Podosomes are multifunctional organelles of invasive cells, including immune cells such as macrophags. Podosomes are cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. In this study, we aimed to identify all protein components that build up these important structures and also enable their multiple functions and regulate their turnover. We used macrophages as a cell system, as these cells form numerous podosomes in the plane of adhesion, which makes them an ideal system for the purification of podosome-enriched cell fractions. We combined isotopic labelling of cells with a differential lysis technique to gain podosome-enriched cell fractions. Identification of protein components in these fractions was performed by mass spectrometry analysis. We could identify 203 proteins, comprising 33 known podosome proteins and 170 potential novel components. Software analyses show that particularly proteins involved in actin cytoskeleton regulation, adhesion mediation and those harboring ATPase or GTPase activity are enriched. Surprisingly, we also identified a variety of ribosomal and RNA binding proteins. This was corroborated by proof-of-principle experiments, in which the newly identified RNA binding protein hnRNP-K, and also the actin binding protein WDR1, could be identified as novel components of podosomes. Our results point to the potential relevance of RNA binding proteins as additional regulators of podosome structure or function. They also indicate that the list of identified proteins should be a useful and relevant source for the identification and study of novel podosome components and thus of novel regulators of macrophage adhesion and invasion in health and disease.
Generation of podosome-enriched cell fractions. 3D reconstructions (upper images) of human macrophages before (left) or after differential cell lysis (right), together with side views of the respective cells (lower images). Cells are stained for podosomal proteins F-actin (red) and vinculin (green). Note the dome-shaped part of the cell (left images), which contains the nucleus and most of the cytoplasm, and the remaining adhesive part (“footplate”; right images), which remains after lysis and contains podosomes. White bars: 5 µm.
Macpherson L, Monypenny J, Blundell MP, Cory GO, Tomé-García J, Thrasher AJ, Jones GE, Calle Y. Haematologica. 2012 May;97(5):687-91. Epub 2011 Dec 1.
Cells of the myeloid lineage assemble characteristic highly dynamic actin-based adhesive structures termed podosomes that are thought to be involved in migration of cells that have to cross and invade tissue boundaries. Podosomes cluster behind the extending leading edge of migrating myeloid cells and are required for polarization, persistent migration and chemotaxis.
We and others have identified the Arp2/3 activator Wiskott Aldrich Syndrome Protein (WASP) and the WASP interacting protein(WIP) in the podosome actin core, where they play a major role in their formation and dynamics. We have previously shown that the rapid turnover of podosomes of migrating myeloid cells involves cleavage of WASP by the protease calpain, supporting a role for WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown.
In this publication we show that tyrosine phosphorylation of human (Y291) and murine (Y293) WASP not only promotes a sustained WASP open conformation required for Arp2/3 binding and actin filament nucleation, but also enhances its susceptibility to calpain-mediated cleavage, thereby promoting disassembly of podosomes as the leading edge of the cell progresses forward.
The f-actin core of podosomes (red) are less stable and have shorter half lives when generated through human phosphomimic (Y291E) WASP (B) and phophodead (Y291F) WASP (C) generates very stable podosomes. Addition of a calpain inhibitor (ALLM) restores the stability of Y291E-generated podosomes (E).
Bhuwania R, Cornfine S, Fang Z, Krüger M, Luna EJ, Linder S. J Cell Sci. 2012 May 1;125(Pt 9):2300-14. Epub 2012 Feb 17.
Podosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations: large precursors at the cell periphery and smaller podosomes, called successors, in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. In this study, we could show that podosome subpopulations differ in their molecular composition. We identified the membrane-associated protein supervillin as the first factor that localizes differentially to successor podosomes, and especially to a newly described “cap” structure that decorates the top of podosomes. We further show that supervillin-mediated coupling of myosin-dependent contractility to podosomes is required for the regulation of podosome dynamics. Consistently, depletion of supervillin from cells resulted in longer life times of podosomes. We further show that supervillin also regulates cell polarization, which is a prerequisite for successful migration. Supervillin is therefore involved in multiple aspects of macrophage adhesion, migration and invasion, which identifies it as an important factor for immune cell regulation in health and disease.
Supervillin localizes to a subpopulation of podosomes. Confocal micrographs of primary human macrophages expressing fluorescently labelled supervillin (green) and stained for F-actin (red) to label podosomes. Note that supervillin localizes to smaller successor podosomes in the inner part of a round, unpolarized cell (upper images) or to the rear of a polarized cell (lower images). Larger podosomes at the cell periphery (upper images) or the cell front (lower images) do mostly not contain supervillin. Upper images: ca. 30 µm, lower images: ca. 40 µm.
