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Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages. [ABSTRACT FROM AUTHOR]

During immune responses against invading pathogenic bacteria, the cytoskeleton network enables macrophages to implement multiple essential functions. To protect the host from infection, macrophages initially polarize to adopt different phenotypes in response to distinct signals from the microenvironment. The extracellular stimulus regulates the rearrangement of the cytoskeleton, thereby altering the morphology and migratory properties of macrophages. Subsequently, macrophages degrade the extracellular matrix (ECM) and migrate toward the sites of infection to directly contact invading pathogens, during which the involvement of cytoskeleton‐based structures such as podosomes and lamellipodia is indispensable. Ultimately, macrophages execute the function of phagocytosis to engulf and eliminate the invading pathogens. Phagocytosis is a complex process that requires the cooperation of cytoskeleton‐enriched super‐structures, such as filopodia, lamellipodia, and phagocytic cup. This review presents an overview of cytoskeletal regulations in macrophage polarization, ECM degradation, migration, and phagocytosis, highlighting the pivotal role of the cytoskeleton in host defense against infection. [ABSTRACT FROM AUTHOR]

Background: Tau protein forms neurofibrillary tangles and becomes deposited in the brain during Alzheimer's disease (AD). Tau oligomers are the most reactive species, mediating neurotoxic and inflammatory activity. Microglia are the immune cells in the central nervous system, sense the extracellular Tau via various cell surface receptors. Purinergic P2Y12 receptor can directly interact with Tau oligomers and mediates microglial chemotaxis via actin remodeling. The disease-associated microglia are associated with impaired migration and express a reduced level of P2Y12, but elevate the level of reactive oxygen species and pro-inflammatory cytokines. Results: Here, we studied the formation and organization of various actin microstructures such as-podosome, filopodia and uropod in colocalization with actin nucleator protein Arp2 and scaffold protein TKS5 in Tau-induced microglia by fluorescence microscopy. Further, the relevance of P2Y12 signaling either by activation or blockage was studied in terms of actin structure formations and Tau deposits degradation by N9 microglia. Extracellular Tau oligomers facilitate the microglial migration via Arp2-associated podosome and filopodia formation through the involvement of P2Y12 signaling. Similarly, Tau oligomers induce the TKS5-associated podosome clustering in microglial lamella in a time-dependent manner. Moreover, the P2Y12 was evidenced to localize with F-actin-rich podosome and filopodia during Tau-deposit degradation. The blockage of P2Y12 signaling resulted in decreased microglial migration and Tau-deposit degradation. Conclusions: The P2Y12 signaling mediate the formation of migratory actin structures like- podosome and filopodia to exhibit chemotaxis and degrade Tau deposit. These beneficial roles of P2Y12 in microglial chemotaxis, actin network remodeling and Tau clearance can be intervened as a therapeutic target in AD. [ABSTRACT FROM AUTHOR]

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-ofplane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. [ABSTRACT FROM AUTHOR]

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrinmediated adhesion structures, such as sealing zones. [ABSTRACT FROM AUTHOR]

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells. • Podosomes are actin-rich structures used for degradation of extracellular matrix. • Dynamin 2 was implicated in podosome formation and function in various cells. • Pacsin 2 interacts with dynamin 2 and they co-localize to the podosomes. • Pacsin 2 and dynamin 2 cooperatively regulate formation/maturation of podosomes. [ABSTRACT FROM AUTHOR]

Macrophages are one of the major targets of human immunodeficiency virus 1 (HIV-1) and play crucial roles in viral dissemination and persistence during AIDS progression. Here, we reveal the dynamic podosome-mediated entry of HIV-1 into macrophages. Inhibition of podosomes prevented HIV-1 entry into macrophages, while stimulation of podosome formation promoted viral entry. Single-virus tracking revealed the temporal and spatial mechanism of the dynamic podosomemediated viral entry process. The core and ring structures of podosomes played complex roles in viral entry. The HIV coreceptor CCR5 was recruited to form specific clusters at the podosome ring, where it participated in viral entry. The podosome facilitated HIV-1 entry with a rotation mode triggered by dynamic actin. Our discovery of this novel HIV-1 entry route into macrophages, mediated by podosomes critical for cell migration and tissue infiltration, provides a new view of HIV infection and pathogenesis, which may assist in the development of new antiviral strategies. [ABSTRACT FROM AUTHOR]

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway. • Podosomes are actin-rich adhesion structures in the sealing zone of osteoclast. • Osteoclasts sense both the RGD and stiffness of calcified collagen by podosomes. • Osteoclast differentiation is associated with Src pathway in podosome. [ABSTRACT FROM AUTHOR]

