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Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin‐based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton. [ABSTRACT FROM AUTHOR]

High wall shear stress (HWSS) contributes to intracranial aneurysm (IA) development. However, the underlying molecular mechanisms remain unclear, in part due to the lack of robust animal models that develop IAs in a HWSS-dependent manner. The current study established a new experimental IA model in mice that was utilized to determine HWSS-triggered downstream mechanisms. By a strategic combination of HWSS and low dose elastase, IAs were induced with a high penetrance in hypertensive mice. In contrast, no IAs were observed in control groups where HWSS was absent, suggesting that our new IA model is HWSS-dependent. IA outcomes were assessed by neuroscores that correlate with IA rupture events. Pathological analyses confirmed these experimental IAs resemble those found in humans. Interestingly, HWSS alone promotes the turnover of collagen IV, a major basement membrane component underneath the endothelium, and the formation of endothelial podosomes, subcellular organelles that are known to degrade extracellular matrix proteins. These induced podosomes are functional as they degrade collagen-based substrates locally in the endothelium. These data suggest that this new murine model develops IAs in a HWSS-dependent manner and highlights the contribution of endothelial cells to the early phase of IA. With this model, podosome formation and function was identified as a novel endothelial phenotype triggered by HWSS, which provides new insight into IA pathogenesis.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase a to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation. [ABSTRACT FROM AUTHOR]

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation. [ABSTRACT FROM AUTHOR]

Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where “phagocytic podosomes” have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 μm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 μm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 μm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-μm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment. [Display omitted] [ABSTRACT FROM AUTHOR]

Cancer cell invasion and metastasis rely on invadopodia, important extensions of the cytoskeleton that initiate degradation of the basement membrane that holds a cell in place. Transforming growth factor-β (TGF-β) is well-known to induce breast cancer migration and invasion, but the mechanism by which TGF-β signaling converts into cell motility is not completely understood. A study from Kiepas et al. revealed a new TGF-β-dependent role for Src homology/collagen adaptor protein (SHCA) in the initiation of dynamic adhesion complexes involved in the formation of invadopodia. These results highlight new therapeutic opportunities for cancer patients that are not sensitive to HER2 antagonists.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed. [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocytophilum protein) and found that it dynamically changed its pattern and subcellular location in cells and enhanced cell adhesion. Tandem affinity purification combined with mass spectrometry identified host nucleolin as an AFAP-interacting protein. Further study showed the disruption of nucleolin by RNA interference, and the treatment of a nucleolin-binding DNA aptamer AS1411 attenuated AFAP-mediated cell adhesion, indicating that AFAP enhanced cell adhesion in a nucleolin-dependent manner. The characterization of cell adhesion-enhancing AFAP and the identification of host nucleolin as its interaction partner may help understand the mechanism underlying A. phagocytophilum-promoting cell adhesion, facilitating the elucidation of HGA pathogenesis. [ABSTRACT FROM AUTHOR]

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2. [ABSTRACT FROM AUTHOR]

Cell fusion (fusogenesis) occurs in natural and pathological conditions in prokaryotes and eukaryotes. Cells of monocyte–macrophage lineage are highly fusogenic. They create syncytial multinucleated giant cells (MGCs) such as osteoclasts (OCs), MGCs associated with the areas of infection/inflammation, and foreign body-induced giant cells (FBGCs). The fusion of monocytes/macrophages with tumor cells may promote cancer metastasis. We describe types and examples of monocyte–macrophage lineage cell fusion and the role of actin-based structures in cell fusion. [ABSTRACT FROM AUTHOR]