Harre U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E, Jakobsson PJ, Baum W, Nimmerjahn F, Szarka E, Sarmay G, Krumbholz G, Neumann E, Toes R, Scherer HU, Catrina AI, Klareskog L, Jurdic P, Schett G. J Clin Invest. 2012 Apr 16. pii: 60975. doi: 10.1172/JCI60975. [Epub ahead of print]
Rheumatoid arthritis is the most severe inflammatory disease and affects 1% of the population worldwide. Increasing evidence describes autoimmunity as the underlying cause of RA-induced bone loss. Indeed, autoantibodies against citrullinated proteins (ACPAs) are among the strongest risk factors for bone destruction and are highly present in serum years before the onset of clinically overt RA. Citrullination is a posttranslational modification of the arginine aminoacid into citrullin. Mechanisms though which ACPAs effect, directly or indirectly, bone homeostasis are still unknown. In this study, we found a significant association in RA-patients between ACPAs and serum markers for osteoclast (OC)-mediated bone resorption. Also, we showed that OCs expressed enzymes that citrullinate proteins and thus giving new targets for ACPAs. In particular, vimentin, a intermediate filament protein, was found to be highly citrullinated in differentiating OCs. We therefore purified an antibody against this mutated citrullinated vimentin (MCV-ACPA) and found it to bind to the surface of OCs and, more importantly, to induce osteoclastogenesis. In addition, the injection of MCV-ACPA into mice caused osteopenia (loss of bone mass) and increased osteoclastogenesis. This was explained by an induction of TNF-α release from OC precursors and its consequent effect on the proliferation of these precursors and their expression of their activation makers (CD11b, CD14, CD115) and cytokine receptors (RANK).
Altogether, our in vitro and in vivo data provide evidence for a new pathological mechanism that links the adaptive immune response to bone remodelling: differentiating OCs increasingly express enzymes that induce protein (such as vimentin) citrullination. ACPAs, in particular anti-MCV, are therefore released and target OC precursors, causing their self-activation and differentiation into OC. This induced osteoclastogenesis is largely responsible for the increase of bone resorption and the decrease of bone mass in inflammatory conditions.
MCV-ACPAs induce bone loss in vivo. (A) 3-dimensional (top) and 2-dimensional (bottom) μCT images of the tibial metaphysis of Rag1–/– mice that were left untreated or treated with control IgG or MCV-ACPAs showing decreased bone volume, trabecular number, trabecular thickness and connectivity density. (B) Microphotographs (original magnification, ×400) of tibial bones of Rag1–/– mice treated with either IgG or MCV-ACPAs, stained for osteoclasts (purple stain and arrows) by histochemical detection of TRAP showing increased osteoclastogenesis following MCV-ACPA injection.
Susan Cox, Edward Rosten, James Monypenny, Tijana Jovanovic-Talisman, Dylan T Burnette, Jennifer Lippincott-Schwartz, Gareth E Jones & Rainer Heintzmann. Nature Methods. 2012, volume 9: 195-200. doi: 10.1038/nmeth.1812.
An analytical approach based on the concept of fluorophore localisation provides dynamic super-resolution data of xFP- labelled live cells using a common arc lamp–based wide-field fluorescence microscope.
One method of achieving fluorescence super-resolution is based around finding the positions of fluorescent molecules that label the cellular structure of interest. In this approach, positions can be determined precisely and accurately using fluorescent probes that can be photoactivated, photoconverted or photoswitched to generate single images with emitter densities of only about one active fluorophore per diffraction-limited area. Many images each containing subsets of active fluorophores are collected and analysed to attain the spatial sampling required for a super-resolution image. One drawback of this approach is that the active density of fluorophores in each frame must be kept low, forcing long imaging times and requiring the use of probes that can be manipulated to a low single-frame density. In our paper we describe a new approach to modelling such datasets using a Bayesian analysis that can work with fluorophore densities as high as those found when using conventional labelling and imaging.
In our analysis, that we term Bayesian blinking and bleaching (3B) analysis, a few seconds of data collection using a typical lamp-based wide-field fluorescence microscope can yield an image with resolution approaching 50 nanometers over time intervals of about 4 seconds (see Fig 1). The 3B analysis provides exciting new possibilities for super-resolution imaging with simple optical setups and probes that are neither photoactivatable nor photoswitchable. This brings the technique of super-resolution imaging to standard microscopy set-ups without the need for expensive new hardware so should be a powerful tool for cell biologists. However, there is a caveat: the 3B approach shifts the complexity of super-resolution from the optical setup onto the post-processing analysis. Without the aid of a competent physicist such post-processing would be a major hindrance to cell biologists so the authors are currently writing a plugin software version suitable to non-mathematicians. The source code for the project has been made available and the plugin, suitable for general use, will become freely available to the academic community once beta-testing is completed.