Migrating precursors of epidermal sensory neurons are a relatively rare cell type in developing amphioxus. Serial blockface scanning electron microscopy (SBSEM) was used here to locate these cells and reconstruct them in three dimensions in the context of the surrounding tissues. The reconstructions showed that, in contrast to some earlier reports, these cells are not located in an appreciable subepidermal space while they are crawling dorsally up the flank of the animal. Instead, they migrate while tightly sandwiched between overlying epidermal cells and the underlying basal lamina. The cytoplasmic region at the leading edge of the crawling cell has some characteristics of a large podosome, an F‐actin‐rich organelle with prominent roles in adhesion, matrix degradation and cell migration. The discussion includes a comparison of the precursors of epidermal sensory cells of amphioxus with migrating neurogenic cells from vertebrate placodes. [ABSTRACT FROM AUTHOR]

• Re -epithelialization is a critical step in the skin wound healing response. • Keratinocytes deposit extracellular matrix laminin 332 along their path while migrating. • Matrix metalloproteinases-9 and −14 exert their proteolytic activity in epithelial podosomes in primary keratinocytes. • The laminin α3 chain globular domains 4 and 5 recruit and merge syndecan-1 and CD44 in podosomes. • The proteoglycan receptor syndecan-1 regulates epithelial podosome formation and proteolytic activity. Re -epithelialization describes the resurfacing of a skin wound with new epithelium. In response to various stimuli including that of growth factors, cytokines and extracellular matrix (ECM), wound edge epidermal keratinocytes undergo cytoskeleton rearrangements compatible with their motile behavior and develop protrusive adhesion contacts. Matrix metalloproteinases (MMP) expression is crucial for proper cell movement and ECM remodeling; however, their deposition mechanism is unknown in keratinocytes. Here, we show that similar to cytokine IL-1ß, the precursor laminin 332 pro-migratory fragment G45 induces expression of the MMP-9 pro-enzyme, which together with MMP-14, further exerts its proteolytic activity within epithelial podosomes. This event strictly depends on the expression of the proteoglycan receptor syndecan-1 that was found in a ring surrounding the podosome core, co-localised with CD44. Our findings uncover that by directly recruiting both syndecan-1 and CD44, the laminin-332 G45 domain plays a major role in regulating mechanisms underlying keratinocyte / ECM remodeling during wound repair. [ABSTRACT FROM AUTHOR]

Actin remodeling plays an essential role in diverse cellular processes such as cell motility, vesicle trafficking or cytokinesis. The scaffold protein and actin nucleation promoting factor Cortactin is present in virtually all actin-based structures, participating in the formation of branched actin networks. It has been involved in the control of endocytosis, and vesicle trafficking, axon guidance and organization, as well as adhesion, migration and invasion. To migrate and invade through three-dimensional environments, cells have developed specialized actin-based structures called invadosomes, a generic term to designate invadopodia and podosomes. Cortactin has emerged as a critical regulator of invadosome formation, function and disassembly. Underscoring this role, Cortactin is frequently overexpressed in several types of invasive cancers. Herein we will review the roles played by Cortactin in these specific invasive structures. [ABSTRACT FROM AUTHOR]

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well‐characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross‐talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO‐1 and ZO‐2 localize at podosomes in response to phorbol‐12‐myristate‐13‐acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO‐1 and ZO‐2 are relocated from a pre‐existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases. [ABSTRACT FROM AUTHOR]

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c‐Src (c‐Src−/−) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk‐associated substrate [Cas]) as one of c‐Src downstream molecule and osteoclast‐specific p130Cas‐deficient (p130CasΔOCL−/−) mice also exhibited a similar phenotype to c‐Src−/− mice, indicating that the c‐Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c‐Src−/− or p130CasΔOCL−/− mice with wild‐type (WT) osteoclasts to identify downstream molecules of c‐Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c‐Src−/− and p130CasΔOCL−/− osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c‐Src−/− and p130CasΔOCL−/− osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL−/− osteoclasts but not c‐Src−/− osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). Significance of the study: We previously showed that the c‐Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c‐Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild‐type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast‐specific p130Cas‐deficient (p130CasΔOCL−/−) osteoclasts but not c‐Src−/− osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas. [ABSTRACT FROM AUTHOR]