Eucalyptus essential oil and its major constituent eucalyptol are extensively employed in the cosmetic, food, and pharmaceutical industries and their clinical use has recently expanded worldwide as an adjuvant in the treatment of infective and inflammatory diseases. We previously demonstrated that essential oil from Eucalyptus globulus (Labill.) (EO) stimulates in vitro the phagocytic activity of human monocyte-derived macrophages and counteracts the myelotoxicity induced by the chemotherapeutic 5-fluorouracil in immunocompetent rats. Here we characterize some mechanistic aspects underlying the immunostimulatory ability exerted by EO on macrophages. The internalization of fluorescent beads, fluorescent zymosan BioParticles, or apoptotic cancer cells was evaluated by confocal microscopy. Pro-inflammatory cytokine and chemokine release was determined by flow cytometry using the BD cytometric bead array. Receptor involvement in EO-stimulated phagocytosis was assessed using complement- or IgG-opsonized zymosan particles. The localization and expression of podosome components was analyzed by confocal microscopy and western blot. The main results demonstrated that: EO-induced activation of a macrophage is ascribable to its major component eucalyptol, as recently demonstrated for other cells of innate immunity; EO implements pathogen internalization and clearance by stimulating the complement receptor-mediated phagocytosis; EO stimulates podosome formation and increases the expression of podosome components. These results confirm that EO extract is a potent activator of innate cell-mediated immunity and thereby increase the scientific evidence supporting an additional property of this plant extract besides the known antiseptic and anti-inflammatory properties. [ABSTRACT FROM AUTHOR]

Angiogenesis involves cell specification orchestrated by regulatory interactions between the vascular endothelial growth factor and Notch signaling pathways. However, the role of microRNAs in these regulations remains poorly explored. Here we show that a controlled level of miR-155 is essential for proper angiogenesis. In the mouse retina angiogenesis model, antimiR-155 altered neovascularization. In vitro assays established that endogenous miR-155 is involved in podosome formation, activation of the proteolytic machinery and cell migration but not in morphogenesis. The role of miR-155 was explored using miR-155 mimics. In vivo, exposing the developing vasculature to miR-155 promoted hypersprouting, thus phenocopying defects associated with Notch deficiency. Mechanistically, miR-155 overexpression weakened Notch signaling by reducing Smad1/5 expression, leading to the formation of tip cell-like cells which did not reach full invasive capacity and became unable to undergo morphogenesis. These results identify miR-155 as a novel regulator of physiological angiogenesis and as a novel actor of pathological angiogenesis. [ABSTRACT FROM AUTHOR]

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.

Atherosclerosis, cancer, and various chronic fibrotic conditions are characterized by an increase in the migratory behavior of resident cells and the enhanced invasion of assorted exogenous cells across a stiffened extracellular matrix (ECM). This stiffened scaffold aberrantly engages cellular mechanosignaling networks in cells, which promotes the assembly of invadosomes and lamellae for cell invasion and migration. Accordingly, deciphering the conserved molecular mechanisms whereby matrix stiffness fosters invadosome and lamella formation could identify therapeutic targets to treat fibrotic conditions, and reducing ECM stiffness could ameliorate disease progression.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell’s microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed.

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2.

Cell invasion is associated with numerous patho-physiologic states including cell development and metastatic dissemination. This process couples the activation of cell motility with the capacity to degrade the extracellular matrix, thereby permitting cells to pass through basal membranes. Invasion is sustained by the actions of invadosomes, an ensemble of subcellular structures with high functional homology. Invadosomes are 3D acto-adhesive structures that can also mediate local extracellular matrix degradation through the controlled delivery of proteases. Intracellular RHO GTPases play a central role in the regulation of invadosomes where their complex interplay regulates multiple invadosome functions. This review aims to provide an overview of the synergistic activities of the small GTPases in invadosome biology. This broad-based review also reinforces the importance of the spatiotemporal regulation of small GTPases and the impact of this process on invadosome dynamics. [ABSTRACT FROM AUTHOR]

