The differential regulation of podosome formation by Cytohesin-2 is mediated via α5β1 integrin and the small GTPase RhoA

The interaction of a cell with the surrounding tissue is crucial not only to provide mechanical stability but also to enable adaptation of cellular responses to the environment. Recognition of extracellular matrix (ECM) is primarily exerted by integrin receptors, which consist of an α and a β chain. The high diversity of these integrin dimers allows for a variety of ligand specificities but differences in intracellular signaling cascades are only partially understood. Within the cell, integrin-mediated adhesions are organized in specific adhesion structures. Among these, podosomes are characterized by their typical organization in an actin-rich core and an adhesive ring structure, which contains integrins and adhesion-related adaptor and signaling molecules. Apart from mediating adhesion, podosomes also are important during cell migration and invasion. Their formation is induced by growth factor or integrin signaling and depends very much on actin remodeling factors. Both actin dynamics and integrin signaling are regulated by numerous factors, including the cytohesin protein family. Cytohesins primarily act as guanine nucleotide exchange factors (GEFs) for Arf GTPases, but they are also involved in activation and recycling of integrins, as well as actin remodeling processes. One member of the cytohesin family, Cyth2, has recently been shown to be important for podosome formation in THP-1 cells. So far, though, it is unclear whether integrin-related functions of cytohesins also affect podosome formation. Therefore, this study aimed at identifying the role of different cytohesins in integrin-dependent podosome formation. Using bone marrow-derived dendritic cells (BMDCs) from several cytohesin KO mice, we found that only Cyth2, but not Cyth1, Cyth3 or Cyth4, regulates podosome formation in a matrix-depending manner. Loss of Cyth2 impaired podosome formation on a fibronectin (FN) matrix, while Cyth2 KO BMDCs on fibrinogen or Icam-1 remained unaffected. Moreover, Cyth2 KO cells cultured on gelatin or collagen even increased their podosome numbers compared to wildtype cells. These effects were dependent on a differential involvement of α5β1 integrin and were mediated via the small GTPase RhoA. Thus, our results show that Cyth2 is involved in regulation of integrin-dependent responses to environmental cues leading to differential formation of podosomes. Such specific adaptations of signaling pathways downstream of different integrins might affect invasion and migration processes of immune cells in certain ECM microenvironments in vivo

Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data

Dendritic Cell Migration and Traction Force Generation in Engineered Microenvironments

Dendritic cells (DCs) are potent initiators of the adaptive immune response. Their trafficking from sites of inflammation to lymphoid tissue is essential to their function. Exactly how dendritic cells integrate multiple chemotactic cues to organize an accurate migratory path is not fully understood. We first characterize DC random motility (chemokinesis) on extracellular matrix proteins in the presence of chemokines. Then, using a microfluidic device, we present both single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. We show that in counter gradients, CCL19 is 10 to 100 fold more potent than other chemokines CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells home to a central region in which the signals from multiple chemokines are balanced. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs. We extended this work to a combination of the microfluidic gradient generator and micropost array detectors to develop a novel method for probing traction forces during chemotaxis. We find DC migration is driven by short-lived traction stresses at the leading edge or filopodia. We illustrate that spatiotemporal pattern of traction stresses can be used to predict changes in the direction of DC motion. Additionally, we determine the characteristic duration of local dendritic cell traction forces and correlate this duration with force. Overall, DCs show a mode of migration distinct from both mesenchymal cells and other leukocytes, characterized by rapid turnover of traction forces in leading filopodia. In this thesis, we extend the current understanding of DC motility to include signal integration and traction forces

VEGF-A/Notch-induced podosomes proteolyse basement membrane collagen-IV during retinal sprouting angiogenesis

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

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

International audiencePodosomes 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

The role of plectin in the regulation of carcinoma cell invasion

The research presented in this thesis endeavours to understand the role of the plakin family of cytoskeletal linker proteins in the migration and invasive potential of epithelial carcinomas. This study focuses on plectin, a plakin family member that has previously been implicated with a role in tissue integrity of the skin and muscle. I investigated the complex gene organisation of the alternative first exons in human plectin, leading to the discovery of a further novel isoform plectin-lk. A panel of colon and breast carcinoma cell lines that vary in their differentiation and metastatic potential were used to investigate the expression of the isoforms at the mRNA and protein levels, showing plectin to be expressed at higher levels in the more invasive cell types. The subcellular localisation of the alternatively spliced plectin isoforms was investigated using green fluorescent protein (GFP) and polyclonal antibodies, revealing isoform specific targeting to different actin structures. Ablation of plectin or vimentin (a major intermediate filament protein of mesenchymal cells that interacts with plectin), by small interfering RNAs suggest these proteins are able to modulate invasion, migration and attachment of the epithelial carcinoma cells. Further investigation into the novel isoform plectin-lk reveals a role in the formation of podosome like adhesion structures in a Rho kinase dependant manner that facilitate migration in the SW480 colon carcinoma cells. The above findings are novel and contribute to the understanding of migration and invasion of cancer cells. Furthermore, this understanding could provide novel targets of cancer cell metastasis

A Src-Tks5 Pathway Is Required for Neural Crest Cell Migration during Embryonic Development

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis