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Nature Communications Nov 2019Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and...
Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.
Topics: Actins; Animals; Cell Adhesion; Cell Membrane; Cell Movement; Cells, Cultured; Dendritic Cells; Humans; Mechanotransduction, Cellular; Mice; Podosomes
PubMed: 31729386
DOI: 10.1038/s41467-019-13123-3 -
The Journal of Cell Biology Oct 2019Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3...
Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3-null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.
Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Chromatography, Liquid; Cytoskeletal Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Paxillin; Podosomes; RAW 264.7 Cells; Signal Transduction; Tandem Mass Spectrometry; Transcription Factors
PubMed: 31537712
DOI: 10.1083/jcb.201903109 -
The International Journal of... Aug 2015Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types.... (Review)
Review
Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types. A podosome is a dynamic actin-based and membrane-bound microdomain and the organelle consists in an interconnected network of such basic units, forming a cytoskeletal superstructure linked to the plasma membrane. At this strategic location, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, density, lifetime, distribution, architecture and functioning. Actin polymerization is the driving force behind most podosome characteristics. In contrast to classical organelles, podosomes are not vital at the cell level but rather serve diverse and often intricate functions of which adhesion, matrix degradation and substrate sensing are the most established. These capabilities involve specific molecules, depend on podosome organization and may vary according to the cell type in which they form. Podosome-associated diseases manifest by loss or gain of podosome functions and include genetic diseases affecting podosome components and various cancers where tumor cells ectopically express podosome equivalents (invadopodia).
Topics: Animals; Cytoskeleton; Humans; Podosomes
PubMed: 26028292
DOI: 10.1016/j.biocel.2015.05.020 -
Cellular Signalling Sep 2019Recent study established the role of integrins in keratinocyte growth factor (KGF)-induced oral epithelial adhesion and rete peg elongation. However, how extracellular...
Recent study established the role of integrins in keratinocyte growth factor (KGF)-induced oral epithelial adhesion and rete peg elongation. However, how extracellular matrix (ECM) remodeling cooperates with the increased epithelial adhesion during rete peg elongation has yet to be determined. Podosomes are cell-matrix contact structures that combine several abilities, including adhesion and matrix degradation. In the present study, we identified podosome formation at the ventral side of human immortalized oral epithelial cells (HIOECs) upon KGF treatment. Moreover, podosomal components including integrin α6,β4,α3,β1 and MMP14 colocalized with the F-actin-cortactin complex and matrix degradation assays demonstrated the ability of the F-actin-cortactin complex to degrade matrix. Inhibition both of integrin subunits β4 and β1 with specific blocking antibodies and inhibition of Erk1/2 abrogated the KGF-induced podosome formation. Notably, knockdown of integrin subunits β4 and β1 with specific small interfering RNA (siRNA) downregulated the phosphorylation levels of Erk1/2. In contrast, inhibition of both Erk1/2 could upregulate the expression of integrin subunits β4 and β1. These results demonstrate that KGF induces podosome formation via integrin-Erk1/2 signaling in HIOECs, suggesting a novel mechanism by which integrins enhance oral epithelial adhesion and rete peg elongation.
Topics: Actins; Cell Adhesion; Cell Line; Cortactin; Epithelial Cells; Extracellular Matrix; Fibroblast Growth Factor 7; Gene Knockdown Techniques; Humans; Integrin beta1; Integrin beta4; MAP Kinase Signaling System; Mouth Mucosa; Phosphorylation; Podosomes; RNA, Small Interfering; Receptor, Fibroblast Growth Factor, Type 2; Transfection
PubMed: 31082464
DOI: 10.1016/j.cellsig.2019.05.007 -
The Journal of Cell Biology Jan 2017Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells...
Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.
Topics: ADP-Ribosylation Factor 1; Actin Cytoskeleton; Actins; Animals; Cell Line, Tumor; Enzyme Inhibitors; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Mice; Microscopy, Fluorescence; Nonmuscle Myosin Type IIA; Podosomes; RNA Interference; Recombinant Fusion Proteins; Signal Transduction; Time Factors; Transfection; rho-Associated Kinases; rhoA GTP-Binding Protein
PubMed: 28007915
DOI: 10.1083/jcb.201605104 -
Cytoskeleton (Hoboken, N.J.) Jun 2016Extracellular matrix (ECM) remodeling during physiological processes is mediated by invasive protrusions called podosomes. Positioning and dynamics of podosomes define...