Different types of multinucleated giant cells (MGCs) of myeloid origin have been described; osteoclasts are the most extensively studied because of their importance in bone homeostasis. MGCs are formed by cell-to-cell fusion, and most types have been observed in pathological conditions, especially in infectious and non-infectious chronic inflammatory contexts. The precise role of the different MGCs and the mechanisms that govern their formation remain poorly understood, likely due to their heterogeneity. First, we will introduce the main populations of MGCs derived from the monocyte/macrophage lineage. We will then discuss the known molecular actors mediating the early stages of fusion, focusing on cell-surface receptors involved in the cell-to-cell adhesion steps that ultimately lead to multinucleation. Given that cell-to-cell fusion is a complex and well-coordinated process, we will also describe what is currently known about the evolution of F-actin-based structures involved in macrophage fusion, i.e., podosomes, zipper-like structures, and tunneling nanotubes (TNT). Finally, the localization and potential role of the key fusion mediators related to the formation of these F-actin structures will be discussed. This review intends to present the current status of knowledge of the molecular and cellular mechanisms supporting multinucleation of myeloid cells, highlighting the gaps still existing, and contributing to the proposition of potential disease-specific MGC markers and/or therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Microtubule-associated protein Tau undergoes aggregation in Alzheimer's disease (AD) and a group of other related diseases collectively known as Tauopathies. In AD, Tau forms aggregates, which are deposited intracellularly as neurofibrillary tangles. Histone deacetylase-6 (HDAC6) plays an important role in aggresome formation, where it recruits polyubiquitinated aggregates to the motor protein dynein. Methods: Here, we have studied the effects of HDAC6 ZnF UBP on Tau phosphorylation, ApoE localization, GSK-3β regulation and cytoskeletal organization in neuronal cells by immunocytochemical analysis. This analysis reveals that the cell exposure to the UBP-type zinc finger domain of HDAC6 (HDAC6 ZnF UBP) can modulate Tau phosphorylation and actin cytoskeleton organization. Results: HDAC6 ZnF UBP treatment to cells did not affect their viability and resulted in enhanced neurite extension and formation of structures similar to podosomes, lamellipodia and podonuts suggesting the role of this domain in actin re-organization. Also, HDAC6 ZnF UBP treatment caused increase in nuclear localization of ApoE and tubulin localization in microtubule organizing centre (MTOC). Therefore, our studies suggest the regulatory role of this domain in different aspects of neurodegenerative diseases. Upon HDAC6 ZnF UBP treatment, inactive phosphorylated form of GSK-3β increases without any change in total GSK-3β level. Conclusions: HDAC6 ZnF UBP was found to be involved in cytoskeletal re-organization by modulating actin dynamics and tubulin localization. Overall, our study suggests that ZnF domain of HDAC6 performs various regulatory functions apart from its classical function in aggresome formation in protein misfolding diseases. BbeLHN_zgyG8Hf4zoSkS1n Video abstract [ABSTRACT FROM AUTHOR]

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, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans-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. [ABSTRACT FROM AUTHOR]

Eucalyptus essential oil and its major constituent eucalyptol are extensively employed in the cosmetic, food, and pharmaceutical industries and their clinical use has recently expanded worldwide as an adjuvant in the treatment of infective and inflammatory diseases. We previously demonstrated that essential oil from Eucalyptus globulus (Labill.) (EO) stimulates in vitro the phagocytic activity of human monocyte-derived macrophages and counteracts the myelotoxicity induced by the chemotherapeutic 5-fluorouracil in immunocompetent rats. Here we characterize some mechanistic aspects underlying the immunostimulatory ability exerted by EO on macrophages. The internalization of fluorescent beads, fluorescent zymosan BioParticles, or apoptotic cancer cells was evaluated by confocal microscopy. Pro-inflammatory cytokine and chemokine release was determined by flow cytometry using the BD cytometric bead array. Receptor involvement in EO-stimulated phagocytosis was assessed using complement- or IgG-opsonized zymosan particles. The localization and expression of podosome components was analyzed by confocal microscopy and western blot. The main results demonstrated that: EO-induced activation of a macrophage is ascribable to its major component eucalyptol, as recently demonstrated for other cells of innate immunity; EO implements pathogen internalization and clearance by stimulating the complement receptor-mediated phagocytosis; EO stimulates podosome formation and increases the expression of podosome components. These results confirm that EO extract is a potent activator of innate cell-mediated immunity and thereby increase the scientific evidence supporting an additional property of this plant extract besides the known antiseptic and anti-inflammatory properties.