To test the method, we imaged podosomes in a human cell line expressing mCherry-tagged truncated talin. Podosomes are cytoskeletal structures involved in cell migration and adhesion and are associated with the degradation of the extracellular matrix. They consist of an actin filament core surrounded by a ring of integrin-associated proteins such as talin and vinculin. Surprisingly we found that podosomes often have a polygonal structure and that they are highly dynamic, dissociating and reforming over a timescale of tens of seconds. These characteristics were previously unappreciated, as the integrin-associated ring was thought to be round and podosomes were thought to dissociate over several minutes.
Thus, the 3B method should readily increase the resolution that is possible with live-cell imaging experiments using fluorescent proteins and reveal previously unappreciated levels of complexity in biological systems.
Scale bar is 2 microns. On the left side is the conventional image and on the right side the 3B analysis of the same image.
Emilie Le Roux-Goglin, Christine Varon, Pirjo Spuul, Corinne Asencio, Francis Mégraud, Elisabeth Génot. Eur. J. Biol. Cell. Vol. 91, March 2012, pages 161-170.
Helicobacter pylori is a bacteria which colonizes the stomach in about 50% of all humans. Well known as a key risk factor in gastric diseases, it may also damage liver, causing cirrhosis and liver cancer. The paper demonstrates that the bacteria promotes podosome formation in murine primary hepatocytes (or cells of hepatoma cell lines) in vitro. Infection of hepatocytes by Helicobacter pylori induces the release of inflammatory cytokines, including TGFb, which trigger podosome assembly. Liver cells with podosomes have reduced self-healing capacities. Although it is not yet clear which role podosomes play in the response to bacterial infection, one may expect that in vivo, podosomes in liver cells infected with Helicobacter pylori contribute to the pathological state.
Hepatocellular carcinoma cells were infected with Helicobacter pylori and cells were stained for F-actin (red), cortactin (green) and vinculin (white). Nuclei of the cells are shown in blue. Infected cells assemble podosome rosettes (large ring appearing in yellow).
Katarina Wolf and Peter Friedl. Trends Cell Biol. 2011 Dec;21(12):736-44. Epub 2011 Oct 27.
In tumor invasion into three-dimensional extracellular matrix (ECM), migrating cells must navigate through connective tissue structures of complex physicochemical properties, i.e. of certain space and stiffness. To successfully migrate, cells adopt two basic strategies that involve (a) deformation of the cell body towards pre-given ECM space and/or (b) the engagement of proteases to degrade ECM structures and generate new space supporting invasion.
We here develop a new concept for the steps a cell must take and can “chose from” to move through 3D tissue. We first review known types of tissue physics and their space determinants that both guide and restrict moving cells. Then, known types of proteolysis against 3D ECM proteolysis are reviewed, including diffuse or contact-dependent extracellular and intracellular proteolysis. Lastly, we show an algorithm of ECM and cell determinants that support migration through (narrow) 3D space (see Figure), including initial physical confrontation, deformation and proteolysis as well as published examples for proteolytic and non-proteolytic migration.
Step-wise physicochemical events and ‘decisions’ during dynamic cell confrontation with 3D ECM depicting the perspectives of both, the tissue structure and cell morphology, leading to an either proteolytic or non-proteolytic migration, or a combination thereof.
Podosomes are specialized actin microdomains of the plasma membrane endowed with matrix degrading activities. Endothelial cells do not usually assemble podosomes but can be induced to do so in vitro or ex vivo. TGFb, a pleiotropic cytokine which exhibits inflammatory or anti-inflammatory properties depending on the context, appears as a pivotal regulator of this process. Given their strategic location, endothelial podosomes are expected to play a role in vessel remodeling and/or in breaching anatomical barriers. Understanding the molecular events involved triggered by TGFb in these cells should help to determine the physiological and pathological role played by endothelial podosomes.
This work stemmed from knowledge that Cdc42, a molecular switch for cytoskeletal reorganization was involved in podosome assembly. In this paper, we present work leading to the identification of the protein linking TGFb to Cdc42. This protein, Fgd1, had not been previously reported in endothelial cells. Our findings suggest that mutations in the Fgd1 gene may have consequences in the vascular system. This is especially interesting since naturally occurring mutations in the Fgd1 gene cause the genetic disease Aarskog-Scott syndrome or faciogenital dysplasia.
