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Biochimica Et Biophysica Acta.... Jan 2024To explore the novel function of MYO6 on Osteoclast differentiation and its joint destruction capacity in Rheumatoid arthritis mice model.
OBJECTIVES
To explore the novel function of MYO6 on Osteoclast differentiation and its joint destruction capacity in Rheumatoid arthritis mice model.
METHODS
We examined joint erosion in a collagen-induced arthritis (CIA) mouse model using micro-CT, with the mice having a MYO6 knockout background. Inflammatory cytokines were analyzed using an enzyme-linked immunosorbent assay (ELISA). In vitro, we investigated the osteoclastogenesis ability of bone marrow-derived macrophages isolated from MYO6 mice and their littermate controls, examining both morphological and functional differences. Furthermore, we explored podosome formation and endosome maturation using immunofluorescence staining.
RESULTS
We found that MYO6 deficiency attenuated arthritis development and bone destruction in CIA mice as well as impaired osteoclast differentiation by inhibiting NFATc1 induction. Our findings indicate that MYO6 is essential for the organization of podosomes by modulating the FAK/AKT and integrin-β3/Src pathways. MYO6 also mediates endosome transportation by regulating the expression of Rab5 and GM130. This may impact the maintenance and functionality of the ruffled border, as well as the regulation of autophagy in osteoclasts.
CONCLUSION
Our results demonstrated a critical function of MYO6 in osteoclast differentiation and its potential relevance in experimental arthritis.
Topics: Animals; Mice; Arthritis, Experimental; Arthritis, Rheumatoid; Disease Models, Animal; Macrophages; Osteoclasts; Osteogenesis
PubMed: 37816396
DOI: 10.1016/j.bbadis.2023.166902 -
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 -
Experimental Cell Research Apr 2016Cells interact with their environment through highly localized contact structures. Podosomes represent a subgroup of cell-matrix contacts, which is especially prominent... (Review)
Review
Cells interact with their environment through highly localized contact structures. Podosomes represent a subgroup of cell-matrix contacts, which is especially prominent in cells of the monocytic lineage such as monocytes, macrophages and dendritic cells, but also in a variety of other cell types. Comparable to other adhesion structures, podosomes feature a complex architecture, which forms the basis for their extensive repertoire of sensory and effector functions. These functions are mainly linked to interactions with the extracellular matrix and comprise well known properties such as cell-matrix adhesion and extracellular matrix degradation. A more recent discovery is the ability of podosomes to act as mechanosensory devices, by detecting rigidity and topography of the substratum. In this review, we focus especially on the molecular events involved in mechanosensing by podosomes, the structural elements of podosomes that enable this function, as well as the intra- and extracellular signals generated downstream of podosome mechanosensing.
Topics: Extracellular Matrix; Humans; Mechanotransduction, Cellular; Models, Biological; Podosomes
PubMed: 26658516
DOI: 10.1016/j.yexcr.2015.11.026 -
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 -
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 -
Advances in Experimental Medicine and... 2019The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis,... (Review)
Review
The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis, allowing the experimentalists to extend their playground to single cells and single molecules, with high throughput and resolution which were inconceivable few decades ago. In particular, microfluidic devices are profoundly changing the conventional way of studying the cell motility and cell migratory dynamics. In this chapter we will furnish a comprehensive view of the advancements made in the research domain of confinement-induced cell migration, thanks to the use of microfluidic devices. The chapter is subdivided in three parts. Each section will be addressing one of the fundamental questions that the microfluidic technology is contributing to unravel: (i) where cell migration takes place, (ii) why cells migrate and, (iii) how the cells migrate. The first introductory part is devoted to a thumbnail, and partially historical, description of microfluidics and its impact in biological sciences. Stress will be put on two aspects of the devices fabrication process, which are crucial for biological applications: materials used and coating methods. The second paragraph concerns the cell migration induced by environmental cues: chemical, leading to chemotaxis, mechanical, at the basis of mechanotaxis, and electrical, which induces electrotaxis. Each of them will be addressed separately, highlighting the fundamental role of microfluidics in providing the well-controlled experimental conditions where cell migration can be induced, investigated and ultimately understood. The third part of the chapter is entirely dedicated to how the cells move in confined environments. Invadosomes (the joint name for podosomes and invadopodia) are cell protrusion that contribute actively to cell migration or invasion. The formation of invadosomes under confinement is a research topic that only recently has caught the attention of the scientific community: microfluidic design is helping shaping the future direction of this emerging field of research.
Topics: Animals; Cell Movement; Chemotaxis; Humans; Lab-On-A-Chip Devices; Microfluidics; Podosomes; Research
PubMed: 31612455
DOI: 10.1007/978-3-030-17593-1_6 -
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