Podosomes are dynamic adhesion structures formed constitutively by macrophages, dendritic cells and osteoclasts and transiently in a wide variety of cells, such as endothelial cells and megakaryocytes. They mediate numerous functions, including cell-matrix adhesion, extracellular matrix degradation, mechanosensing and cell migration. Podosomes present as micron-sized F-actin cores surrounded by an adhesive ring of integrins and integrin-actin linkers, such as talin and vinculin. In this Review, we highlight recent research that has considerably advanced our understanding of the complex architecture-function relationship of podosomes by demonstrating that the podosome ring actually consists of discontinuous nanoclusters and that the actin network in between podosomes comprises two subsets of unbranched actin filaments, lateral and dorsal podosome-connecting filaments. These lateral and dorsal podosomeconnecting filaments connect the core and ring of individual podosomes and adjacent podosomes, respectively. We also highlight recent insights into the podosome cap as a novel regulatory module of actomyosin-based contractility. We propose that these newly identified features are instrumental for the ability of podosomes to generate protrusion forces and to mechanically probe their environment. Furthermore, these new results point to an increasing complexity of podosome architecture and have led to our current view of podosomes as autonomous force generators that drive cell migration. [ABSTRACT FROM AUTHOR]

Background: Podosomes are membrane‐bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion. Methods: This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA‐MB‐231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP)  mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far‐western blotting. Results: This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short‐hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc‐Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far‐western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells. Conclusions: Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy. [ABSTRACT FROM AUTHOR]

Aims: Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2 either stimulatory or repressive influence on angiogenesis. Our findings unveil the mechanism of Nrf2-dependent vessel formation, which reaches beyond transactivation of gene expression and reconciles previous discrepancies. Results: We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system. [ABSTRACT FROM AUTHOR]

Successful pregnancy requires normal placentation, which largely depends on the tight regulation of proliferation, invasion, and migration of trophoblast cells. Abnormal functioning of trophoblast cells may cause failure of uterine spiral artery remodeling, which may be related to pregnancy-related disorders, such as preeclampsia. Here, we reported that an actin-binding protein, α-actinin (ACTN)4, was dysregulated in placentas from early onset preeclampsia. Moreover, knockdown of ACTN4 markedly inhibited trophoblast cell proliferation by reducing AKT membrane translocation. Furthermore, E-cadherin regulated ACTN4 and ß-catenin colocalization on trophoblast cell podosomes, and ACTN4 down-regulation suppressed the E-cadherin-induced cell invasion increase via depolymerizing actin filaments. Moreover, loss of ACTN4 recapitulated a number of the features of human preeclampsia. Therefore, our data indicate that ACNT4 plays a role in trophoblast function and is required for normal placental development. [ABSTRACT FROM AUTHOR]

Abstract Macrophage functions in the immune response depend on their ability to infiltrate tissues and organs. The penetration between and within the tissues requires degradation of extracellular matrix (ECM), a function performed by the specialized, endopeptidase- and actin filament- rich organelles located at the ventral surface of macrophage, called the podosomes. Podosome formation requires local inhibition of small GTPase RhoA activity, and depends on Rac 1/Rho guanine nucleotide exchange factor 7, β-PIX and its binding partner the p21-activated kinase (PAK-1). The activity of RhoA and Rac 1 is in turn regulated by mTOR/mTORC2 pathway. Here we showed that a fungus metabolite Fingolimod (FTY720, Gilenya), which is clinically approved for the treatment of multiple sclerosis, down-regulates Rictor, which is a signature molecule of mTORC2 and dictates its substrate (actin cytoskeleton) specificity, down-regulates RhoA, up-regulates PAK-1, and causes amplification of podosomes in mouse peritoneal macrophages. [ABSTRACT FROM AUTHOR]

Abstract Transgelin-1 (SM22α) has been recognized as a smooth muscle marker and a tumor suppressor, but many details of the working mechanisms remain unclear. Transgelin-1 belongs to the calponin family of actin-binding proteins with an N-terminal calponin homology domain (CH-domain) and a C-terminal calponin-like module (CLIK23). Here, we demonstrate that transgelin-1 interacts with actin stress fibers and podosomes in smooth muscle cells via its type-3 CH-domain, while CLIK23 is dispensable for the binding to the actin structures. We further suggest that the EF-hand motif in transgelin-1 contributes to proper folding of the CH-domain and in turn to the interaction with the actin structures. These results are in contrast to the ones reported in in vitro studies that demonstrated CLIK23 was necessary for the transgelin-1–actin binding, while the CH-domain was not. Besides, within cells, transgelin-1 phosphorylation at Ser181 in CLIK23 did not affect its colocalization with the actin structures, while the same phosphorylation was reported in in vitro studies to negatively regulate actin binding. Thus, our results suggest the molecular basis of intracellular interaction between transgelin-1 and actin, distinct from that in vitro. The actin binding capability intrinsic to CLIK23 may not appear within cells probably because of the weaker competition for actin binding compared to other actin binding molecules. Highlights • Transgelin-1 interacts with stress fibers and podosomes via its type-3 CH-domain. • EF-hand affects the transgelin-1–actin interaction cooperatively with CLIK23. • Phosphorylation in CLIK23 does not affect the transgelin-1–actin interaction. • Increased numbers of CLIK23 do not compensate for the deletion of the CH-domain. [ABSTRACT FROM AUTHOR]

The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro , two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering. [ABSTRACT FROM AUTHOR]

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. UponRANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Osteoclasts form a specialized cell-matrix adhesion structure, known as the "sealing zone", during bone resorption. The sealing zone is a dynamic actin-rich structure that defines the resorption area of the bone. The detailed dynamics and fine structure of the sealing zone have been elusive. Osteoclasts plated on glass do not form a sealing zone, but generate a separate supra-molecular structure called the "podosome belt". Podosomes are integrin-based adhesion complexes involved in matrix adhesion, cell migration, matrix degradation, and mechanosensing. Invadopodia, podosome-like protrusions in cancer cells, are involved in cell invasion into other tissues by promoting matrix degradation. Both podosomes and invadopodia exhibit actin pattern transitions during maturation. We previously found that Arp2/3-dependent actin flow occurs in all observed assembly patterns of podosomes in osteoclasts on glass. It is known that the actin wave in Dictyostelium cells exhibits a similar pattern transition in its evolution. Because of significant advances in our understanding regarding the mechanism of podosomes/invadopodia formation over the last decade, we revisited the structure and function of the sealing zone in this review, highlighting the possible involvement of self-organized actin waves in the organogenesis of the sealing zone. [ABSTRACT FROM AUTHOR]

Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-β plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling. [Display omitted] • The extracellular Tau regulates the Arp2 and TKS5-associated podosomes and filopodia formation. • Tau aggregates can act as dual inducers of P2Y12R signaling and the podosomes and filopodia-associated actin remodeling. • The activation of actin remodeling pathways and associated proteins signaling by Tau aggregates evoke new insights of beneficial activation of microglia in disease condition. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated bone-resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin-based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho-specific guanine-nucleotide exchange factor (Rho-GEF) Plekhg5 as an up-regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end-binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5-depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5-depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts. [ABSTRACT FROM AUTHOR]

At the early stage of atherosclerosis, neointima is formed due to the migration of vascular smooth muscle cells (VSMCs) from the media to the intima. VSMCs are surrounded by highly adhesive 3D matrices. They take specific strategies to cross various 3D matrices in the media, including heterogeneous collagen and mechanically strong basement membrane. Migration of VSMCs is potentially caused by biomechanical mechanism. Most in vitro studies focus on cell migration on 2D substrates in response to biochemical factors. How the cells move through 3D matrices under the action of mechanosensing machineries remains unexplored. In this review, we propose that several interesting tension-dependent machineries act as 'tractor'-posterior myosin II accumulation, and 'wrecker'-anterior podosome maintaining, to power VSMCs ahead. VSMCs embedded in 3D matrices may accumulate a minor myosin II isoform, myosin IIB, at the cell rear. Anisotropic myosin IIB distribution creates cell rear, polarizes cell body, pushes the nucleus and reshapes the cell body, and cooperates with a uniformly distributed myosin IIA to propel the cell forward. On the other hand, matrix digestion by podosome further promote the migration when the matrix becomes denser. Actomyosin tension activates Src to induce podosome in soft 3D matrices and retain the podosome integrity to steadily digest the matrix. [ABSTRACT FROM AUTHOR]

SLC4A2 belongs to the Na+-independent solute carrier family 4 (SLC4) of anion exchangers, which regulate electroneutral exchange of Cl− for HCO 3 − and mediate intra- and extra-cellular pH, chloride concentration and cell volume. Slc4a2 also participates in gastric acid secretion, spermatogenesis and osteoclastogenesis. During osteoclast differentiation, Slc4a2 is exclusively expressed at the contra-lacunar membrane and is up-regulated with osteoclast maturation. Bi-allelic Slc4a2 loss-of-function mutations have been known to cause osteopetrosis in mice and cattle, but not in human. Recently, we have identified bi-allelic pathogenic variants in SLC4A2 in a patient affected by osteopetrosis with severe renal insufficiency, suggesting SLC4A2 deficiency causes a new type of autosomal recessive osteopetrosis (osteopetrosis, Ikegawa type). In this article, we review the advances in exploring the multiple functions of SLC4A2 with emphasis on its roles in osteoclast. Our review would contribute to understanding of the phenotypic spectrum and the pathomechanism of SLC4A2 -associated osteopetrosis. • SLC4A2 deficiency causes osteopetrosis in cattle, mice and humans. • SLC4A2 is involved in acid-base balancing machinery of osteoclasts. • SLC4A2 regulates osteoclast differentiation, apoptosis and maturation related to dynamic organization of podosomes. [ABSTRACT FROM AUTHOR]

