24301979
Podosomes in migrating microglia: components and matrix degradation
BackgroundTo 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.MethodsUsing rat microglia, we performed several functional assays: live cell imaging for chemokinesis, degradation of the ECM component, fibronectin, and chemotactic invasion through Matrigel™, a basement membrane type of ECM. Fluorescent markers were used with high-resolution microscopy to identify podosomes and their components.ResultsThe fan-shaped lamella at the leading edge of migrating microglia contained a large F-actin-rich superstructure composed of many tiny (<1 μm) punctae that were adjacent to the substrate, as expected for cell–matrix contact points. This superstructure (which we call a podonut) was restricted to cells with lamellae, and conversely almost every lamella contained a podonut. Each podonut comprised hundreds of podosomes, which could also be seen individually adjacent to the podonut. Microglial podosomes contained hallmark components of these structures previously seen in several cell types: the plaque protein talin in the ring, and F-actin and actin-related protein (Arp) 2 in the core. In microglia, podosomes were also enriched in phosphotyrosine residues and three tyrosine-kinase-regulated proteins: tyrosine kinase substrate with five Src homology 3 domains (Tks5), phosphorylated caveolin-1, and Nox1 (nicotinamide adenine dinucleotide phosphate oxidase 1). When microglia expressed podonuts, they were able to degrade the ECM components, fibronectin, and Matrigel™.ConclusionThe discovery of functional podosomes in microglia has broad implications, because migration of these innate immune cells is crucial in the developing brain, after damage, and in disease states involving inflammation and matrix remodeling. Based on the roles of invadosomes in peripheral tissues, we propose that microglia use these complex structures to adhere to and degrade the ECM for efficient migration.
37501756
Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages
Primitive Origins for Microglia Microglia are the resident macrophages of the central nervous system and are associated with neurodegeneration and brain inflammatory diseases. Although the developmental origins of other tissue macrophage populations are well established, the origins of microglia remain controversial. Ginhoux et al. (p. 841, published online 21 October) used in vivo lineage tracing studies to show that microglia arise early in mouse development and derive from primitive macrophages in the yolk sac. This is in contrast to other cells of the mononuclear phagocyte system, which arise later in development from a distinct progenitor population. The developmental origins of adult microglia are revealed. Microglia are the resident macrophages of the central nervous system and are associated with the pathogenesis of many neurodegenerative and brain inflammatory diseases; however, the origin of adult microglia remains controversial. We show that postnatal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain. In contrast to many macrophage populations, we show that microglia develop in mice that lack colony stimulating factor-1 (CSF-1) but are absent in CSF-1 receptor–deficient mice. In vivo lineage tracing studies established that adult microglia derive from primitive myeloid progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically derived microglial progenitors for the treatment of various brain disorders.
28302415
Microglia in neurodegenerative disease
Microglia, the resident macrophages of the CNS, are exquisitely sensitive to brain injury and disease, altering their morphology and phenotype to adopt a so-called activated state in response to pathophysiological brain insults. Morphologically activated microglia, like other tissue macrophages, exist as many different phenotypes, depending on the nature of the tissue injury. Microglial responsiveness to injury suggests that these cells have the potential to act as diagnostic markers of disease onset or progression, and could contribute to the outcome of neurodegenerative diseases. The persistence of activated microglia long after acute injury and in chronic disease suggests that these cells have an innate immune memory of tissue injury and degeneration. Microglial phenotype is also modified by systemic infection or inflammation. Evidence from some preclinical models shows that systemic manipulations can ameliorate disease progression, although data from other models indicates that systemic inflammation exacerbates disease progression. Systemic inflammation is associated with a decline in function in patients with chronic neurodegenerative disease, both acutely and in the long term. The fact that diseases with a chronic systemic inflammatory component are risk factors for Alzheimer disease implies that crosstalk occurs between systemic inflammation and microglia in the CNS.
25629076
A rose by any other name? the potential consequences of microglial heterogeneity during CNS health and disease
SummaryMicroglial activation and macrophage infiltration into the CNS are common features of CNS autoimmune disease and of chronic neurodegenerative diseases. Because these cells largely express an overlapping set of common macrophage markers, it has been difficult to separate their respective contributions to disease onset and progression. This problem is further confounded by the many types of macrophages that have been termed microglia. Several approaches, ranging from molecular profiling of isolated cells to the generation of irradiation chimeric rodent models, are now beginning to generate rudimentary definitions distinguishing the various types of microglia and macrophages found within the CNS and the potential roles that these cells may play in health and disease.
31454645
From bone marrow to microglia: barriers and avenues.
Microglia form a unique population of brain-resident macrophages. Although microglia have been involved in multiple disorders of the central nervous system (CNS), the issue of microglial renewal, under normal or pathological conditions, has been controversial. In mice, results from bone marrow chimera studies indicated that microglia are slowly but continuously replenished by bone marrow-derived cells. Moreover, such a microglial turnover was found to be greatly accelerated under multiple neurological conditions. However, recent works questioned the use of irradiation/reconstitution experiments to assess microglial turnover. Based on these different studies, we propose here a re-evaluation of microglia origin(s) in the inflamed CNS. We also discuss the therapeutic perspectives offered by the demonstration of an adult microglial lineage, from bone marrow to brain.
11055292
Microglia in the CNS: Immigrants from another world
Microglia are immunocompetent cells of the brain that continuously survey their environment with highly motile extensions. Although first described almost a century ago, the developmental origin of microglia is still debated. Besides early myeolomonocytic cell populations that colonize the developing neuroectoderm already during embryogenesis, newly migrated myeloid cells are considered important disease modulators in the adult brain. Thus, understanding the mechanisms of myeloid cell recruitment from the periphery and their development into bone marrow‐derived phagocytes in the adult brain is pivotal for manipulating immune cell entry into the CNS and potentially reducing disease burden. A major question is whether bone marrow‐derived mononuclear phagocytes have similar features and fates as their endogenous counterparts. Significant advances in our understanding of microglia biology under physiological as well as under pathological conditions have been made in the last few years, especially by combining novel gene‐targeting techniques that allow cell manipulation and trafficking analysis. These new aspects will be discussed in the present review. ©2010 Wiley‐Liss, Inc.
8995430
Physiology of microglia.
Microglial cells are the resident macrophages in the central nervous system. These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system, disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed "resting microglia." Recent studies indicate that even in the normal brain, microglia have highly motile processes by which they scan their territorial domains. By a large number of signaling pathways they can communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells express receptors classically described for brain-specific communication such as neurotransmitter receptors and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the "activated microglial cell." This cell form has the capacity to release a large number of substances that can act detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury, proliferate, and phagocytose cells and cellular compartments.
340861
Microglia Biology in Health and Disease
Microglia cells are resident central nervous system (CNS) leukocytes that regulate innate immunity and participate in adaptive immune responses in CNS tissue. However, microglia cells also appear to play an important role during normal function of the mature nervous system. In response to injury, ischemia, and inflammatory stimuli, microglia cells assume an activated phenotype associated with proliferation, migration to the site of injury, phagocytosis of cellular debris, and elaboration (Power and Proudfoot 2001) of both neurotoxic and neurotrophic factors. Recent reports strongly suggest that regulating microglia function may be a fruitful future therapeutic target for the prevention of neurological dysfunction in a variety of CNS injuries and chronic diseases. Thus, developing a thorough understanding of extracellular signals that activate microglia as well as a complete catalogue of microglia responses to activating stimuli in both the healthy and diseased state are crucial scientific endeavors. This review presents the current understanding of the biology of microglia during normal CNS function as well as in response to CNS injury or neurodegenerative disease. In addition, microglia modulate both the activation and down-regulation of the adaptive immune response in the CNS. Evidence that microglia cells play a primary role in regulating CNS immune responses will also be discussed.
195689705
Targeting K(V) channels rescues retinal ganglion cells in vivo directly and by reducing inflammation.
Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment, and results in part from microglia-mediated inflammation. Numerous experimental studies have focused on identifying drug targets to rescue these neurons. We recently showed that K(V)1.1 and K(V)1.3 channels are expressed in adult rat RGCs and that siRNA-mediated knockdown of either channel reduces RGC death after optic nerve transection. Earlier we found that K(V)1.3 channels also contribute to microglial activation and neurotoxicity; raising the possibility that these channels contribute to neurodegeneration through direct roles in RGCs and through inflammatory mechanisms. Here, RGC survival was increased by combined siRNA-mediated knockdown of K(V)1.1 and K(V)1.3 in RGCs, but survival was much greater when knockdown of either channel was combined with intraocular injection of a K(V)1.3 channel blocker (agitoxin-2 or margatoxin). After axotomy, increased expression of several inflammation-related molecules preceded RGC loss and, consistent with a dual mechanism, their expression was differentially affected when channel knockdown in RGCs was combined with K(V)1.3 blocker injection. K(V)1.3 blockers reduced activation of retinal microglia and their tight apposition along RGC axon fascicles after axotomy, but did not prevent their migration from the inner plexiform to the damaged ganglion cell layer. Expression of several growth factors increased after axotomy; and again, there were differences following blocker injection compared with RGC-selective channel knockdown. These results provide evidence that K(V)1.3 channels play important roles in apoptotic degeneration of adult RGCs through cell-autonomous mechanisms mediated by channels in the neurons, and nonautonomous mechanisms mediated by microglia and inflammation.
8537820
Neutrophil Depletion Reduces Blood-Brain Barrier Breakdown, Axon Injury, and Inflammation After Intracerebral Hemorrhage
Neutrophils are thought to contribute to damage after intracerebral hemorrhage (ICH), but there is little direct evidence for this. We depleted circulating blood neutrophils with an anti-polymorphonuclear leukocyte antibody (anti-PMN) before inducing ICH in the rat striatum. Neutrophil infiltration, which was mainly at the edge of the hematoma, was decreased by more than 60% by anti-PMN mediated depletion. We then analyzed neutrophil contributions to BBB breakdown, white matter damage (axons and myelin), and glial and inflammatory responses, both spatially and temporally. Neutrophil depletion reduced BBB leakiness in the peri-hematoma region. Matrix metalloprotease 9, which is thought to contribute to BBB breakdown, was restricted to neutrophils after ICH and was thus reduced by neutrophil depletion. Early perihematomal axonal injury seen at 1 and 3 days after ICH was decreased by depleting neutrophils, and at later times (7 and 14 days), the astrocytic and microglia/macrophage responses were reduced in the perihematoma region and the surrounding striatum. Detailed spatial analysis showed that neutrophil depletion reduced infiltration of activated microglia/macrophages in the peri-hematoma white matter tracts and decreased myelin fragmentation and axon damage. These results show that, in experimental ICH, neutrophils produce matrix metalloprotease9and contribute to blood vessel disruption, BBB breakdown, axon damage, and astrocytic and microglial/macrophage responses that evolve after ICH.
14115010
Evolution of Inflammation and White Matter Injury in a Model of Transient Focal Ischemia
After an ischemic stroke, there is a prolonged inflammatory response and secondary phase of injury that is more amenable to treatment than acute neurotoxicity. Surprisingly, little is known about temporal and spatial relationships between inflammation and white matter injury. Here, we quantified development of white matter damage, inflammation, and a glial limitans at 1, 3, and 7 days after transient ischemia in the rat striatum using immunohistochemistry. Quantitative analysis showed that decreased staining for myelin basic protein and increased staining for damaged myelin basic protein began in the core, coincided with neutrophil infiltration, and progressed outward over time. Axon damage (i.e. accumulation of amyloid precursor protein) began at the edge of the lesion, coinciding with substantial microglia/macrophage activation, and progressed into the core. During the 7 days, activated microglia/macrophages dramatically increased only in the core and edge of the lesion. Detailed spatial analyses revealed that activated microglia/macrophages that surrounded undamaged axon bundles did not express ED1, a marker of phagocytic cells, whereas those inside damaged bundles expressed ED1. These results imply different contributions of neutrophils and microglia/macrophages to white matter injury after ischemic stroke. The distinct localizations of activated microglia/macrophages imply complex signals that regulate their migration toward and infiltration of damaged white matter.
205403918
Compartmentalization from the outside: the extracellular matrix and functional microdomains in the brain
The extracellular matrix (ECM) of the central nervous system is well recognized as a migration and diffusion barrier that allows for the trapping and presentation of growth factors to their receptors at the cell surface. Recent data highlight the importance of ECM molecules as synaptic and perisynaptic scaffolds that direct the clustering of neurotransmitter receptors in the postsynaptic compartment and that present barriers to reduce the lateral diffusion of membrane proteins away from synapses. The ECM also contributes to the migration and differentiation of stem cells in the neurogenic niche and organizes the polarized localization of ion channels and transporters at contacts between astrocytic processes and blood vessels. Thus, the ECM contributes to functional compartmentalization in the brain.
5764565
Random versus directionally persistent cell migration
Directional migration is an important component of cell motility. Although the basic mechanisms of random cell movement are well characterized, no single model explains the complex regulation of directional migration. Multiple factors operate at each step of cell migration to stabilize lamellipodia and maintain directional migration. Factors such as the topography of the extracellular matrix, the cellular polarity machinery, receptor signalling, integrin trafficking, integrin co-receptors and actomyosin contraction converge on regulation of the Rho family of GTPases and the control of lamellipodial protrusions to promote directional migration.
135981
Cell Migration: A Physically Integrated Molecular Process
The authors are grateful for financial support from the National Institutes of Health (grants GM23244 and GM53905), and to very helpful comments on the manuscript from Elliot Elson, Vlodya Gelfand, Paul Matsudaira, Julie Theriot, and Sally Zigmond. D. A. L. and A. F. H. would also like to thank Alan Wells, and Anna Huttenlocher and Rebecca Sandborg, respectively, for stimulating conversations on this subject, and Sean Palecek for Figure 2Figure 2. Finally, we extend our apologies to all our colleagues in the field whose work we were unable to cite formally because of imposed reference limitations.
22379697
Invadosomes at a glance
This article is part of a Minifocus on invadopodia and podosomes. For further reading, please see related articles: `Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia' by Renaud Poincloux et al. (J. Cell Sci. 122 , [3015-3024][1]), \`Mechanisms for transcellular
29124197
The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function
Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.