The regulatory processes involved in eosinophil trafficking into tissues are poorly understood; therefore, it is crucial to elucidate these mechanisms to advance the quality of clinical care for patients with eosinophil‐mediated diseases. The complex interactions between eosinophil integrin receptors and their corresponding ligands on the post‐capillary venules of the bronchial endothelium result in distinct modifications to the cytoskeletal architecture that occur in coordinated, temporally regulated sequences. The current study utilizes real‐time confocal microscopy and time‐based immunofluorescence staining to further characterize the effects of physiologically relevant fluid shear stress on this novel phenomenon of perfusion‐induced calcium response. We found that the mere perfusion of fluid over adhered human eosinophils induced a release of intracellular calcium observed in conjunction with changes in cell morphology (flattening onto the coverslip surface, an increase in surface area, and a loss of circularity), suggesting a previously unknown mechanosensing aspect of eosinophil migration out of the vasculature. Although changes in morphology and degree of calcium release remained consistent across varying perfusion rates, the latency of the response was highly dependent on the degree of shear stresses. Eosinophils were fixed post‐perfusion at specific timepoints for immunofluorescence staining to track proteins of interest over time. The distribution of proteins was diffuse throughout the cell prior to perfusion; however, they quickly localized to the periphery of the cell within 5 min. The actin cytoskeleton became markedly built up at the cell edges rapidly after stimulation, forming punctate dots by 4 min, suggesting a pivotal role in directed cell motility. [ABSTRACT FROM AUTHOR]

In the ageing skeleton, the balance of bone reconstruction could commonly be broken by the increasing of bone resorption and decreasing of bone formation. Consequently, the bone resorption gradually occupies a dominant status. During this imbalance process, osteoclast is unique cell linage act the bone resorptive biological activity, which is a highly differentiated ultimate cell derived from monocyte/macrophage. The erosive function of osteoclasts is that they have to adhere the bone matrix and migrate along it, in which adhesive cytoskeleton recombination of osteoclast is essential. In that, the podosome is a membrane binding microdomain organelle, based on dynamic actin, which forms a cytoskeleton superstructure connected with the plasma membrane. Otherwise, as the main adhesive protein, integrin regulates the formation of podosome and cytoskeleton, which collaborates with the various molecules including: c‐Cbl, p130Cas, c‐Src and Pyk2, through several signalling cascades cross talking, including: M‐CSF and RANKL. In our current study, we discuss the role of integrin and associated molecules in osteoclastogenesis cytoskeletal, especially podosomes, regulation and relevant signalling cascades cross talking. [ABSTRACT FROM AUTHOR]

A dynamic, properly organised actin cytoskeleton is critical for the production and haemostatic function of platelets. The Wiskott Aldrich Syndrome protein (WASp) and Actin-Related Proteins 2 & 3 Complex (Arp2/3 complex) are critical mediators of actin polymerisation and organisation in many cell types. In platelets and megakaryocytes, these proteins have been shown to be important for proper platelet production and function. The cortactin family of proteins (Cttn & HS1) are known to regulate WASp-Arp2/3-mediated actin polymerisation in other cell types and so here we address the role of these proteins in platelets using knockout mouse models. We generated mice lacking Cttn and HS1 in the megakaryocyte/platelet lineage. These mice had normal platelet production, with platelet number, size and surface receptor profile comparable to controls. Platelet function was also unaffected by loss of Cttn/HS1 with no differences observed in a range of platelet function assays including aggregation, secretion, spreading, clot retraction or tyrosine phosphorylation. No effect on tail bleeding time or in thrombosis models was observed. In addition, platelet actin nodules, and megakaryocyte podosomes, actin-based structures known to be dependent on WASp and the Arp2/3 complex, formed normally. We conclude that despite the importance of WASp and the Arp2/3 complex in regulating F-actin dynamics in many cells types, the role of cortactin in their regulation appears to be fulfilled by other proteins in platelets.

It has been widely accepted that periodontitis is a result of the host immune inflammatory response caused by various periodontal microorganisms leading to tissue degradation and bone resorption. One important mechanism of the inflammatory mediators present in periodontal tissue is the stimulation and formation of osteoclasts that are believed to be the major cell type responsible for bone resorption. This review article comprehensively explains the various molecular activities of osteoclasts and their role in periodontitis.