Bone resorption by osteoclasts is mediated by a typical adhesion structure called the sealing zone or actin ring, whose architecture is based on a belt of podosomes. The molecular mechanisms driving podosome organization into superstructures remain poorly understood to date, in particular at the osteoclast podosome belt. We performed proteomic analyses in osteoclasts and found that the adaptor protein tensin 3 is a partner of Dock5, a Rac exchange factor necessary for podosome belt formation and bone resorption. Expression of tensin 3 and Dock5 concomitantly increase during osteoclast differentiation. These proteins associate with the osteoclast podosome belt but not with individual podosomes, in contrast to vinculin. Super-resolution microscopy revealed that, even if they colocalize in the x-y plane of the podosome belt, Dock5 and tensin 3 differentially localize relative to vinculin in the z-axis. Tensin 3 increases Dock5 exchange activity towards Rac, and suppression of tensin 3 in osteoclasts destabilizes podosome organization, leading to delocalization of Dock5 and a severe reduction in osteoclast activity. Our results suggest that Dock5 and tensin 3 cooperate for osteoclast activity, to ensure the correct organization of podosomes. [ABSTRACT FROM AUTHOR]

Bone resorption is an important cellular function in skeletal development and remodeling of the adult skeleton. Most of the pathological bone disease conditions like osteoporosis reflect increased osteoclast activity; hence, increased bone resorption. Researchers have unraveled most of the intracellular mechanisms responsible for osteoclast bone-resorbing activity in last few decades. Therefore, understanding the fundamentals of osteoclast-induced bone resorption and the cytokines and other substances that modulate the osteoclast activity unequivocally provide insights into the development of drugs to ameliorate pathological bone diseases with enhanced bone resorption. The aim of this review is to examine the literature on osteoclast function and bone-resorbing activity. [ABSTRACT FROM AUTHOR]

Invadosomes are actin-based structures involved in extracellular matrix degradation. Invadosomes is a term that includes podosomes and invadopodia, which decorate normal and tumour cells, respectively. They are mainly organised into dots or rosettes, and podosomes and invadopodia are often compared and contrasted. Various internal or external stimuli have been shown to induce their formation and/or activity. In this Commentary, we address the impact of the microenvironment and the role of matrix receptors on the formation, and dynamic and degradative activities of invadosomes. In particular, we highlight recent findings regarding the role of type I collagen fibrils in inducing the formation of a new linear organisation of invadosomes. We will also discuss invadosome plasticity more generally and emphasise its physio-pathological relevance. [ABSTRACT FROM AUTHOR]

We studied podosome formation and development in activated monocytes (THP1) at ICAM1 (intercellular adhesion molecule 1) nanopatterns of circular and ring-shaped domains and show that cellular binding to a preclustered ICAM1 nanopattern requires ligand patches of at least 200 nm (corresponding to 14 or more integrins). Podosome-like adhesion formation depends on the structure of the ligand pattern under the developing podosome with larger single domains promoting adhesion in a single patch and multiple smaller domains allowing podosome formation by integration of at least 2 smaller domains on either side of the podosome core. Maturation to rosette structures and recruitment of proteases were only observed with macroscopic ICAM1 presentation. [ABSTRACT FROM AUTHOR]

Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 ;µg/ml) or control BSA (100 ;µg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs. [ABSTRACT FROM AUTHOR]

Osteoclasts are highly polarized cells from both morphological and functional points of view. Using quick-freeze, rotary-replication methods combined with cell-shearing, we clarified the variability of cytoplasmic surface of the polarized membranes of osteoclasts seeded on apatite. As to the organization of actin filaments and clathrin sheets, we confirmed almost the same ventral membrane specializations of osteoclasts on apatite as seen on glass plates. The organized actin filaments and membrane-associated particles supported the ruffled border membranes. Inside the actin sealing zone, membrane specializations were not always occupied with the ruffled border but also with other types of membranes. Some osteoclasts formed an actin ring but lacked the ruffled border projections. We report a unique and distinctive membrane modification of apatite-attached osteoclasts, i.e., the presence of dense aggregates of membrane-associated particles and related structures not found in the osteoclasts seeded on glass plates. Actin filament polarity in the podosomes was determined by decoration with myosin S1. The actin filament polarity within podosome appears to be oriented predominantly with its barbed ends toward the core, whereas the interconnecting F-actin appears to be mixed oriented. Two different types of clathrin plaques displayed different distributions: clathrin-dependent endocytosis was observed in the ruffled border regions, whereas flat clathrin sheets were found in the leading edge of lamellipodia and near podosomes. The clathrin sheets adhered to the apatite surface tightly on the ventral membranes overlaying the resorption lacunae. All these membrane specializations as mentioned above may indicate the functional variability of osteoclasts seeded on apatite. [ABSTRACT FROM AUTHOR]