25315905
Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus.
It was recently shown by combined immunofluorescence and interference reflection microscopy that a protein named vinculin, along with alpha-actinin, is concentrated at focal adhesion plaques inside cultured normal fibroblasts [Geiger, B. (1979) Cell 18, 193-205]. These plaques are the discrete, isolated sites of strong adhesions formed between the ventral surfaces of the cells and the substrata on which they are grown. We show that after transformation of fibroblasts by Rous sarcoma virus a majority of the cells have many fewer focal adhesion plaques and now exhibit a cluster of small patches that are immunolabelled for both vinculin and alpha-actinin. Such a cluster (rosette) is located near the ventral surface of the cell, usually partly under the nucleus. The significance that these altered distributions of vinculin and alpha-actinin may have for the rounding up and loss of adherence of transformed cells is discussed.
25588790
Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes.
Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.
46034121
Assembly and biological role of podosomes and invadopodia.
Regulated tissue invasion via motile and lytic events is critical for physiological processes such as immune system function and inflammatory responses, wound healing, and organ development, but pathological subversion of this process drives tumour cell invasion and metastasis. Cell migration and invasion require the integration of several processes that include: first, the local modulation of cytoskeleton structure and contractile forces; second, the turnover of substrate adhesions and their associated microfilaments; and third, the generation of specialised, transient domains that mediate the protease-dependent focal degradation of the extracellular matrix. Recent work has re-discovered prominent actin-based cellular structures, termed invadopodia and podosomes, as unique structural and functional modules through which major invasive mechanisms are regulated. The stage is now set to unravel their roles in the physiology and pathology of tissue plasticity and repair.
33985085
The matrix corroded: podosomes and invadopodia in extracellular matrix degradation.
Podosomes and invadopodia are unique actin-rich adhesions that establish close contact to the substratum but can also degrade components of the extracellular matrix. Accordingly, matrix degradation localized at podosomes or invadopodia is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia have been mostly observed in carcinoma cells. This review highlights important new developments in the field, discusses the common and divergent features of podosomes and invadopodia and summarizes current knowledge about matrix-degrading proteinases at these structures.
15938763
The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro
BackgroundSmall-conductance Ca2+ activated K+ channels are expressed in the CNS, where KCNN2/SK2/KCa2.2 and KCNN3/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of KCNN3/SK3 in CNS microglia in vivo and in vitro, and to use an established in vitro model to determine if this channel contributes to the neurotoxic capacity of activated microglia.MethodsKCNN3 mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-κB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke).ResultsKCNN3 mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells.ConclusionsSK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic in vitro studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.
23164946
Dominance of E. coli phagocytosis over LPS in the inflammatory response of microglia
CNS bacterial infections are prevalent in neonates, the immune-compromised and elderly. During peripheral infections, macrophages employ multiple pattern recognition receptors (PRRs) to respond to pathogens, but less is known about brain microglia. We assessed microglial expression of PRRs, compared responses to whole E. coli and LPS, and tested the hypothesis that bacteria modulate the response to LPS. LPS increased the microglial phagocytic capacity, and changed expression of CD14, CR3, Fcgr1, Fcgr3a, TLR4, MARCO, MHCII, NOD2, TLR9 and SR-A, differently from stimulation with whole E. coli. Importantly, when added with LPS, E. coli dominated the microglial responses for 11/13 genes examined.
2590751
Seeing is believing? A beginners' guide to practical pitfalls in image acquisition
Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data. This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word “confocal pinhole” or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!
7885663
Blind image deconvolution: theory and applications
BLIND IMAGE DECONVOLUTION: PROBLEM FORMULATION AND EXISTING APPROACHES Tom E. Bishop, S. Derin Babacan, Bruno Amizic, Aggelos K. Katsaggelos, Tony Chan, and Rafael Molina Introduction Mathematical Problem Formulation Classification of Blind Image Deconvolution Methodologies Bayesian Framework for Blind Image Deconvolution Bayesian Modeling of Blind Image Deconvolution Bayesian Inference Methods in Blind Image Deconvolution Non-Bayesian Blind Image Deconvolution Models Conclusions References BLIND IMAGE DECONVOLUTION USING BUSSGANG TECHNIQUES: APPLICATIONS TO IMAGE DEBLURRING AND TEXTURE SYNTHESIS Patrizio Campisi, Alessandro Neri, Stefania Colonnese, Gianpiero Panci, and Gaetano Scarano Introduction Bussgang Processes Single-Channel Bussgang Deconvolution Multichannel Bussgang deconvolution Conclusions References BLIND MULTIFRAME IMAGE DECONVOLUTION USING ANISOTROPIC SPATIALLY ADAPTIVE FILTERING FOR DENOISING AND REGULARIZATION Vladimir Katkovnik, Karen Egiazarian, and Jaakko Astola Introduction Observation Model and Preliminaries Frequency Domain Equations Projection Gradient Optimization Anisotropic LPA-ICI Spatially Adaptive Filtering Blind Deconvolution Algorithm Identifiability and Convergence Simulations Conclusions Acknowledgments References BAYESIAN METHODS BASED ON VARIATIONAL APPROXIMATIONS FOR BLIND IMAGE DECONVOLUTION Aristidis Likas and Nikolas P. Galatsanos Introduction Background on Variational Methods Variational Blind Deconvolution Numerical Experiments Conclusions and Future Work APPENDIX A: Computation of the Variational Bound F(q,?) APPENDIX B: Maximization of F(q,?) References DECONVOLUTION OF MEDICAL IMAGES FROM MICROSCOPIC TO WHOLE BODY IMAGES Oleg V. Michailovich and Dan R. Adam Introduction Nonblind Deconvolution Blind Deconvolution in Ultrasound Imaging Blind Deconvolution in SPECT Blind Deconvolution in Confocal Microscopy Summary References BAYESIAN ESTIMATION OF BLUR AND NOISE IN REMOTE SENSING IMAGING Andre Jalobeanu, Josiane Zerubia, and Laure Blanc-Feraud Introduction The Forward Model Bayesian Estimation: Invert the Forward Model Possible Improvements and Further Development Results Conclusions Acknowledgments References DECONVOLUTION AND BLIND DECONVOLUTION IN ASTRONOMY Eric Pantin, Jean-luc Starck, and Fionn Murtagh Introduction The Deconvolution Problem Linear Regularized Methods CLEAN Bayesian Methodology Iterative Regularized Methods Wavelet-Based Deconvolution Deconvolution and Resolution Myopic and Blind Deconvolution Conclusions and Chapter Summary Acknowledgments References MULTIFRAME BLIND DECONVOLUTION COUPLED WITH FRAME REGISTRATION AND RESOLUTION ENHANCEMENT Filip Sroubek, Jan Flusser, and Gabriel Cristobal Introduction Mathematical Model Polyphase Formulation Reconstruction of Volatile Blurs Blind Superresolution Experiments Conclusions Acknowledgments References BLIND RECONSTRUCTION OF MULTIFRAME IMAGERY BASED ON FUSION AND CLASSIFICATION Dimitrios Hatzinakos, Alexia Giannoula, and Jianxin Han Introduction System Overview Recursive Inverse Filtering with Finite Normal-Density Mixtures (RIF-FNM) Optimal Filter Adaptation Effects of Noise The Fusion and Classification Recursive Inverse Filtering Algorithm (FAC-RIF) Experimental Results Final Remarks References BLIND DECONVOLUTION AND STRUCTURED MATRIX COMPUTATIONS WITH APPLICATIONS TO ARRAY IMAGING Michael K. Ng and Robert J. Plemmons Introduction One-Dimensional Deconvolution Formulation Regularized and Constrained TLS Formulation Numerical Algorithms Two-Dimensional Deconvolution Problems Numerical Examples Application: High-Resolution Image Reconstruction Concluding Remarks and Current Work Acknowledgments References INDEX
27791602
Podosomes: adhesion hot-spots of invasive cells.
Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.
36552424
The Adaptor Protein Fish Associates with Members of the ADAMs Family and Localizes to Podosomes of Src-transformed Cells*
Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.
38485364
The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.
Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.
3225580
Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases
Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1–deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities. Directional persistency of migration is lost, and the cells show an impaired response to external directional stimuli. Both Src inactivation and p190RhoGAP knockdown restore the wild-type phenotype to caveolin-1–deficient cells, suggesting that caveolin-1 stimulates normal Rho GTP loading through inactivation of the Src–p190RhoGAP pathway. These findings highlight the importance of caveolin-1 in the establishment of cell polarity during directional migration through coordination of the signaling of Src kinase and Rho GTPases.
8603155
Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.
Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.
43387935
ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells.
Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.
8878582
The involvement of the tyrosine kinase c-Src in the regulation of reactive oxygen species generation mediated by NADPH oxidase-1.
NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.
42231397
Novel p47phox-Related Organizers Regulate Localized NADPH Oxidase 1 (Nox1) Activity
Tks proteins, which are Src substrates, control the localized production of reactive oxygen species by Nox. Localizing ROS Localized formation of reactive oxygen species (ROS) by NADPH oxidase (Nox) complexes is crucial to various physiological and pathological processes; however, the mechanisms targeting ROS production to particular cellular locales have been unclear. Here, Gianni et al. identify Tks4 and Tks5 as members of a family of organizer proteins that regulate assembly of the Nox complex in response to various signals. They found that Tks4 and Tks5 supported ROS production by Nox1 and Nox3 and that, in a line of human colon cancer cells, Nox1 localized to invadopodia, actin-rich structures that facilitate degradation of the extracellular matrix and cancer invasion. Furthermore, Tks4 was critical to Nox1-dependent ROS production by these cells and in their ability to degrade the extracellular matrix. The authors thus propose that Tks4 targets Nox1 to invadopodia and, more generally, that Tks organizer proteins may play an important role in the localized ROS production by Nox complexes. The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47phox-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain–mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase– and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation.
25032364
Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases
Chemokines were originally described as chemotactic cytokines involved in leukocyte trafficking. Research over the last decade, however, has shown that chemokine receptors are not restricted to leukocytes. In the brain, chemokine receptors are not only found in microglia (a brain macrophage), but also in astrocytes, oligodendrocytes and neurons. In this review, we describe the spatial and cellular distribution of chemokine receptors in the brain, distinguishing between constitutively and inducibly expressed receptors. We then discuss possible physiological functions, including neuronal migration, cell proliferation and synaptic activity. Evidence is emerging that chemokine receptors are also involved in neuronal death and hence neurodegenerative diseases. Chemokines may induce neuronal death either indirectly (e.g. through activation of microglia killing mechanisms) or directly through activation of neuronal chemokine receptors. Disease processes in which chemokines and their receptors are likely to be involved include multiple sclerosis (MS), Alzheimer's disease (AD), HIV-associated dementia (HAD) and cerebral ischemic disease. The study of chemokines and their receptors in the central nervous system (CNS) is not only relevant for the understanding of brain physiology and pathophysiology, but may also lead to the development of targeted treatments for neurodegenerative diseases.
9270237
Extracellular ATP or ADP Induce Chemotaxis of Cultured Microglia through Gi/o-Coupled P2Y Receptors
The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid, suramin, or adenosine-3′-phosphate-5′-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by Gi/o-coupled P2Y receptors.
13913614
The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system
Chondroitin sulfate proteoglycans (CSPGs) consist of a core protein and glycosaminoglycan (GAG) chains. There is enormous structural diversity among CSPGs due to variation in the core protein, the number of GAG chains and the extent and position of sulfation. Most CSPGs are secreted from cells and participate in the formation of the extracellular matrix (ECM). CSPGs are able to interact with various growth-active molecules and this may be important in their mechanism of action. In the normal central nervous system (CNS), CSPGs have a role in development and plasticity during postnatal development and in the adult. Plasticity is greatest in the young, especially during critical periods. CSPGs are crucial components of perineuronal nets (PNNs). PNNs have a role in closure of the critical period and digestion of PNNs allows their re-opening. In the adult, CSPGs play a part in learning and memory and the hypothalamo-neurohypophysial system. CSPGs have an important role in CNS injuries and diseases. After CNS injury, CSPGs are the major inhibitory component of the glial scar. Removal of CSPGs improves axonal regeneration and functional recovery. CSPGs may also be involved in the pathological processes in diseases such as epilepsy, stroke and Alzheimer's disease. Several possible methods of manipulating CSPGs in the CNS have recently been identified. The development of methods to remove CSPGs has considerable therapeutic potential in a number of CNS disorders.
36412733
Brain Extracellular Matrix in Neurodegeneration
The role of extracellular matrix (ECM) in neurological development, function and degeneration has evolved from a simplistic physical adhesion to a system of intricate cellular signaling. While most cells require ECM adhesion to survive, it is now clear that differentiated function is intimately dependent upon cellular interaction with the ECM. Therefore, it is not surprising that the ECM is increasingly found to be involved in the enigmatic process of neurodegeneration. Descriptive studies of human neurodegenerative disorders and experimental studies of animal models of neurodegeneration have begun to define potential mechanisms of ECM disruption that can lead to synaptic and neuronal loss.
23758712
The role of extracellular matrix in CNS regeneration
Chondroitin sulfate proteoglycans are the principal inhibitory component of glial scars, which form after damage to the adult central nervous system and act as a barrier to regenerating axons. Recent findings have furthered our understanding of the mechanisms that result in a failure of regeneration after spinal cord injury and suggest that a multipartite approach will be required to facilitate long-distance regeneration and functional recovery.
28534752
Modulation of the immune response by extracellular matrix proteins
Abstract.Inflammation entrains a focused and coordinated response from many different elements. Soluble factors such as chemokines and cytokines direct the recruitment, differentiation, and fate of leukocytes. Cells and pathogens are killed and consumed, yet where the response is effective, inflammation will melt away, leaving a healthy functioning tissue. All this commonly takes place in an environment known as the extracellular matrix (ECM). The ECM is not a passive partner in the process and recent work demonstrates the important role that proteins found in this environment play in connecting different parts of the immune response together. In this review we will focus on these connections and the proteins that make them. One emerging trend that we will highlight is the ability of endogenous molecules to interact with receptors that are better known as sensors of the molecular fingerprints of infection. We propose that this may be particularly relevant in the context of autoimmunity, since the provision of such signals may be crucial in breaking tolerance.