The molecular mechanism of action of Fgd1 involves its phosphorylation on tyrosine residues by a Src-like kinase, another well-known player of the podosome assembly cascade. This post-translational modification is associated with Fgd1 translocation to the cell membrane and, later on, to podosomes. These results identify Fgd1 as the first Cdc42 regulator to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to understand the role of endothelial podosomes in vascular pathophysiology.
TGFb is a key regulator of vascular homeostasis. We first discovered that TGFb induces podosome formation in endothelial cells. We establish that Fgd1 is a regulator of endothelial podosome formation and, by fluorescence microscopy, we show that it is a component of endothelial podosomes. Actin staining reveals podosomes (labeled in red) which contains the Fgd1 protein (green). The scale bar is 10 mm.
Osteoclasts are huge cells containing several nuclei formed by fusion of mononucleated cells belonging to the monocyte/macrophage lineage. They are found on the bone surface, where they remove the old bone matrix in order to allow bone rejuvenation by osteoblasts. In order to adhere to their substrates, to migrate and to resorb, they are equipped of structures called podosomes, which are loosely organized on artificial substrates (plastic or glass) but condensed on calcium apatite mineral. Podosomes are made of polymerized actin organized as dense dots, and are surrounded by a loose network of polymerized actin. They are very dynamic structures with a short life-time (2 to 3 minutes), that self organize with different patterns during osteoclast life.
Podosomes are thought to be responsible for cell adhesion and migration. In this article we show that osteoclast podosomes are major anchoring sites exerting tensions on the substrate. They appear in areas where membranes are expanding and disappear in areas where membranes retract. Videomicroscopy has shown that podosome dynamics can move latex beads dispersed in a soft substrate, thus demonstrating podosome-mediated tension forces. Finally, analyzing the migration dynamic of several osteoclasts, we were able to show that actin-containing podosomes are major driving forces for osteoclast migration. A mathematical model of osteoclast migration provides unexpected results: osteoclasts are moving by jumps and this is reminiscent of the way that osteoclasts are moving to resorb bone surfaces.
Dynamic images of an osteoclast spreading on the plastic surface of a petri dish. Podosomes are in green; they are present at the periphery of the cell in the spreading area.
t= 0 rounded non-adherent osteoclasts are seeded in the petri dish; Then images of the same cells 12minutes (t= 12 mn) and 20 minutes (t=20mn) after seeding.
Novel invadopodia components revealed by differential proteomic analysis
Francesca Attanasio, Giusi Caldieri, Giada Giacchettia, Remcovan Horssen, Bé Wieringa, Roberto Buccione, 2010. European Journal of Cell Biology Vol. 90, Issues 2-3, February-March 2011, Pages 115-127.
Tumour growth and dissemination throughout the body to form metastases, the main cause of cancer-related mortality, depend on the ability of tumour cells to move through/invade the very dense meshwork formed by the extracellular matrix (ECM), in which the cells that make up tissues and organs are embedded. The controlled degradation of specific components of the ECM is an essential step in tumour cell invasion. This process can be recapitulated in the “test tube” using tumour cell lines that form protrusive structures called invadopodia that have the ability to locally degrade and penetrate the ECM.
Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete. In this paper, we describe a novel approach to accelerate the discovery process. In detail, we were able to prepare tumour cell fragments enriched in invadopodia which were then analyzed by proteomic techniques. This led to the discovery of new protein components belonging to different functional groups including those related to the production of energy, cell movement and secretion of proteins.
We expect these findings to open further avenues for the molecular study of invasive growth behaviour of cancer cells.
The G protein Beta subunit is a very important protein that mediates communication between the extracellular and intracellular environments. We first discovered it to be associated with invadopodia in our proteomics analysis and then validated the finding by fluorescence microscopy as shown here. Invadopodia (labeled in blue) are positioned over areas of digested extracellular matrix (dark holes in the green background) and contain the G protein Beta subunit (red).
Invadosomes: Intriguing structures with promise
Frédéric Saltel, Thomas Daubon, Amélie Juina, Isabel Egańa Ganuza, Véronique Veillat, Elisabeth Génot, 2010. European Journal of Cell Biology Vol. 90, Issues 2-3, February-March 2011, Pages 100-107.
