peptides and Proteins; Macrophages; Macrophages, Peritoneal; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Metastasis; Podosomes; RAW 264.7 Cells
PubMed: 26577701
DOI: 10.1158/0008-5472.CAN-15-1879 -
Proceedings of the National Academy of... Dec 2010Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond...
Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.
Topics: Actins; Cytoplasmic Structures; Cytoskeleton; Extracellular Matrix; Hardness; Humans; Macrophages; Microscopy, Atomic Force; Myosin Type II
PubMed: 21081699
DOI: 10.1073/pnas.1007835107 -
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