15028111
Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection
Microglial activation in response to pathological stimuli is characterized by increased migratory activity and potential cytotoxic action on injured neurons during later stages of neurodegeneration. The initial molecular changes in the CNS favoring neuronofugal migration of microglia remain, however, largely unknown. We report that the extracellular matrix protein tenascin-R (TN-R) present in the intact CNS is antiadhesive for activated microglia, and its downregulation after facial nerve axotomy may account for the loss of motoneuron protection and subsequent neurodegeneration. Studies on the protein expression in the facial and hypoglossal nucleus in rats demonstrate that TN-R is a constituent of the perineuronal net of motoneurons and 7 d after peripheral nerve injury becomes downregulated in the corresponding motor nucleus. This downregulation is reversible under regenerative (nerve suture) conditions and irreversible under degenerative (nerve resection) conditions. In short-term adhesion assays, the unlesioned side of brainstem cryosections from unilaterally operated animals is nonpermissive for activated microglia, and this nonpermissiveness is almost abolished by a monoclonal antibody to TN-R. Microglia-conditioned media and tumor necrosis factor-α downregulate TN-R protein and mRNA synthesis by cultured oligodendrocytes, which are one of the sources for TN-R in the brainstem. Our findings suggest a new role for TN-R in neuronal protection against activated microglia and the participation of the latter in perineuronal net destruction, e.g., downregulation of TN-R.
7743721
SC1/Hevin and Reactive Gliosis After Transient Ischemic Stroke in Young and Aged Rats
SC1 is a member of the SPARC family of glycoproteins that regulate cell-matrix interactions in the developing brain. SC1 is expressed in astrocytes, but nothing is known about the expression in the aged or after stroke. We found that after focal striatal ischemic infarction in adult rats, SC1 increased in astrocytes surrounding the infarct and in the glial scar, but in aged rats, SC1 was lower at the lesion edge. Glial fibrillary acidic protein (GFAP) also increased, but it was less prominent in reactive astrocytes further from the lesion in the aged rats. On the basis of their differential expression of several molecules, 2 types of reactive astrocytes with differing spatiotemporal distributions were identified. On Days 3 and 7, SC1 was prevalent in cells expressing markers of classic reactive astrocytes (GFAP, vimentin, nestin, S100&bgr;), as well as apoliprotein E (ApoE), interleukin 1&bgr;, aggrecanase 1 (ADAMTS4), and heat shock protein 25 (Hsp25). Adjacent to the lesion on Days 1 and 3, astrocytes with low GFAP levels and a "starburst" SC1 pattern expressed S100&bgr;, ApoE, and Hsp32 but not vimentin, nestin, interleukin 1&bgr;, ADAMTS4, or Hsp25. Neither cell type was immunoreactive for NG2,CC-1, CD11b, or ionized calcium-binding adapter-1. Their differing expression of inflammation-related and putatively protective molecules suggests different roles for starburst and classic reactive astrocytes in the early glial responses to ischemia.
1931864
Microglial Activation and Matrix Protease Generation During Focal Cerebral Ischemia
Local environmental conditions contribute to the activation state of cells. Extracellular matrix glycoproteins participate in cell-cell boundaries within the microvascular and extravascular tissues of the central nervous system and provide a scaffold for the local environment. These conditions are altered during focal cerebral ischemia (and other central nervous system disorders) when extracellular matrix boundaries are degraded or when matrix proteins in the vascular circulation enter the neuropil as the microvascular permeability barrier is degraded. Microglia in the resting state become activated after the onset of ischemia. During activation these cells can express a number of factors and proteases, including latent matrix metalloproteinase-9 (pro–MMP-9). Whereas MMP-9 and MMP-2 are generated early during focal ischemia in select models, their cellular sources in vivo are still under study. In vitro microglia cells activate and respond to exposure to specific matrix proteins (eg, vitronectin, fibronectin) that circulate. Certain MMP inhibitors, specifically tetracycline derivatives, can modulate microglial activation and reduce injury volume in limited studies. But, the injury reduction relies on preinjury exposure to the tetracycline. Other studies underway suggest the hypothesis that microglial cell activation and pro-MMP-9 generation during focal cerebral ischemia is promoted in part by matrix proteins in the circulation that extravasate into the neuropil when the blood-brain barrier is compromised. These matrix proteins are known to activate microglia through their specific cell surface matrix receptors.
2363377
Invadosomes: intriguing structures with promise.
Podosomes and invadopodia are highly dynamic, actin-rich adhesion structures and represent the two founding members of the invadosome family. Podosomes form spontaneously in cells of the myelomonocytic lineage but a plethora of other cells are endowed with this capacity, under appropriate stimulation, such as a soluble factor, matrix receptor, or cell stress. Related structures called invadopodia are detected in some cancer cells or appear on cells upon oncogenic transformation. In contrast to other cell adhesion devices, invadosomes harbour metalloproteases which degrade components of the extracellular matrix. Because of this distinctive feature, invadosomes have been systematically linked with invasion processes. However, it now appears that these intriguing structures are endowed with other functions and are therefore expected to contribute to a wider range of biological processes. The invadosome field has been progressing for thirty years, expanding exponentially during the last decade, where tremendous advances have been made regarding the molecular mechanism underlying their formation, dynamics and function. Invadosomes are involved in human diseases but the causative link remains to be established. To this end, 3D analysis of invadosomes is now being actively developed in ex vivo and in vivo models to demonstrate their occurrence and establish their role in pathological and physiological processes.
7085813
Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells.
Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.
31346091
Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages.
The actin-bundling protein fimbrin is homologous to 1-plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069-1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton-X-100 (soluble fraction), Tween 40-deoxy-cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone-extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC-extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins.
7096752
MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro
PDGF enhances podosome formation and cell migration by regulating expression of the microRNAs miR-143 and -145, which target PDGF-R, PKC-ε, and fascin.
564175
Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling.
Adhesion, migration and invasion of endothelial cells are prerequisites for the formation of blood vessels and have to be controlled on a subcellular level. We report that subconfluent human umbilical vein endothelial cells (HUVEC) are able to constitutively form podosomal adhesions that are sites of matrix metalloprotease concentration and matrix degradation. Importantly, incubation of serum-starved cells with VEGF or TNFalpha revealed the dependence of podosomes on cytokine signaling. Podosome formation was also stimulated by addition of monocytes to HUVEC. Microinjection/application of specific inhibitors or active/inactive mutants showed that regulatory pathways include Src kinase and RhoGTPase signaling, N-WASP activation and Arp2/3 complex-dependent actin nucleation. In sum, our data show that HUVEC displaying a migratory phenotype constitutively form f-actin-rich adhesions with podosomal characteristics downstream of cytokine signaling. We propose that HUVEC podosomes play an important role in endothelial cell migration and invasion.
7224989
Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis.
Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck(-/-) mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck(-/-) BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.