Matrix stiffness potently promotes the malignant phenotype in various biological contexts. Therefore, identification of gene expression to participate in mechanical force signals transduced into downstream biochemical signaling will contribute substantially to the advances in nasopharyngeal carcinoma (NPC) treatment. In the present study, we detected that cortactin (CTTN) played an indispensable role in matrix stiffness-induced cell migration, invasion, and invadopodia formation. Advances in cancer research have highlighted that dysregulated alternative splicing contributes to cancer progression as an oncogenic driver. However, whether WT-CTTN or splice variants (SV1-CTTN or SV2-CTTN) regulate matrix stiffness-induced malignant phenotype is largely unknown. We proved that alteration of WT-CTTN expression modulated matrix stiffness-induced cell migration, invasion, and invadopodia formation. Considering that splicing factors might drive cancer progression through positive feedback loops, we analyzed and showed how the splicing factor PTBP2 and TIA1 modulated the production of WT-CTTN. Moreover, we determined that high stiffness activated PTBP2 expression. Taken together, our findings showed that the PTBP2-WT-CTTN level increases upon stiffening and then promotes cell migration, invasion, and invadopodia formation in NPC., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRβ) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Multicellular organisms consist of cells and extracellular matrix (ECM). ECM creates a cellular microenvironment, and cells locally degrade the ECM according to their cellular activity. A major group of enzymes that modify ECM belongs to matrix metalloproteinases (MMPs) and play major roles in various pathophysiological events. ECM degradation by MMPs does not occur in all cellular surroundings but only where it is necessary, and cells achieve this by directionally secreting these proteolytic enzymes. Recent studies have indicated that such enzyme secretion is achieved by targeted vesicle transport along the microtubules, and several kinesin superfamily proteins (KIFs) have been identified as responsible motor proteins involved in the processes. This chapter discusses recent findings of the vesicle transport of MMPs and their roles., (Copyright © 2024. Published by Elsevier Inc.)

Cytoskeletal dysregulation forms an important aspect of many neurodegenerative diseases such as Alzheimer's disease. Cytoskeletal functions require the dynamic activity of the cytoskeletal proteins-actin, tubulin, and the associated proteins. One of such important phenomena is that of actin remodeling, which helps the cell to migrate, navigate, and interact with extracellular materials. Podosomes are complex actin-rich cytoskeletal structures, abundant in proteins that interact and degrade the extracellular matrix, enabling cells to displace and migrate. The formation of podosomes requires extensive actin networks and remodeling. Here we present a novel immunofluorescence-based approach to study actin remodeling in neurons through the medium of podosomes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recent study established the role of integrins in keratinocyte growth factor (KGF)-induced oral epithelial adhesion and rete peg elongation. However, how extracellular matrix (ECM) remodeling cooperates with the increased epithelial adhesion during rete peg elongation has yet to be determined. Podosomes are cell-matrix contact structures that combine several abilities, including adhesion and matrix degradation. In the present study, we identified podosome formation at the ventral side of human immortalized oral epithelial cells (HIOECs) upon KGF treatment. Moreover, podosomal components including integrin α6，β4，α3，β1 and MMP14 colocalized with the F-actin-cortactin complex and matrix degradation assays demonstrated the ability of the F-actin-cortactin complex to degrade matrix. Inhibition both of integrin subunits β4 and β1 with specific blocking antibodies and inhibition of Erk1/2 abrogated the KGF-induced podosome formation. Notably, knockdown of integrin subunits β4 and β1 with specific small interfering RNA (siRNA) downregulated the phosphorylation levels of Erk1/2. In contrast, inhibition of both Erk1/2 could upregulate the expression of integrin subunits β4 and β1. These results demonstrate that KGF induces podosome formation via integrin-Erk1/2 signaling in HIOECs, suggesting a novel mechanism by which integrins enhance oral epithelial adhesion and rete peg elongation. • KGF induces the assembly of podosome structures in HIOECs via KGFR. • Integrins and Erk1/2 play essential role in podosome formation within HIOECs. • Integrins are key mediators between KGF and Erk1/2 activation in HIOECs. [ABSTRACT FROM AUTHOR]