Podosomes are dynamic degrading devices present in myeloid cells among other cell types. They consist of an actin core with associated regulators, surrounded by an adhesive ring. Both fascin and cortactin are known constituents but the role of fascin actin bundling is still unclear and cortactin research rather focuses on its homologue hematopoietic lineage cell-specific protein-1 (HS1). A fascin nanobody (FASNb5) that inhibits actin bundling and a cortactin nanobody (CORNb2) specifically targeting its Src-homology 3 (SH3) domain were used as unique tools to study the function of these regulators in podosome dynamics in both THP-1 macrophages and dendritic cells (DC). Upon intracellular FASNb5 expression, the few podosomes present were aberrantly stable, long-living and large, suggesting a role for fascin actin bundling in podosome turnover and disassembly. Fascin modulates this by balancing the equilibrium between branched and bundled actin networks. In the presence of CORNb2, the few podosomes formed show disrupted structures but their dynamics were unaffected. This suggests a role of the cortactin SH3 domain in podosome assembly. Remarkably, both nanobody-induced podosome-losses were compensated for by focal adhesion structures. Furthermore, matrix degradation capacities were altered and migratory phenotypes were lost. In conclusion, the cortactin SH3 domain contributes to podosome assembly while fascin actin bundling is a master regulator of podosome disassembly in THP-1 macrophages and DC. [ABSTRACT FROM AUTHOR]

Mesenchymal cells employ actin-based membrane protrusions called podosomes and invadopodia for cross-tissue migration during normal human development such as embryogenesis and angiogenesis, and in diseases such as atherosclerosis plaque formation and cancer cell metastasis. The Akt isoforms, downstream effectors of phosphatidylinositol 3 kinase (PI3K), play crucial roles in cell migration and invasion, but their involvement in podosome formation and cell invasion is not known. In this study, we have used Akt1 and/or Akt2 knockout mouse embryonic fibroblasts and Akt3-targeted shRNA to determine the roles of the three Akt isoforms in Src and phorbol ester-induced podosome formation, and extracellular matrix (ECM) digestion. We found that deletion or knockdown of Akt1 significantly reduces Src-induced formation of podosomes and rosettes, and ECM digestion, while suppression of Akt2 has little effect. In contrast, Akt3 knockdown by shRNA increases Src-induced podosome/rosette formation and ECM invasion. These data suggest that Akt1 promotes, while Akt3 suppresses, podosome formation induced by Src, and Akt2 appears to play an insignificant role. Interestingly, both Akt1 and Akt3 suppress, while Akt2 enhances, phorbol ester-induced podosome formation. These data show that Akt1, Akt2 and Akt3 play different roles in podosome formation and ECM invasion induced by Src or phorbol ester, thus underscoring the importance of cell context in the roles of Akt isoforms in cell invasion. [ABSTRACT FROM AUTHOR]

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca 2+ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30 ng/mL) and M-CSF (25 ng/mL). The cells were pretreated with Bapta-AM (5 μM), U0126 (5 μM), or SB202190 (10 μM) for 30 min, followed by 40 ng/mL OPG for 3 h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca 2+ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca 2+ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix. [ABSTRACT FROM AUTHOR]

Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP-WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP-WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP-WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor. [ABSTRACT FROM AUTHOR]

The kinesin KIF1C is known to regulate podosomes, actin-rich adhesion structures that remodel the extracellular matrix during physiological processes. Here, we show that KIF1C is a player in the podosome-inducing signaling cascade. Upon induction of podosome formation by protein kinase C (PKC), KIF1C translocation to the cell periphery intensifies and KIF1C accumulates both in the proximity of peripheral microtubules that show enrichment for the plus-tip-associated proteins CLASPs and around podosomes. Importantly, without CLASPs, both KIF1C trafficking and podosome formation are suppressed. Moreover, chimeric mitochondrially targeted CLASP2 recruits KIF1C, suggesting a transient CLASP-KIF1C association. We propose that CLASPs create preferred microtubule tracks for KIF1C to promote podosome induction downstream of PKC. [ABSTRACT FROM AUTHOR]

