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There and back again: Intracellular trafficking, release and recycling of matrix metalloproteinases.Biochimica Et Biophysica Acta.... Apr 2022Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological... (Review)
Review
Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological scenarios, including immune cell surveillance, tissue homeostasis, or tumor cell metastasis. This is based on their ability to cleave a plethora of substrates that include components of the extracellular matrix, but also cell surface-associated and intracellular proteins. Accordingly, a tight regulatory web has evolved that closely regulates spatiotemporal activity of specific MMPs. An often underappreciated mechanism of MMP regulation involves their trafficking to and from specific subcellular sites that require MMP activity only for a certain period. In this review, we focus on the current knowledge of MMP intracellular trafficking, their secretion or surface exposure, as well as their recycling back from the cell surface. We discuss molecular mechanisms that enable these steps, in particular microtubule-dependent motility of vesicles that is driven by molecular motors and directed by vesicle regulatory proteins. Finally, we also point out open questions in the field of MMP motility that may become important in the future.
Topics: Endocytosis; Endoplasmic Reticulum; Extracellular Vesicles; Golgi Apparatus; Humans; Matrix Metalloproteinases; Microtubules; Podosomes; Protein Transport
PubMed: 34973301
DOI: 10.1016/j.bbamcr.2021.119189 -
Experimental Cell Research Feb 2021Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures...
Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures including podosome belts and sealing zones, and adjacent clathrin sheets. Clathrin-coated structures are exclusively localized at the podosome belts and sealing zone, in both of which the plasma membrane forms a tight attachment to the substrate surface. When cultured on apatite osteoclasts can degrade the apatite leading to the formation of resorption lacunae. The sealing zone divides the ventral membrane into different domains, outside and inside of the sealing zones. The former facing the smooth-surfaced intact apatite contains relatively solitary or networks of larger flat clathrin structures; and the latter, facing the rough-surfaced degraded apatite in the resorption lacunae contain clathrin in various shapes and sizes. Clathrin assemblies on the membrane domain facing not only a resorption lacuna, or trails but also intact apatite indeed were observed to be heterogeneous in size and intensity, suggesting that they appeared to follow variations in the surface topography of the apatite surface. These results provide a detailed insight into the flat clathrin sheets that have been suggested to be the sites of adhesion and mechanosensing in co-operation with podosomes.
Topics: Actins; Animals; Animals, Newborn; Bone Resorption; Cell Membrane; Cells, Cultured; Clathrin; Cytoskeleton; Osteoclasts; Protein Multimerization; Rabbits; Tissue Distribution
PubMed: 33359468
DOI: 10.1016/j.yexcr.2020.112433 -
Physical Biology Nov 2022Cancer invasion and metastasis require remodeling of the adjacent extracellular matrix (ECM). In this mini review, we will cover the mechanisms of proteolytic... (Review)
Review
Cancer invasion and metastasis require remodeling of the adjacent extracellular matrix (ECM). In this mini review, we will cover the mechanisms of proteolytic degradation and the mechanical remodeling of the ECM by cancer cells, with a focus on invadopodia. Invadopodia are membrane protrusions unique to cancer cells, characterized by an actin core and by the focal degradation of ECM via matrix metalloproteases (MMPs). While ECM can also be remodeled, at lower levels, by focal adhesions, or internal collagen digestion, invadopodia are now recognized as the major mechanism for MMP-dependent pericellular ECM degradation by cancer cells. Recent evidence suggests that the completion of epithelial-mesenchymal transition may be dispensable for invadopodia and metastasis, and that invadopodia are required not only for mesenchymal, single cell invasion, but also for collective invasion. During collective invasion, invadopodia was then shown to be located in leader cells, allowing follower cells to move via cooperation. Collectively, this suggests that invadopodia function may be a requirement not only for later steps of metastasis, but also for early invasion of epithelial cells into the stromal tissue. Over the last decade, invadopodia studies have transitioned into in 3D andsettings, leading to the confirmation of their essential role in metastasis in preclinical animal models. In summary, invadopodia may hold a great potential for individual risk assessment as a prognostic marker for metastasis, as well as a therapeutic target.