It has been widely accepted that periodontitis is a result of the host immune inflammatory response caused by various periodontal microorganisms leading to tissue degradation and bone resorption. One important mechanism of the inflammatory mediators present in periodontal tissue is the stimulation and formation of osteoclasts that are believed to be the major cell type responsible for bone resorption. This review article comprehensively explains the various molecular activities of osteoclasts and their role in periodontitis. [ABSTRACT FROM AUTHOR]

Frank‐Ter Haar syndrome (FTHS), Winchester syndrome (WS), and multicentric osteolysis, nodulosis, and arthropathy (MONA) are ultra‐rare multisystem disorders characterized by craniofacial malformations, reduced bone density, skeletal and cardiac anomalies, and dermal fibrosis. These autosomal recessive syndromes are caused by homozygous mutation or deletion of respectively SH3PXD2B (SH3 and PX Domains 2B), MMP14 (matrix metalloproteinase 14), or MMP2. Here, we give an overview of the clinical features of 63 previously reported patients with an SH3PXD2B, MMP14, or MMP2 mutation, demonstrating considerable clinical overlap between FTHS, WS, and MONA. Interestingly, the protein products of SH3PXD2B, MMP14, and MMP2 directly cooperate in collagen remodeling. We review animal models for these three disorders that accurately reflect the major clinical features and likewise show significant phenotypical similarity with each other. Furthermore, they demonstrate that defective collagen remodeling is central in the underlying pathology. As such, we propose a nosological revision, placing these SH3PXD2B, MMP14, and MMP2 related syndromes in a novel "defective collagen‐remodelling spectrum (DECORS)". In our opinion, this revised nosology better reflects the central role for impaired collagen remodeling, a potential target for pharmaceutical intervention. [ABSTRACT FROM AUTHOR]

• The significantly different ectoplasmic side of mononuclear-macrophage membrane. • The lipid bilayer and the lipid rafts on the protein-covered cytoplasmic side. • The characteristic podosome-associated structures were directly examined. • The independent localization of podosome domains and the lipid rafts was revealed. Mononuclear macrophages are important immune cells in the organisms. The complicated membrane structure underlying the diverse functions of mononuclear-macrophage has been largely unresolved. As a representative of monocyte-derived macrophages, the membrane structure of PMA differentiated THP-1 cells was comprehensively investigated by AFM-based single molecule approaches. The rugged ectoplasmic side of mononuclear-macrophage membrane are significantly different from erythrocytes and mammalian somatic cell membranes. But the smooth lipid bilayer and the branched lipid raft domains obtained by proteinase K and MβCD treatment of the protein-covered cytoplasmic side, are common characteristics among all the studied cell membranes. This discovery of distinct organization of membrane proteins on both sides of mononuclear-macrophage membranes provides additional evidence for the asymmetry of membrane structure. The podosome-associated structures of mononuclear-macrophage were directly examined, and the independent localization of podosome domains and the lipid rafts was verified by in situ AFM, giving new insight into this multifunctional organelle. [ABSTRACT FROM AUTHOR]

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. For instance, increased arterial stiffness can lead to atherosclerosis, while stiffening of the heart due to fibrosis can increase the chances of heart failure. Cells can sense the stiffness of the extracellular matrix through integrin adhesions and other mechanosensitive structures and in response to this initiate mechanosignalling pathways that ultimately change the cellular behaviour. Over the past decades, interest in mechanobiology has steadily increased and with this also our understanding of the molecular basis of mechanosensing and transduction. However, much of our knowledge about the mechanisms is derived from studies investigating focal adhesions in non-muscle cells, which are distinct in several regards from the cell–matrix adhesions in cardiomyocytes (costameres) or vascular smooth muscle cells (dense plaques or podosomes). Therefore, we will look here first at the evidence for mechanical sensing in the cardiovascular system, before comparing the different cytoskeletal arrangements and adhesion sites in cardiomyocytes and vascular smooth muscle cells and what is known about mechanical sensing through the various structures. [ABSTRACT FROM AUTHOR]