Podosomes and invadopodia, collectively known as invadosomes, are specialized cell–matrix contacts with an inherent ability to degrade extracellular matrix. Their occurrence in either normal (podosomes) or cancer cells (invadopodia) is thus traditionally associated with cell invasiveness and tissue remodelling. These specialized micro-domains of the plasma membrane are characterized by enrichment of F-actin, cortactin and metalloproteases. Recent developments in the field show that, under some circumstances, vascular endothelial cells (ECs) can be induced to form this kind of peculiar structures. Cultured ECs contain either 0.5–1-μm-wide individual podosomes or 5 to 10 μm wide ring-like clusters of podosomes (podosome rosettes). The formation of individual podosomes or podosome rosettes in ECs can be induced by soluble factors, such as TGFβ, VEGF, TNFα or pharmacological agents, such as phorbol esters. Recently, the evidence of the existence of such structures in vascular endothelium has been provided by ex vivo observation. Endothelial podosome rosettes have recently been functionally linked to arterial remodelling and sprouting angiogenesis. Concerted efforts aim now at confirming the relevance of endothelial podosomes in these patho-physiological processes in vivo . In the current review, we will introduce some general considerations regarding ECs in the vascular system. From there on, we will review the various EC types where podosomes have been described and the state-of-art knowledge hitherto generated regarding endothelial podosome features. [ABSTRACT FROM AUTHOR]

We recently identified a novel role for podosomes in antigen sampling. Podosomes are dynamic cellular structures that consist of point-like concentrations of actin surrounded by integrins and adaptor proteins such as vinculin and talin. Podosomes establish cellular contact with the extracellular matrix (ECM) and facilitate cell migration via ECM degradation. In our recent paper, we studied podosomes of human dendritic cells (DCs), major antigen presenting cells (APC) that take-up, process, and present foreign antigen to naive T-cells. We employed gelatin-impregnated porous polycarbonate filters to demonstrate that the mechanosensitive podosomes of DCs selectively localize to regions of low-physical resistance such as the filter pores. After degradation of the gelatin, podosomes increasingly protrude into the lumen of these pores. These protrusive podosome-derived structures contain several endocytic and early endosomal markers such as clathrin, Rab5, and VAMP3, and, surprisingly, also contain C-type lectins, a type of pathogen recognition receptors (PRRs). Finally, we performed functional uptake experiments to demonstrate that these PRRs facilitate uptake of antigen from the opposite side of the filter. Our data provide mechanistic insight in how dendritic cells sample for antigen across epithelial barriers for instance from the lumen of the lung and gut. [ABSTRACT FROM PUBLISHER]

The occurrence of invadopodia has been, since its characterization, a hallmark of cancerous cell invasion and metastasis. These structures are now the subject of a controversy concerning their cellular function, molecular regulation, and assembly. The terms invadopodia and podosomes have been used interchangeably since their discovery back in 1980. Since then, these phenotypes are now more established and accepted by the scientific community as vital structures for 3D cancer cell motility. Many characteristics relating to invadopodia and podosomes have been elucidated, which might prove these structures as good targets for metastasis treatment. In this review, we briefly review the actin reorganization process needed in most types of cancer cell motility. We also review the important characteristics of invadopodia, including molecular components, assembly, markers, and the signaling pathways, providing a comprehensive model for invadopodia regulation. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Bone is a dynamic tissue constantly renewed through a regulated balance between bone formation and resorption. excessive bone degradation by osteoclasts leads to pathological decreased bone density characteristic of osteolytic diseases such as post-menopausal osteoporosis or bone metastasis. Osteoclasts are multinucleated cells derived from hematopoietic stem cells via a complex differentiation process. Their unique ability to resorb bone is dependent on the formation of the actin-rich sealing zone. within this adhesion structure, the plasma membrane differentiates into the ruffled border where protons and proteases are secreted to demineralize and degrade bone, respectively. On the bone surface, mature osteoclasts alternate between stationary resorptive and migratory phases. These are associated with profound actin cytoskeleton reorganization, until osteoclasts die of apoptosis. in this review, we highlight the role of Rho GTPases in all the steps of osteoclasts differentiation, function and death and conclude on their interest as targets for treatment of osteolytic pathologies. [ABSTRACT FROM AUTHOR]

Cell motility is defined as cell movement in the three-dimensional space leading to repositioning of the cell. Atypical protein kinase C (aPKC, including ζ and λ/ι) are a subfamily of PKC. Different from classic PKC and novel PKC, the activation of atypical PKC is not dependent on diacylglycerol or calcium. PKCζ can be activated by lipid components, such as phosphatidylinositols, phosphatidic acid, arachidonic acid, and ceramide. Both phosphatidylinositol (3,4,5)-trisphosphate and PDK1 are necessary for the complete and stable activation of PKCζ. Atypical PKC is involved in the regulation of cell polarization, directional sensing, formation of filopodia, and cell motility. It is essential for migration and invasion of multiple cancer cell types. Particularly, atypical PKC has been found in the regulation of the motility of hematopoietic cells. It also participates in the regulation of proteolytic activity of podosomes and invadopodia. It has been found that atypical PKC can work coordinately with other PKC subfamily members and other signaling pathways. Research on the roles of atypical PKC in cell motility may lead to new therapeutic strategies for cancer and other diseases. [ABSTRACT FROM AUTHOR]