Topics: Animals; Podosomes; Proteolysis; Extracellular Matrix; Neoplasms; Focal Adhesions
PubMed: 36343366
DOI: 10.1088/1478-3975/aca0d8 -
European Journal of Cell Biology Sep 2020The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor...
The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native collagen-I, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.
Topics: Animals; Cell Line, Tumor; Cell Proliferation; Extracellular Matrix; Humans; Mice; Podosomes; Protein Isoforms
PubMed: 33070041
DOI: 10.1016/j.ejcb.2020.151122 -
Proceedings of the National Academy of... Apr 2021Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal...
Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of -macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.
Topics: AMP-Activated Protein Kinase Kinases; Actomyosin; Animals; CD18 Antigens; Candida albicans; Cell Adhesion Molecules; Cells, Cultured; Humans; Hyphae; Lectins, C-Type; Macrophages; Mice; Phagocytosis; Protein Kinases; RAW 264.7 Cells
PubMed: 33876755
DOI: 10.1073/pnas.2020484118 -
IScience Dec 2022Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural...
Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50 nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes at the layer of myosin IIA. The microtubule disruption-caused podosome dissolution is previously ascribed to Rho/ROCK-myosin signaling, yet inhibiting this pathway with Y27632 or blebbistatin only partially recovers podosome assembly, thus suggesting the contribution of the physical supporting of microtubules in stabilizing podosome structures. Through improved substrate-coating technique, we further corroborate that the matrix-degrading capability of macrophages depends on the formation of podosome clusters. Together, 3D-STORM super-resolution microscopy reveals the nanoscale spatial arrangement and the microtubule-dependent regulation of the matrix-degrading podosome clusters in macrophages.
PubMed: 36425766
DOI: 10.1016/j.isci.2022.105514 -
Methods in Molecular Biology (Clifton,... 2023Cancer cells possess a remarkable capacity to dissociate from a primary tumor, invade the surrounding tissues and vasculature, and eventually form metastases in distant...
Cancer cells possess a remarkable capacity to dissociate from a primary tumor, invade the surrounding tissues and vasculature, and eventually form metastases in distant organs. This complex and multistep process remains one of the major causes of mortality in cancer patients worldwide. Multiple studies have highlighted the role of actin-rich structures called invadopodia ("invasive feet"), which adhere to the matrix, contain and secrete matrix-degrading proteinases, and apply protrusive forces generated by the actin cytoskeleton, which drive the invasive process. Here, we describe a fluorescent microscopy-based protocol for imaging and quantifying both invadopodia formation and matrix degradation.
Topics: Humans; Podosomes; Extracellular Matrix; Actins; Actin Cytoskeleton; Cell Line, Tumor; Neoplasm Invasiveness
PubMed: 36587098
DOI: 10.1007/978-1-0716-2851-5_12 -
The Journal of Cell Biology Feb 2020Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by...
Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.
Topics: Actin Cytoskeleton; Adaptor Proteins, Vesicular Transport; Animals; Cell Cycle Proteins; Cell Movement; Cells, Cultured; Endothelial Cells; Extracellular Matrix; Humans; Morphogenesis; Neovascularization, Physiologic; Podosomes; Septins
PubMed: 31865373
DOI: 10.1083/jcb.201903023 -
Cell Death & Disease Apr 2023Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the...
Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Toward that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that the knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.
Topics: Humans; Actins; Cell Line, Tumor; Hippo Signaling Pathway; Podosomes; Proteomics; YAP-Signaling Proteins
PubMed: 37185904
DOI: 10.1038/s41419-023-05769-1 -
Blood Advances Dec 2022Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to...
Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.
Topics: Mice; Animals; Megakaryocytes; Actins; Thrombocytopenia; Mice, Knockout; Disease Models, Animal; Adaptor Proteins, Signal Transducing; Integrins
PubMed: 36251748
DOI: 10.1182/bloodadvances.2022007824