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Abstract 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.

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]

Abstract 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.

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]

Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility.Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL−/− mice, using Airyscan confocal microscopy.Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates.Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.

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]

Abstract 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.

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.

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]

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors. [ABSTRACT FROM AUTHOR]

At vertebrate neuromuscular junctions (NMJs), the synaptic basal lamina contains different extracellular matrix (ECM) proteins and synaptogenic factors that induce and maintain synaptic specializations. Here, we report that podosome-like structures (PLSs) induced by ubiquitous ECM proteins regulate the formation and remodeling of acetylcholine receptor (AChR) clusters via focal ECM degradation. Mechanistically, ECM degradation is mediated by PLS-directed trafficking and surface insertion of membrane-type 1 matrix metalloproteinase (MT1-MMP) to AChR clusters through microtubule-capturing mechanisms. Upon synaptic induction, MT1-MMP plays a crucial role in the recruitment of aneural AChR clusters for the assembly of postsynaptic specializations. Lastly, the structural defects of NMJs in embryonic MT1-MMP-/- mice further demonstrate the physiological role of MT1-MMP in normal NMJ development. Collectively, this study suggests that postsynaptic MT1-MMP serves as a molecular switch to synaptogenesis by modulating local ECM environment for the deposition of synaptogenic signals that regulate postsynaptic differentiation at developing NMJs.

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 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]

The aim of this study was to elucidate the role of the zipper-like structure (ZLS), a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3). Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116–127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.

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]

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC. [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]

Arf GTPase-activating proteins (Arf GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf GAPs. Arf GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.

Proinflammatory cytokine production, cell chemotaxis, and osteoclastogenesis can lead to inflammatory bone loss. Previously, we showed that sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor, regulates inflammatory cytokine production and osteoclastogenesis. However, the signaling pathways regulated by S1PR2 in modulating inflammatory bone loss have not been elucidated. Herein, we demonstrated that inhibition of S1PR2 by a specific S1PR2 antagonist (JTE013) suppressed phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB) induced by an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans, and inhibited the release of IL-1β, IL-6, TNF-α, and S1P in murine bone marrow cells. In addition, shRNA knockdown of S1PR2 or treatment by JTE013 suppressed cell chemotaxis induced by bacteria-stimulated cell culture media. Furthermore, JTE013 suppressed osteoclastogenesis and bone resorption induced by RANKL in murine bone marrow cultures. ShRNA knockdown of S1PR2 or inhibition of S1PR2 by JTE013 suppressed podosome components, including PI3K, Src, Pyk2, integrin β3, filamentous actin (F-actin), and paxillin levels induced by RANKL in murine bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production, cell chemotaxis, osteoclastogenesis, and bone resorption. Inhibition of S1PR2 signaling could be a novel therapeutic strategy for bone loss associated with skeletal diseases.

Macrophages' phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs' expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes' mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes' expression and PLC proteins' presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have complicated membrane systems. The outermost plasma membrane contains various substructures, such as invaginations and protrusions, which are involved in endocytosis and cell migration. Moreover, the intracellular membrane compartments, such as autophagosomes and endosomes, are essential for cellular viability. The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily proteins are important players in membrane remodeling through their structurally determined membrane binding surfaces. A variety of BAR domain superfamily proteins exist, and each family member appears to be involved in the formation of certain subcellular structures or intracellular membrane compartments. Most of the BAR domain superfamily proteins contain SH3 domains, which bind to the membrane scission molecule, dynamin, as well as the actin regulatory WASP/WAVE proteins and several signal transduction molecules, providing possible links between the membrane and the cytoskeleton or other machineries. In this review, we summarize the current information about each BAR superfamily protein with an SH3 domain(s). The involvement of BAR domain superfamily proteins in various diseases is also discussed.

Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility. Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL -/- mice, using Airyscan confocal microscopy. Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates. Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lin, Krishnamoorthy, Walker, Joshi and Morley.)

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]

ABSTRACT The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research. [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]

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]