Abstract: Recently developed super-resolution techniques in optical microscopy have pushed the length scale at which cellular structure can be observed down to tens of nanometres. A wide array of methods have been described that fall under the umbrella term of super-resolution microscopy and each of these methods has different requirements for acquisition speed, experimental complexity, fluorophore requirements and post-processing of data. For example, experimental complexity can be decreased by using a standard widefield microscope for acquisition, but this requires substantial processing of the data to extract the super-resolution information. These powerful techniques are bringing new insights into the nanoscale structure of sub-cellular assemblies such as podosomes, which are an ideal system to observe with super-resolution microscopy as the structures are relatively thin and they form and dissociate over a period of several minutes. Here we discuss the major classes of super-resolution microscopy techniques, and demonstrate their relative performance by imaging podosomes. [Copyright &y& Elsevier]

Remodeling of extracellular matrix (ECM) is a fundamental cell property that allows cells to alter their microenvironment and move through tissues. Invadopodia and podosomes are subcellular actin-rich structures that are specialized for matrix degradation and are formed by cancer and normal cells, respectively. Although initial studies focused on defining the core machinery of these two structures, recent studies have identified inputs from both growth factor and adhesion signaling as crucial for invasive activity. This Commentary will outline the current knowledge on the upstream signaling inputs to invadopodia and podosomes and their role in governing distinct stages of these invasive structures. We discuss invadopodia and podosomes as adhesion structures and highlight new data showing that invadopodia-associated adhesion rings promote the maturation of already-formed invadopodia. We present a model in which growth factor stimulation leads to phosphoinositide 3-kinase (PI3K) activity and formation of invadopodia, whereas adhesion signaling promotes exocytosis of proteinases at invadopodia. [ABSTRACT FROM AUTHOR]

Neuromuscular junctions (NMJs) in mammalian skeletal muscle undergo a postnatal topological transformation from a simple oval plaque to a complex branched structure. We previously showed that podosomes, actin-rich adhesive organelles, promote the remodeling process, and demonstrated a key role for one podosome component, LL5β. To further investigate molecular mechanisms of postsynaptic maturation, we purified LL5β-associated proteins from myotubes and showed that three regulators of the actin cytoskeleton - Amotl2, Asef2 and Flii - interact with LL5β. These and other LL5β-interacting proteins are associated with conventional podosomes in macrophages and podosome-like invadopodia in fibroblasts, strengthening the close relationship between synaptic and non-synaptic podosomes. We then focused on Amotl2, showing that it is associated with synaptic podosomes in cultured myotubes and with NMJs in vivo. Depletion of Amotl2 in myotubes leads to increased size of synaptic podosomes and corresponding alterations in postsynaptic topology. Depletion of Amotl2 from fibroblasts disrupts invadopodia in these cells. These results demonstrate a role for Amotl2 in synaptic maturation and support the involvement of podosomes in this process. [ABSTRACT FROM AUTHOR]

Highlights: [•] WASP family proteins, including WASPs and WAVEs, are the best characterized nucleation promoting factors, i.e. the activators of the Arp2/3 complex, and new members are still being identified. [•] WASP is a prototype of the WASP family. Very recently, the loss of WIP, a WASP chaperon, led to development of a WAS like genetic disorder. [•] WAVEs1–3 markedly differ in their tissue distribution. In addition, WAVE2 may selectively prefer IRSp53 as a ligand, potentially its unique indispensable role in lamellipodia of mammalian cells. [•] The essential roles of WASP/N-WASP in the development of podosomes, invadopodia, and the related structures have been well recognized. [•] N-WASP reportedly is critical in vesicular trafficking. So is a new WASP family member WASH. [ABSTRACT FROM AUTHOR]

Abstract: Podosomes are adhesion structures characteristic of the myeloid cell lineage, encompassing osteoclasts, dendritic cells and macrophages. Podosomes are actin-based structures that are dynamic and capable of self-organization. In particular in the osteoclast, podosomes densely pack into a thick ring called the sealing zone. This adhesion structure is typical of osteoclasts and necessary for the resorption of the bone matrix. We thought to explore in more details the role of podosomes during osteoclast differentiation and migration. To this end, we made from soft to stiff substrates that had not been functionalized with extracellular matrix proteins. Such substrates did not support podosome formation in osteoclasts. With such devices, we could show that integrin activation was sufficient to drive podosome assembly, in a substrate stiffness independent fashion. We additionally report here that osteoclast differentiation is a podosome-independent process. Finally, we show that osteoclasts devoid of podosomes can migrate efficiently. Our study further illustrates the great capacity of myeloid cells to adapt to the different environments they encounter during their life cycle. [Copyright &y& Elsevier]