22607798
TGFβ-induced endothelial podosomes mediate basement membrane collagen degradation in arterial vessels
Podosomes are specialized plasma-membrane actin-based microdomains that combine adhesive and proteolytic activities to spatially restrict sites of matrix degradation in in vitro assays, but the physiological relevance of these observations remain unknown. Inducible rings of podosomes (podosome rosettes) form in cultured aortic cells exposed to the inflammatory cytokine TGFβ. In an attempt to prove the existence of podosomes in living tissues, we developed an ex vivo endothelium observation model. This system enabled us to visualize podosome rosettes in the endothelium of native arterial vessel exposed to biologically active TGFβ. Podosomes induced in the vessel appear similar to those formed in cultured cells in terms of molecular composition, but in contrast to the latter, arrange in a protruding structure that is similar to invadopodia. Local degradation of the basement membrane scaffold protein collagen-IV, is observed underneath the structures. Our results reveal for the first time the presence of podosome rosettes in the native endothelium and provide evidence for their capacity to degrade the basement membrane, opening up new avenues to study their role in vascular pathophysiology. We propose that podosome rosettes are involved in arterial vessel remodeling.
30794665
Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice
To understand the normal, physiological role of the c-src proto-oncogene, a null mutation was introduced into the gene by homologous recombination in mouse embryonic stem cells. Two independent targeted clones were used to generate chimeras that transmitted the mutated allele to their offspring. Intercrossing of heterozygotes gave rise to live born homozygotes, but most of these mice died within the first few weeks of birth. Histological and hematological examination of the homozygous mutants did not reveal detectable abnormalities in the brain or platelets, where src is most highly expressed. However, these mutants were deficient in bone remodeling, indicating impaired osteoclast function, and developed osteopetrosis. These results demonstrate that src is not required for general cell viability (possibly because of functional overlap with other tyrosine kinases related to src) and uncover an essential role for src in bone formation.
5620213
A partial down-regulation of WASP is sufficient to inhibit podosome formation in dendritic cells.
The Wiskott Aldrich syndrome protein (WASP) is a hematopoietic-specific cytoskeletal regulator that is necessary for induction of normal immunity. In the context of effective gene therapy for WAS, cellular models of human WASP deficiency are important for definition of the threshold of protein expression required for optimal activity. Using lentiviral vector-mediated RNA interference (RNAi), we were able to down-regulate the levels of human WASP in cell lines and primary cells. In dendritic cells (DC), RNAi-induced WASP deficiency did not impair phenotypic maturation but perturbed cytoskeletal organization. As a result, podosomes, which are actin-rich structures present in immature adherent DC, were formed less efficiently and motility was disturbed. Overall, treatment of cells with RNAi recapitulated the phenotype of cells derived from patients or animals with inactivating mutations of the WAS gene. Interestingly, reduction of the levels of WASP to about 60% of normal was sufficient to inhibit the formation of podosomes in DC, implying that this cell type requires near-normal levels of WASP to sustain physiological cytoskeleton-dependent activities.
39753048
N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis.
Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.
43351678
Chemotaxis of macrophages is abolished in the Wiskott‐Aldrich syndrome
Wiskott‐Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct‐viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42‐WASp‐mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen‐presenting cells may play a significant part in the immune deficiency and eczema of WAS.
42728755
SRC: a century of science brought to the clinic.
The SRC family kinases are the largest family of nonreceptor tyrosine kinases and one of the best-studied targets for cancer therapy. SRC, arguably the oldest oncogene, has been implicated in pathways regulating proliferation, angiogenesis, invasion and metastasis, and bone metabolism. More recently, researchers have proposed that the transforming ability of SRC is linked to its ability to activate key signaling molecules in these pathways, rather than through direct activity. It has been hypothesized that blocking SRC activation may inhibit these pathways, resulting in antitumor activity. However, successfully targeting SRC in a clinical setting remains a challenge, and SRC inhibitors have only recently begun to move through clinical development. Preclinical studies have identified specific molecular "subgroups" and histologies that may be more sensitive to SRC inhibition. In addition, other studies have demonstrated synergistic interactions between SRC inhibitors and other targeted therapies and cytotoxics. In this review, we summarize SRC biology and how it has been applied to the clinical development of SRC inhibitors. The status of SRC inhibitors, including dasatinib, saracatinib, and bosutinib, which are in phase 1, 2, and 3 trials, is highlighted.
28655834
The Polarization Defect of Wiskott-Aldrich Syndrome Macrophages Is Linked to Dislocalization of the Arp2/3 Complex1
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally characterized by the clinical triad eczema, thrombocytopenia, and severe immunodeficieny, with recurrent bacterial and viral infections, indicating a profound immune cell defect. Such altered immune cells include monocytes, macrophages, and dendritic cells, which were reported to display disturbed cell polarization or chemotaxis. WAS is caused by mutations in the WAS protein (WASp), which is thought to organize the actin cytoskeleton through the Arp2/3 complex. Here we show that the Arp2/3 complex is an integral part of podosomes, actin-rich adhesion structures of macrophages, and that WAS macrophages fail to organize the Arp2/3 complex into podosomes. We also demonstrate that microinjection of a C-terminal acidic stretch of WASp into normal macrophages displaces Arp2/3 from podosomes and, in combination with chemoattractant stimulation of cells, induces a phenotype resembling the polarization-defective phenotype of stimulated WAS macrophages. These findings point to an important role of the Arp2/3 complex in polarization and migration of immune cells.
24335450
Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption
Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast‐like multinucleated cells prepared from co‐cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25‐dihydroxyvitamin D3. When osteoclast‐like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti‐phosphotyrosine antibody. Western blot analysis revealed that 115‐to 130‐kD proteins were tyrosine‐phosphorylated in osteoclast‐like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti‐p 125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast‐like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F‐actin‐containing podosomes in osteoclast‐like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast‐like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.
5943359
Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation.
The cellular mechanisms that configure the cytoskeleton during migration of dendritic cells (DCs) are poorly understood. Immature DCs assemble specialized adhesion structures known as podosomes at their leading edge; these are associated with the localized recruitment of the Wiskott-Aldrich Syndrome protein (WASp) and the actin organizing actin-related protein 2/3 complex. In immature DCs lacking WASp, podosomes are absent, residual dysmorphic lamellipodia and filopodia are nonpolarized, and migration is severely compromised. Microinjection studies indicate that podosome assembly and polarization require concerted action of Cdc42, Rac, and Rho, thereby providing a link between sequential protrusive and adhesive activity. Formation of podosomes is restricted to cells with an immature phenotype, indicating a specific role for these structures during the early migratory phase. (Blood. 2001;98:1142-1149)
31328641
Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages.
Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific, multidomain protein whose mutation is responsible for the immunodeficiency disorder Wiskott-Aldrich syndrome. WASp contains a binding motif for the Rho GTPase CDC42Hs as well as verprolin/cofilin-like actin-regulatory domains, but no specific actin structure regulated by CDC42Hs-WASp has been identified. We found that WASp colocalizes with CDC42Hs and actin in the core of podosomes, a highly dynamic adhesion structure of human blood-derived macrophages. Microinjection of constitutively active V12CDC42Hs or a constitutively active WASp fragment consisting of the verprolin/cofilin-like domains led to the disassemly of podosomes. Conversely, macrophages from patients expressing truncated forms of WASp completely lacked podosomes. These findings indicate that WASp controls podosome assembly and, in cooperation with CDC42Hs, podosome disassembly in primary human macrophages.