Podosomes and invadopodia are dynamic, actin-rich adhesion structures and represent the two founding members of the invadosome family. These plasma membrane microdomains are commonly found in cells of the immune system or in osteoclasts (podosomes) as well as in cancer cells (invadopodia), where they have been extensively described. More recently, it was realized that other cells which do not spontaneously assemble invadosomes can be induced to do so under selective stimulation, such as a soluble factor, matrix receptor, cell stress or oncogenes. Invadosomes harbour metalloproteases which degrade components of the extracellular matrix. Because of this distinctive feature, invadosomes have been systematically linked with invasion processes but hints for other functions are now emerging. During the last decade, tremendous advances have been made regarding the molecular mechanism underlying their formation, dynamics and function. 3D analysis of invadosomes is now being actively developed in ex vivo and in vivo models to demonstrate their involvement in physiological and pathological processes.
Figure legend: Schematic representation of the evolution of models for invadosome analysis. (C) In vitro invadosome observation in 3D and their presumed role are reported in a few publications. (D) The same conclusion holds true for ex vivo or in vivo observation.
Susanne Cornfine, Mirko Himmel, Petra Kopp, Karim el Azzouzi, Christiane Wiesner, Marcus Krüger, Thomas Rudel, Stefan Linder, 2010. Molecular Biology of the Cell Vol. 22, Issue 2, January 15, 2011, Pages 202-215.
Macrophages are immune cells that form the first line of defense against infectious agents such as bacteria or viruses. To get to sites of infection, macrophages have to migrate through the dense meshwork of the extracellular matrix that connects cells within tissues. Podosomes, adhesion structures with the ability to locally degrade matrix material, play a key role in this migration.
We show here that matrix degradation by macrophage podosomes depends on contact with the microtubule network of cells. Microtubules form a “cellular railway system”, which enables the delivery of cargo molecules through transport by tiny motors walking on these fibers. This study identifies the motor protein KIF9 as an essential regulator of matrix degradation at podosomes. KIF9 delivers cargo vesicles, which contain proteins of the reggie family, a group of membrane-associated proteins. We could also identify a distinct region of KIF9 as the binding site for reggie-1. Interaction of both proteins through this region is critical for regulating matrix degradation at podosomes.
Vesicles containing the motor protein KIF9 (green) are associated with microtubules (red), which form the "cellular railway system".
Martín-Villar E, Fernández-Muńoz B, Parsons M, Yurrita MM, Megías D, Pérez-Gómez E, Jones GE, Quintanilla M. Mol Biol Cell. 2010 Dec;21(24):4387-99
Podoplanin is a small transmembrane glycoprotein whose expression is up-regulated in different types of cancer, especially in squamous cell carcinomas (SCCs). Podoplanin up-regulation in tumour cells has been linked to enhanced cell motility and invasiveness; however, the mechanisms underlying this process remain poorly understood. Since podoplanin lacks any obvious enzymatic motif within its structure, all these activities have to be mediated by protein–protein interactions, highlighting the need to identify its binding partners. To further shed light into the biology and function of this glycoprotein, we attempted to identify the key podoplanin binding partners involved in podoplanin-mediated cancer cell migration. In this article, we show that CD44s, the standard isoform of the major hyaluronan (HA) receptor, is a novel partner for podoplanin during podoplanin-mediated migration. We first show that the expression of both molecules is co-ordinately up-regulated in a mouse skin model of carcinogenesis during progression to highly aggressive cell carcinomas. Second, we demonstrate that podoplanin interacts with CD44s both in vitro, by co-immunoprecipitation assays, and in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM). FRET/FLIM demonstrated that the association of these two proteins in the plasma membrane increases in cells with a migratory phenotype. Importantly, we also show for the first time that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in squamous carcinoma cells.
Podoplanin–CD44s complexes at the plasma membrane are up-regulated in cells with a migratory phenotype. Multiphoton FLIM was used to image FRET between podoplanin-eGFP (donor) and CD44s-mRFP (acceptor) in MDCK cells. This technique enables visualisation and quantification of protein–protein interactions by analysis of the donor lifetime decay kinetics. Interaction of podoplanin-eGFP and CD44s-mRFP was observed in single polarized cells, as measured by decreases in eGFP donor fluorescence lifetime (red) relative to its lifetime in control cells (blue) expressing podoplanin-eGFP alone (A). FRET was localised at the trailing edge during rear retraction, and on small foci that were distributed throughout the apical surface of the cell across the lamellae (B). Note that although co-localisation between podoplanin–eGFP and CD44s–mRFP was always detected, FRET was recorded mainly in isolated cells (i.e., those that had detached from their neighbours).