Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin are characterized.

Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the "transcellular" route) or between them (the "paracellular" route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended "invasive podosomes" to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.

To perform their functions during development and after central nervous system injury, the brain's immune cells (microglia) must migrate through dense neuropil and extracellular matrix (ECM), but it is not known how they degrade the ECM. In several cancer cell lines and peripheral cells, small multi-molecular complexes (invadopodia in cancer cells, podosomes in nontumor cells) can both adhere to and dissolve the ECM. Podosomes are tiny multi-molecular structures (0.4 to 1 μm) with a core, rich in F-actin and its regulatory molecules, surrounded by a ring containing adhesion and structural proteins.

Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Podosomes and invadopodia, collectively termed invadosomes, are adhesive and degradative membrane structures formed in many types of cells and are well known for recruiting various proteases. However, another major class of degradative enzymes, deoxyribonuclease (DNase), remains unconfirmed and not studied in invadosomes. Here, using surface-immobilized nuclease sensor (SNS), we demonstrated that invadosomes recruit DNase to their core regions, which degrade extracellular double-stranded DNA. We further identified the DNase as GPI-anchored membrane-bound DNase X. DNase recruitment is ubiquitous and consistent in invadosomes of all tested cell types. DNase activity exhibits within a minute after actin nucleation, functioning concomitantly with protease in podosomes but preceding it in invadopodia. We further showed that macrophages form DNase-active podosome rosettes surrounding bacteria or micropatterned antigen islets, and the podosomes directly degrade bacterial DNA on a surface, exhibiting an apparent immunological function. Overall, this work reports DNase in invadosomes for the first time, suggesting a richer arsenal of degradative enzymes in invadosomes than known before.

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA-induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures.

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) is a multistage process that requires many adhesion-dependent steps and involves the rearrangement of the actin cytoskeleton. The diversity of actin-based structures and their role in macrophage fusion is poorly understood. In this study, we revealed hitherto unrecognized actin-based zipper-like structures (ZLSs) that arise between MGCs formed on the surface of implanted biomaterials. We established an in vitro model for the induction of these structures in mouse macrophages undergoing IL-4-mediated fusion. Using this model, we show that over time MGCs develop cell-cell contacts containing ZLSs. Live-cell imaging using macrophages isolated from mRFP- or eGFP-LifeAct mice demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. Immunostaining experiments showed that vinculin, talin, integrin αMβ2, and other components of podosomes are present in ZLSs. Macrophages deficient in WASp or Cdc42, two key molecules involved in actin core organization in podosomes, as well as cells treated with the inhibitors of the Arp2/3 complex, failed to form ZLSs. Furthermore, E-cadherin and nectin-2 were found between adjoining membranes, suggesting that the transition of podosomes into ZLSs is induced by bridging plasma membranes by junctional proteins.

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

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.

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.

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E(2) (PGE(2)). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE(2) causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE(2)-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses.

Subcellular fine-tuning of the actomyosin cytoskeleton is a prerequisite for polarized cell migration. We identify LSP (lymphocyte-specific protein) 1 as a critical regulator of actomyosin contractility in primary macrophages. LSP1 regulates adhesion and migration, including the parameters cell area and speed, and also podosome turnover, oscillation and protrusive force. LSP1 recruits myosin IIA and its regulators, including myosin light chain kinase and calmodulin, and competes with supervillin, a myosin hyperactivator, for myosin regulators, and for actin isoforms, notably β-actin. Actin isoforms are anisotropically distributed in myosin IIA-expressing macrophages, and contribute to the differential recruitment of LSP1 and supervillin, thus enabling an actomyosin symmetry break, analogous to the situation in cells expressing two myosin II isoforms. Collectively, these results show that the cellular pattern of actin isoforms builds the basis for the differential distribution of two actomyosin machineries with distinct properties, leading to the establishment of discrete zones of actomyosin contractility.