Podosome Core Research Articles

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

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

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages

Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes at the layer of myosin IIA. The microtubule disruption-caused podosome dissolution is previously ascribed to Rho/ROCK-myosin signaling, yet inhibiting this pathway with Y27632 or blebbistatin only partially recovers podosome assembly, thus suggesting the contribution of the physical supporting of microtubules in stabilizing podosome structures. Through improved substrate-coating technique, we further corroborate that the matrix-degrading capability of macrophages depends on the formation of podosome clusters. Together, 3D-STORM super-resolution microscopy reveals the nanoscale spatial arrangement and the microtubule-dependent regulation of the matrix-degrading podosome clusters in macrophages.

Indoxyl sulfate- and P-cresol-induced monocyte adhesion and migration is mediated by integrin-linked kinase-dependent podosome formation

Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvβ3 integrin and core-linked CD44

BackgroundSince enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PurposeThis study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. MethodsThis study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. ResultsLinarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. ConclusionThese observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvβ3 integrin and core-linked CD44.

A novel mechanism in wound healing: Laminin 332 drives MMP9/14 activity by recruiting syndecan-1 and CD44

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.

Abl-mediated PI3K activation regulates macrophage podosome formation.

Podosomes play crucial roles in macrophage adhesion and migration. Wiskott-Aldrich syndrome protein (WASP; also known as WAS)-mediated actin polymerization is one of the key events initiating podosome formation. Nevertheless, membrane signals to trigger WASP activation at macrophage podosomes remain unclear. Here, we show that phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] lipids are enriched at the podosome and stably recruit WASP rather than the WASP-5KE mutant. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit β (PIK3CB) is spatially located at the podosome core. Inhibition of PIK3CB and overexpression of phosphatase and tensin homolog (PTEN) impede F-actin polymerization of the podosome. PIK3CB activation is regulated by Abl1 and Src family kinases. At the podosome core, Src and Hck promote the phosphorylation of Tyr488 in the consensus Y-x-x-M motif of Abl1, which enables the association of phosphoinositide 3-kinase (PI3K) regulatory subunits. Knockdown of Abl1 rather than Abl2 suppresses the PI3K/Akt pathway, regardless of Src and Hck activities. Reintroduction of wild-type Abl1 rather than the Abl1-Y488F mutant rescues PI3KR1 recruitment and PI3K activation. When PIK3CB, Abl1 or Src/Hck is suppressed, macrophage podosome formation, matrix degradation and chemotactic migration are inhibited. Thus, Src/Hck-mediated phosphorylation of Abl1 Tyr488 triggers PIK3CB-dependent PI(3,4,5)P3 production and orchestrates the assembly and function of macrophage podosomes.

Cirsium Setidens Extracts Abrogate Actin Ring Formation and Bone-Resorbing Osteoclasts Through Inhibition of Core-Linked CD44 and Diffuse Cloud-Associated αvβ3 Integrin

Abstract Objectives Since enhanced bone resorption cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Cirsium setidens (Korean thistle) contains linarin, luteolin, pectolinarin, hispidulin, and apigenin with antioxidant and hepatoprotective effects. This study examined whether 1–20 μg/ml Cirsium setidens extracts (CSE) inhibited osteoclastogenesis of RANKL-exposed RAW 264.7murine. Methods RAW 264.7 murine macrophages were incubated with 1–20 μg/ml CSE for 5 days in the presence of 50 ng/ml RANKL. Tartrate-resistance acid phosphatase (TRAP) staining and its activity measurement were performed. Western blotting was done with target proteins involved in the osteoclast activation. Results Nontoxic CSE attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed bone resorption through reducing lacunar acidification and bone matrix degradation. Linarin and pectolinarin were identified as major components of CSE, where linarin but not pectolinarin were effective in inhibiting formation of TRAP-positive osteoclasts. Linarin-rich CSE diminished the induction of αvβ3 integrin-associated proteins of paxillin, Pyk2 and gelsolin. Additionally, CSE deterred actin ring formation with attenuation of induction of F-actin-enriched podosome core proteins of CD44, Arp2/3 and cortactin. Nontoxic linarin and its aglycone acacetin reduced focal contact of osteoclasts to RGD peptide. The inhibition of integrin-mediated actin ring formation by CSE entailed disruption of TRAF6-c-Src-PI3K signaling of osteoclasts. Conclusions CSE was effective in retarding focal adhesion to bone matrix and active bone resorption of osteoclast via inhibition of core-linked CD44 and diffuse cloud-associated αvβ3 integrin. Funding Sources This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2019R1A2C1003218).