Extracellular matrix (ECM) remodeling during physiological processes is mediated by invasive protrusions called podosomes. Positioning and dynamics of podosomes define the extent of ECM degradation. Microtubules are known to be involved in podosome regulation, but the role of microtubule (MT) network configuration in podosome dynamics and positioning is not well understood. Here, we show that the arrangement of the microtubule network defines the pattern of podosome formation and relocation in vascular smooth muscle cells (VSMCs). We show that microtubule plus-end targeting facilitates de novo formation of podosomes, in addition to podosome remodeling. Moreover, specialized bent microtubules with plus ends reversed towards the cell center promote relocation of podosomes from the cell edge to the cell center, resulting in an evenly distributed podosome pattern. Microtubule bending is induced downstream of protein kinase C (PKC) activation and requires microtubule-stabilizing proteins known as cytoplasmic linker associated proteins (CLASPs) and retrograde actin flow. Similar to microtubule depolymerization, CLASP depletion by siRNA blocks microtubule bending and eliminates centripetal relocation of podosomes. Podosome relocation also coincides with translocation of podosome-stimulating kinesin KIF1C, which is known to move preferentially along CLASP-associated microtubules. These findings indicate that CLASP-dependent microtubule network configuration is critical to the cellular location and distribution of KIF1C-dependent podosomes. © 2016 Wiley Periodicals, Inc.
Topics: Actins; Animals; Cell Line; Kinesins; Microtubule-Associated Proteins; Microtubules; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Podosomes; Rats
PubMed: 27105779
DOI: 10.1002/cm.21302 -
ACS Nano Feb 2022Podosomes are integrin-mediated cell adhesion units involved in many cellular and physiological processes. Integrins likely transmit tensions critical for podosome...
Podosomes are integrin-mediated cell adhesion units involved in many cellular and physiological processes. Integrins likely transmit tensions critical for podosome functions, but such force remains poorly characterized. DNA-based tension sensors are powerful in visualizing integrin tensions but subject to degradation by podosomes which ubiquitously recruit DNase. Here, using a DNase-resistant tension sensor based on a DNA/PNA (peptide nucleic acid) duplex, we imaged podosomal integrin tensions (PIT) in the adhesion rings of podosomes on solid substrates with single molecular tension sensitivity. PIT was shown to be generated by both actomyosin contractility and actin polymerization in podosomes. Importantly, by monitoring PIT and podosome structure in parallel, we showed that extracellular integrin-ligand tensions, despite being critical for the formation of focal adhesions, are dispensable for podosome formation, as PIT reduction or elimination has an insignificant impact on structure formation and FAK (focal adhesion kinase) phosphorylation in podosomes. We further verified that even integrin-ligand interaction is dispensable for podosome formation, as macrophages form podosomes normally on passivated surfaces that block integrin-ligand interaction but support macrophage adhesion through electrostatic adsorption or Fc receptor-immunoglobin G interaction. In contrast, focal adhesions are unable to form on these passivated surfaces.
Topics: Actins; Cell Adhesion; Focal Adhesions; Integrins; Ligands; Podosomes
PubMed: 35073043
DOI: 10.1021/acsnano.1c09105 -
PloS One 2012Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions,...
Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Cells, Cultured; Cytoskeletal Proteins; Cytoskeleton; Focal Adhesions; Humans; LIM Domain Proteins; Macrophages; Microfilament Proteins; Nerve Tissue Proteins; Pseudopodia; RNA, Small Interfering; Rats; Vinculin; Zyxin
PubMed: 22514729
DOI: 10.1371/journal.pone.0035340 -
Methods (San Diego, Calif.) Feb 2017Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an...
Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied. Here, we present a pipeline for podosome identification, protein position calculation, and creating a podosome ring model for use with localization microscopy data.
Topics: Actin Cytoskeleton; Carbocyanines; Cell Movement; Cells, Cultured; Dendritic Cells; Extracellular Matrix; Fibroblasts; Fluorescent Dyes; Gene Expression; Genes, Reporter; Humans; Luminescent Proteins; Macrophages; Microscopy, Fluorescence; Osteoclasts; Paxillin; Podosomes; Staining and Labeling; Talin; Vinculin; Red Fluorescent Protein
PubMed: 27840289
DOI: 10.1016/j.ymeth.2016.11.005 -
Microscopy and Microanalysis : the... Feb 2013Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are...
Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.
Topics: Actins; Automation; Cell Surface Extensions; Cells, Cultured; Dendritic Cells; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Phalloidine; Staining and Labeling
PubMed: 23347434
DOI: 10.1017/S1431927612014018