13052818
Gelsolin-Independent Podosome Formation in Dendritic Cells
Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.
24256344
The leukocyte podosome.
Myeloid leukocytes are the first line of host defence. When they sense perturbations in tissue homeostasis such as infection, inflammation and ischemia, they respond by trafficking. Whilst neutrophils and macrophages migrate to sites of infection, dendritic cells (DC) migrate from tissue-resident sites back into lymph nodes where they activate T and B lymphocytes. The directed migration of these leukocytes through peripheral tissues is thus crucial for their function. This article considers recent advances in our understanding of the adhesive and motile behaviour of macrophages and DC, with particular emphasis on the podosomes that appear to be required for normal migration through extracellular matrices.
31772217
Podosome and sealing zone: specificity of the osteoclast model.
The bone resorption function of osteoclasts is dependent on the integrity of the actin cytoskeleton. Depending on the substratum upon which the osteoclasts are spread, there are two different structures of actin known as podosomes and the sealing zone. To understand the specific properties and relationship of podosomes and the sealing zone, we used live-cell imaging of cultured osteoclasts. When cultured on extracellular matrix components, podosomes in these cells are organized in higher-ordered structures. These are clustered podosomes that will arrange later into dynamic short-lived rings which finally expand to the cell periphery to form a stable long-lived podosome belt in fully differentiated cells. In osteoclasts, this specific podosome patterning is under the control of microtubules (MTs). Indeed, nocodazole treatment does not affect podosome formation but only the transition between clusters/rings and belts. During this transition, MTs accumulate a specific post-translational modification of tubulin by acetylation. This process is repressed by an inhibitory pathway involving the GTPase Rho, its effector mDIA2 and the recently discovered tubulin deacetylase HDAC6. The specific function of this acetylation is still unknown but is also observed in active osteoclasts forming a sealing zone which is also MT dependent. Thus, it appears that the podosome belt is reminiscent of the sealing zone. Indeed, podosome belts and sealing zones are characterized by their overall stability. Despite their similar behavior, a sealing zone is not formed by fusion of podosomes. The formation of a podosome belt or a sealing zone is controlled by the external environment. Indeed, only the bone mineral fraction, known as apatite crystal, is able to induce sealing zone formation in mature osteoclasts. Contact of osteoclasts with apatite stimulates the non-receptor tyrosine kinase c-Src and the GTPase Rho in order to form the sealing zone. As we will discuss in this review, it appears that podosomes and the sealing zone are strikingly linked.
3841330
Rho GTPases in osteoclasts: orchestrators of podosome arrangement.
Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts, develop podosomes instead of stress fibers and focal adhesions to adhere and migrate. Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.
205643127
The podosome marker protein Tks5 regulates macrophage invasive behavior
Tks5 is a Src substrate and adaptor protein previously recognized for its regulation of cancer cell invasion through modulation of specialized adhesion structures called podosomes/invadopodia. Here we show for the first time that Tks5 localizes to the podosomes of primary macrophages, and that Tks5 protein levels increase concurrently with podosome deposition during the differentiation of monocytes into macrophages. Similar results are reported for model THP‐1 cells, which differentiate into macrophages and form proteolytically active podosomes in response to a PKC signaling agonist (PMA) and with sensitivity to a PKC inhibitor (bisindolylmaleimide). Genetic manipulation of Tks5 expression (silencing and overexpression) in stable THP‐1 cell lines does not independently alter this macrophage differentiation process. Nor do these cells lose the ability to focalize F‐actin and its accessory proteins into podosome‐like structures following PMA treatment. However, Tks5 directly controls podosome‐associated gelatin degradation and invasion through collective changes in adhesion, chemotaxis, and the expression/proteolytic activity of MMP9. The Src family kinase‐dependent phosphorylation of Tks5 is also implicated in the regulation of THP‐1 macrophage invasive behavior. These results therefore define a previously unappreciated function of Tks5 signaling specific to the functional attributes of the macrophage podosome in adhesion, motility, and extracellular matrix‐remodeling. © 2011 Wiley Periodicals, Inc.
14447915
Dystroglycan, Tks5 and Src Mediated Assembly of Podosomes in Myoblasts
Background Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion. Principal Findings We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with β-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation. Conclusions We propose therefore, that Src-dependent phosphorylation of β-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.
205762426
Tks5 recruits AFAP-110, p190RhoGAP, and cortactin for podosome formation.
Podosome formation in vascular smooth muscle cells is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the molecular mechanism that underlies their specific recruitment to podosomes remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. We show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5 mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. By expressing a catalytically inactive point mutant and by siRNA-mediated expression knock-down we also provide evidence that p190RhoGAP is required for podosome formation. Together our findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation.
33034667
The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion.
Some years ago, we employed a screen of phage cDNA expression libraries to identify novel substrates of the protein tyrosine kinase Src. One of these, Tks5 (previously known as Fish), is a large scaffolding protein with an amino-terminal PX domain and five SH3 domains. In normal fibroblasts, Tks5 is cytoplasmic, but the protein is found in podosomes when the cells are transformed with Src. Using short interfering RNA technology, we have shown that Tks5 is required for podosome formation. Furthermore, cells with reduced Tks5 expression are poorly invasive through Matrigel. Tks5 is expressed and localized to podosomes in invasive human cancer cell lines and in tumor tissue, particularly breast cancers and melanomas. In these cells too, Tks5 is required for invasion. Our future work will focus on the identification of the binding partners of Tks5 that are responsible for podosome formation and invasion, and on determining the role of Tks5 in animal models of metastasis.
2296730
Src-mediated Tyrosine Phosphorylation of Caveolin-1 Induces Its Association with Membrane Type 1 Matrix Metalloproteinase*
We have recently shown that stimulation of endothelial cells with vascular endothelial growth factor (VEGF) induces dissociation of caveolin-1 from the VEGFR-2 receptor, followed by Src family kinase-dependent tyrosine phosphorylation of the protein (Labrecque, L., Royal, I., Surprenant, D. S., Patterson, C., Gingras, D., and Béliveau, R. (2003) Mol. Biol. Cell 14, 334–347). In this study, we provide evidence that the VEGF-dependent tyrosine phosphorylation of caveolin-1 induces interaction of the protein with the membrane-type 1 matrix metalloproteinase (MT1-MMP). This interaction requires the phosphorylation of caveolin-1 on tyrosine 14 by members of the Src family of protein kinases, such as Src and Fyn, because it is completely abolished by expression of a catalytically inactive Src mutant or by site-directed mutagenesis of tyrosine 14 of caveolin-1. Most interestingly, the association of MT1-MMP with phosphorylated caveolin-1 induced the recruitment of Src and a concomitant inhibition of the kinase activity of the enzyme, suggesting that this complex may be involved in the negative regulation of Src activity. The association of MT1-MMP with phosphorylated caveolin-1 occurs in caveolae membranes and involves the cytoplasmic domain of MT1-MMP because it was markedly reduced by mutation of Cys574 and Val582 residues of the cytoplasmic tail of the enzyme. Most interestingly, the reduction of the interaction between MT1-MMP and caveolin-1 by using these mutants also decreases MT1-MMP-dependent cell locomotion. Overall these results indicate that MT1-MMP associates with tyrosine-phosphorylated caveolin-1 and that this complex may play an important role in MT1-MMP regulation and function.