Podosome formation promotes plasma membrane invagination and integrin-\u03b23 endocytosis on a viscous RGD-membrane

Integrin receptors orchestrate cell adhesion and cytoskeletal reorganization. The endocytic mechanism of integrin-β3 receptor at the podosome remains unclear. Using viscous RGD-membrane as the model system, here we show that the formation of podosome-like adhesion promotes Dab2/clathrin-mediated endocytosis of integrin-β3. Integrin-β3 and RGD ligand are endocytosed from the podosome and sorted into the endosomal compartment. Inhibitions of podosome formation and knockdowns of Dab2 and clathrin reduce RGD endocytosis. F-actin assembly at the podosome core exhibits protrusive contact towards the substrate and results in plasma membrane invaginations at the podosome ring. BIN1 specifically associates with the region of invaginated membrane and recruits DNM2. During the podosome formation, BIN1 and DNM2 synchronously enrich at the podosome ring and trigger clathrin dissociation and RGD endocytosis. Knockdowns of BIN1 and DNM2 suppress RGD endocytosis. Thus, plasma membrane invagination caused by F-actin polymerization promotes BIN1-dependent DNM2 recruitment and facilitate integrin-β3 endocytosis at the podosome.

DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.

Tail domains of myosin-1e regulate phosphatidylinositol signaling and F-actin polymerization at the ventral layer of podosomes.

During podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3-dependent manner. The combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Overexpression of TH12, not TH12 KR2A, impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome.

Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes.

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

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Podosome Formation and Development in Monocytes Restricted by the Nanoscale Spatial Distribution of ICAM1.

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.

The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes.

Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.

Tyrosine phosphorylation of WIP releases bound WASP and impairs podosome assembly in macrophages.

ABSTRACTPodosomes 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.

Automated Podosome Identification and Characterization in Fluorescence Microscopy Images

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

Inhibition of Contractility and RhoA Deactivation Trigger Podosome Formation

Development of integrin-mediated cell-matrix adhesions is important in the regulation of cell growth and differentiation. Activated integrin clusters are sites of actin polymerization, and the physical properties of extracellular matrix ligands play critical roles in the development of adhesion structures. Previously, we have utilized RGD-membranes, continuous supported bilayers with freely diffusing lipid-anchored RGD ligands, to activate integrins and investigate force dependence of initial integrin clustering during early adhesion formation (Yu et al., PNAS 2011). At later times (>45 minutes) on RGD-membranes, we found that fibroblasts natively form podosomes as adhesion structures when cell-matrix traction force cannot be stabilized. Podosome core assembly was initiated by Arp2/3-mediated actin polymerization locally within integrin clustering sites that were characterized by integrin-associated proteins, such as vinculin, paxillin, and talin. In addition, myosin-1E/1F and CARMIL1 were found as new markers at podosome cores. We utilized a FRET-based RhoA biosensor to measured differential RhoA activities in cells forming podosomes on RGD-membranes versus focal adhesions on RGD-glass. RhoA-GTP levels were indeed low in cells forming podosomes on RGD-membranes, whereas RhoA-GTP levels were high in the cells on RGD-glass. Constitutively active RhoA-Q63L mutant blocked podosome formation on RGD-membranes. Furthermore, fibroblasts on RGD-membranes with dense nano-partitions (1μm-pitch line partitions) in RGD-membranes provided mechanical barriers that enabled adhesion maturation that was not possible with wider line-partitions (4μm) in RGD-membrane. When a single cell adhered over both regions of high and low density, podosomes formed over low-density regions only. From our data, we suggest that development of podosomes as adhesion structures requires inhibition of traction forces between integrin receptors and matrix ligands. Activation of myosin contractility at integrin clusters activates focal adhesion formation and inhibits podosome formation at integrin clusters, giving rise to a mutually exclusive behavior of focal adhesions and podosomes.

Cofilin Activation during Podosome Belt Formation in Osteoclasts

Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.

Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott–Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.

Role of WASP in cell polarity and podosome dynamics of myeloid cells

The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.