Abstract Extensive in vitro studies have described podosomes as actin-based structures at the plasma membrane, connecting the cell with its extracellular matrix and endowed with multiple capabilities. Contractile actin-myosin cables assemble them into a network that constitutes a multifaceted cellular superstructure taking different forms – with common characteristics – but manifesting different properties depending on the context of study. Their morphology and their role in cell functioning and behavior are therefore now apprehended in in vivo or in vitro situations relevant to physiological processes. We focus here on three of them, namely: macrophage migration, antigen presentation by dendritic cells and endothelial cell sprouting during angiogenesis to highlight the characteristics of podosomes and their functioning shaped by the microenvironment. Highlights • Podosomes are adhesion sites that connect the actin cytoskeleton to the extracellular matrix. • Podosomes construction and functioning are shaped by the microenvironment. • Podosomes are extracellular matrix degrading and mechanosensory organelles. • Actin networks orchestrate podosome architecture, dynamics and distribution. • Podosomes may be involved in a wide range of physiological processes in vivo. [ABSTRACT FROM AUTHOR]

Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where "phagocytic podosomes" have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes. [Display omitted] • Phagocytosing immune cells form F-actin-rich structures with podosomal characteristics. • Phagocytic podosomes are present in a variety of cell types and on several phagocytic targets. • Phagocytic podosomes share components, architecture and dynamics with matrix-attached podosomes. • Phagocytic podosomes enable close attachment to the phagocytic target. • Phagocytic podosomes are mechanosensitive and can contribute to particle disruption. [ABSTRACT FROM AUTHOR]

During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble podosomes during physiological angiogenesis in vivo. In the retina, podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in podosome areas. Moreover, Notch inhibition exacerbates podosome formation and collagen-IV loss. We propose that the localized proteolytic action of podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically.

Cells adhered to an external solid substrate are observed to exhibit rich dynamics of actin structures on the basal membrane, which are distinct from those observed on the dorsal (free) membrane. Here we explore the dynamics of curved membrane proteins, or protein complexes, that recruit actin polymerization when the membrane is confined by the solid substrate. Such curved proteins can induce the spontaneous formation of membrane protrusions on the dorsal side of cells. However, on the basal side of the cells, such protrusions can only extend as far as the solid substrate and this constraint can convert such protrusions into propagating wave-like structures. We also demonstrate that adhesion molecules can stabilize localized protrusions that resemble some features of podosomes. This coupling of curvature and actin forces may underlie the differences in the observed actin-membrane dynamics between the basal and dorsal sides of adhered cells.

Podosomes are multimolecular cytoskeletal structures that coordinate the migration of tissue-resident dendritic cells (DCs). They consist of a protrusive actin-rich core and an adhesive integrin-rich ring that contains adaptor proteins such as vinculin and zyxin. Individual podosomes are typically interconnected by a dense network of actin filaments giving rise to large podosome clusters. The actin density in podosome clusters complicates the analysis of podosomes by light microscopy alone. Here, we present an optimized procedure for performing super-resolution correlative light and electron microscopy (SR-CLEM) to study the organization of multiple proteins with respect to actin in podosome clusters at the ventral plasma membrane of DCs. We demonstrate that our procedure is suited to correlate at least three colors in super-resolution Airyscan microscopy with scanning electron microscopy (SEM). Using this procedure, we first reveal an intriguing complexity in the organization of ventral and radiating actin filaments in clusters formed by DCs which was not properly detected before by light microscopy alone. Next, we demonstrate a differential organization of vinculin and zyxin with respect to the actin filaments at podosomes. While vinculin mostly resides at sites where the actin filaments connect to the cell membrane, zyxin is primarily associated with filaments close to and on top of the core. Finally, we reveal a novel actin-based structure with SEM that connects closely associated podosome cores and which may be important for podosome topography sensing. Interestingly, these interpodosomal connections, in contrast to the radiating and ventral actin filaments appear to be insensitive to inhibition of actin polymerization suggesting that these pools of actin are not dynamically coupled. Together, our work demonstrates the power of correlating different imaging modalities for studying multimolecular cellular structures and could potentially be further exploited to study processes at the ventral plasma membrane of immune cells such as clathrin-mediated endocytosis or immune synapse formation.

Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo, or in collagen matrices in vitro. However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam3CSK4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE2, known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to migrate in 3D dense environments, which relies on their capacity to form podosomes independent of their maturation status, paving the way of further investigations on in vivo DC migration in dense tissues and its regulation during infections.