33489984
Oxidative stress, caveolae and caveolin-1.
Oxidative stress underlies a range of pathophysiological conditions. Reactive oxygen species are also generated intracellularly to serve as second messengers and some are linked to caveolae/raft signalling systems. The effect of oxidative stress on caveolin-1 expression, post-translational modifications, membrane trafficking and function are described.
37353231
A model for phospho-caveolin-1-driven turnover of focal adhesions
The regulation of Focal Adhesion (FA) dynamics is a key aspect of cellular motility. FAs concentrate integrins and associated cytoskeletal elements as well as a large number of regulatory proteins, including adapters, kinases and small GTPases of the Rho Family. We have recently shown that activated Rac1 can localize to FAs and can initiate the accumulation of the adapter protein Caveolin1 (Cav1) at FAs. As reported by several groups including ours, this translocation requires Cav1 phosphorylation at Tyr14, presumably by Src. Here we provide additional data regarding this process and briefly review recent literature. Finally, we incorporated the different pieces of available information into a mechanistic model. This model proposes that local Rac1 activation initiates a series of events that involve endosomal traffic of Cav1 and Src, targeting these proteins to or near FAs. Next, within specific membrane domains, Src can mediate the phosphorylation of Cav1 at Tyr 14, which is important for the stable FA localization of Cav1. Finally, dephosphorylation of Cav1 may represent a key step required for internalization, FA turnover and cell motility.
207548172
Lipid rafts and caveolin-1 are required for invadopodia formation and extracellular matrix degradation by human breast cancer cells.
Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.
2086599
Invadopodia biogenesis is regulated by caveolin‐mediated modulation of membrane cholesterol levels
Invadopodia are proteolytically active protrusions formed by invasive tumoural cells when grown on an extracellular matrix (ECM) substratum. Clearly, invadopodia are specialized membrane domains acting as sites of signal transduction and polarized delivery of components required for focalized ECM degradation. For these reasons, invadopodia are a model to study focal ECM degradation by tumour cells. We investigated the features of invadopodia membrane domains and how altering their composition would affect invadopodia biogenesis and function. This was achieved through multiple approaches including manipulation of the levels of cholesterol and other lipids at the plasma membrane, alteration of cholesterol trafficking by acting on caveolin 1 expression and phosphorylation. We show that cholesterol depletion impairs invadopodia formation and persistence, and that invadopodia themselves are cholesterol‐rich membranes. Furthermore, the inhibition of invadopodia formation and ECM degradation after caveolin 1 knock‐down was efficiently reverted by simple provision of cholesterol. In addition, the inhibitory effect of caveolin 3DGV expression, a mutant known to block cholesterol transport to the plasma membrane, was similarly reverted by provision of cholesterol. We suggest that invadopodia biogenesis, function and structural integrity rely on appropriate levels of plasma membrane cholesterol, and that invadopodia display the properties of cholesterol‐rich membranes. Also, caveolin 1 exerts its function in invadopodia formation by regulating cholesterol balance at the plasma membrane. These findings support the connection between cholesterol, cancer and caveolin 1, provide further understanding of the role of cholesterol in cancer progression and suggest a mechanistic framework for the proposed anti‐cancer activity of statins, tightly related to their blood cholesterol‐lowering properties.
206869062
A novel and specific NADPH oxidase-1 (Nox1) small-molecule inhibitor blocks the formation of functional invadopodia in human colon cancer cells.
The NADPH oxidase (Nox) proteins catalyze the regulated formation of reactive oxygen species (ROS), which play key roles as signaling molecules in several physiological and pathophysiological processes. ROS generation by the Nox1 member of the Nox family is necessary for the formation of extracellular matrix (ECM)-degrading, actin-rich cellular structures known as invadopodia. Selective inhibition of Nox isoforms can provide reversible, mechanistic insights into these cellular processes in contrast to scavenging or inhibition of ROS production. Currently no specific Nox inhibitors have been described. Here, by high-throughput screening, we identify a subset of phenothiazines, 2-acetylphenothiazine (here referred to as ML171) (and its related 2-(trifluoromethyl)-phenothiazine) as nanomolar, cell-active, and specific Nox1 inhibitors that potently block Nox1-dependent ROS generation, with only marginal activity on other cellular ROS-producing enzymes and receptors including the other Nox isoforms. ML171 also blocks the ROS-dependent formation of ECM-degrading invadopodia in colon cancer cells. Such effects can be reversed by overexpression of Nox1 protein, which is suggestive of a selective mechanism of inhibition of Nox1 by this compound. These results elucidate the relevance of Nox1-dependent ROS generation in mechanisms of cancer invasion and define ML171 as a useful Nox1 chemical probe and potential therapeutic agent for inhibition of cancer cell invasion.
1989875
c-Src–Mediated Phosphorylation of NoxA1 and Tks4 Induces the Reactive Oxygen Species (ROS)–Dependent Formation of Functional Invadopodia in Human Colon Cancer Cells
Reactive oxygen species (ROS) generated by the NADPH oxidase system have been shown to be necessary for the invadopodia formation and function. We show here that the abolishment of Src-mediated phosphorylation of NoxA1 and Tks4 blocks their binding, decreases Nox1-dependent ROS generation, and inhibits the invadopodia formation and ECM degradation.
15133665
Microglia Kv1.3 Channels Contribute to Their Ability to Kill Neurons
Many CNS disorders involve an inflammatory response that is orchestrated by cells of the innate immune system: macrophages, neutrophils, and microglia (the endogenous CNS immune cell). Hence, there is considerable interest in anti-inflammatory strategies that target these cells. Microglia express Kv1.3 (KCNA3) channels, which we showed previously are important for their proliferation and the NADPH-mediated respiratory burst. Here, we demonstrate the potential for targeting Kv1.3 channels to control CNS inflammation. Rat microglia express Kv1.2, Kv1.3, and Kv1.5 transcripts and protein, but only a Kv1.3 current was detected. When microglia were activated with lipopolysaccharide or a phorbol ester, only the Kv1.3 transcript (but not protein) expression changed. Using a Transwell cell-culture system that allows separate drug treatment of microglia or neurons, we found that activated microglia killed postnatal hippocampal neurons through a process that requires Kv1.3 channel activity in microglia but not in neurons. A major neurotoxic molecule in this model was peroxynitrite, which is formed from superoxide and nitric oxide; thus, it is significant that Kv1.3 channel blockers reduced the respiratory burst, but not nitric oxide production, by the activated microglia. In addressing the biochemical pathway affected by Kv1.3 channel activity, we found that Kv1.3 acts via a different cellular mechanism from the broad-spectrum drug minocycline, which is often used in animal models of neuroinflammation. That is, the dose-dependent reduction in neuron killing by minocycline corresponded with a reduction in p38 mitogen-activated protein kinase activation in microglia; however, none of the Kv1.3 blockers affected p38 activation.