Publications by authors named "Athanassios Dovas"

24 Publications

  • Page 1 of 1

Single-cell characterization of macrophages in glioblastoma reveals MARCO as a mesenchymal pro-tumor marker.

Genome Med 2021 05 19;13(1):88. Epub 2021 May 19.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA.

Background: Macrophages are the most common infiltrating immune cells in gliomas and play a wide variety of pro-tumor and anti-tumor roles. However, the different subpopulations of macrophages and their effects on the tumor microenvironment remain poorly understood.

Methods: We combined new and previously published single-cell RNA-seq data from 98,015 single cells from a total of 66 gliomas to profile 19,331 individual macrophages.

Results: Unsupervised clustering revealed a pro-tumor subpopulation of bone marrow-derived macrophages characterized by the scavenger receptor MARCO, which is almost exclusively found in IDH1-wild-type glioblastomas. Previous studies have implicated MARCO as an unfavorable marker in melanoma and non-small cell lung cancer; here, we find that bulk MARCO expression is associated with worse prognosis and mesenchymal subtype. Furthermore, MARCO expression is significantly altered over the course of treatment with anti-PD1 checkpoint inhibitors in a response-dependent manner, which we validate with immunofluorescence imaging.

Conclusions: These findings illustrate a novel macrophage subpopulation that drives tumor progression in glioblastomas and suggest potential therapeutic targets to prevent their recruitment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00906-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136167PMC
May 2021

Deconvolution of cell type-specific drug responses in human tumor tissue with single-cell RNA-seq.

Genome Med 2021 05 11;13(1):82. Epub 2021 May 11.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Background: Preclinical studies require models that recapitulate the cellular diversity of human tumors and provide insight into the drug sensitivities of specific cellular populations. The ideal platform would enable rapid screening of cell type-specific drug sensitivities directly in patient tumor tissue and reveal strategies to overcome intratumoral heterogeneity.

Methods: We combine multiplexed drug perturbation in acute slice culture from freshly resected tumors with single-cell RNA sequencing (scRNA-seq) to profile transcriptome-wide drug responses in individual patients. We applied this approach to drug perturbations on slices derived from six glioblastoma (GBM) resections to identify conserved drug responses and to one additional GBM resection to identify patient-specific responses.

Results: We used scRNA-seq to demonstrate that acute slice cultures recapitulate the cellular and molecular features of the originating tumor tissue and the feasibility of drug screening from an individual tumor. Detailed investigation of etoposide, a topoisomerase poison, and the histone deacetylase (HDAC) inhibitor panobinostat in acute slice cultures revealed cell type-specific responses across multiple patients. Etoposide has a conserved impact on proliferating tumor cells, while panobinostat treatment affects both tumor and non-tumor populations, including unexpected effects on the immune microenvironment.

Conclusions: Acute slice cultures recapitulate the major cellular and molecular features of GBM at the single-cell level. In combination with scRNA-seq, this approach enables cell type-specific analysis of sensitivity to multiple drugs in individual tumors. We anticipate that this approach will facilitate pre-clinical studies that identify effective therapies for solid tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00894-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114529PMC
May 2021

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

iScience 2020 Dec 13;23(12):101802. Epub 2020 Nov 13.

Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2020.101802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702012PMC
December 2020

Glioma-Induced Alterations in Neuronal Activity and Neurovascular Coupling during Disease Progression.

Cell Rep 2020 04;31(2):107500

Laboratory for Functional Optical Imaging, Zuckerman Mind Brain Behavior Institute, Departments of Biomedical Engineering and Radiology, Columbia University, New York, NY 10027, USA. Electronic address:

Diffusely infiltrating gliomas are known to cause alterations in cortical function, vascular disruption, and seizures. These neurological complications present major clinical challenges, yet their underlying mechanisms and causal relationships to disease progression are poorly characterized. Here, we follow glioma progression in awake Thy1-GCaMP6f mice using in vivo wide-field optical mapping to monitor alterations in both neuronal activity and functional hemodynamics. The bilateral synchrony of spontaneous neuronal activity gradually decreases in glioma-infiltrated cortical regions, while neurovascular coupling becomes progressively disrupted compared to uninvolved cortex. Over time, mice develop diverse patterns of high amplitude discharges and eventually generalized seizures that appear to originate at the tumors' infiltrative margins. Interictal and seizure events exhibit positive neurovascular coupling in uninfiltrated cortex; however, glioma-infiltrated regions exhibit disrupted hemodynamic responses driving seizure-evoked hypoxia. These results reveal a landscape of complex physiological interactions occurring during glioma progression and present new opportunities for exploring novel biomarkers and therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.03.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443283PMC
April 2020

Radiation-Induced Lipid Peroxidation Triggers Ferroptosis and Synergizes with Ferroptosis Inducers.

ACS Chem Biol 2020 02 14;15(2):469-484. Epub 2020 Jan 14.

Department of Biological Sciences , Columbia University , New York , New York 10027 , United States.

Although radiation is widely used to treat cancers, resistance mechanisms often develop and involve activation of DNA repair and inhibition of apoptosis. Therefore, compounds that sensitize cancer cells to radiation via alternative cell death pathways are valuable. We report here that ferroptosis, a form of nonapoptotic cell death driven by lipid peroxidation, is partly responsible for radiation-induced cancer cell death. Moreover, we found that small molecules activating ferroptosis through system x inhibition or GPX4 inhibition synergize with radiation to induce ferroptosis in several cancer types by enhancing cytoplasmic lipid peroxidation but not increasing DNA damage or caspase activation. Ferroptosis inducers synergized with cytoplasmic irradiation, but not nuclear irradiation. Finally, administration of ferroptosis inducers enhanced the antitumor effect of radiation in a murine xenograft model and in human patient-derived models of lung adenocarcinoma and glioma. These results suggest that ferroptosis inducers may be effective radiosensitizers that can expand the efficacy and range of indications for radiation therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.9b00939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180072PMC
February 2020

Ex vivo multi-electrode analysis reveals spatiotemporal dynamics of ictal behavior at the infiltrated margin of glioma.

Neurobiol Dis 2020 02 12;134:104676. Epub 2019 Nov 12.

Department of Neurology, Columbia University Medical Center, New York, NY, USA.

The purpose of this study is to develop a platform in which the cellular and molecular underpinnings of chronic focal neocortical lesional epilepsy can be explored and use it to characterize seizure-like events (SLEs) in an ex vivo model of infiltrating high-grade glioma. Microelectrode arrays were used to study electrophysiologic changes in ex vivo acute brain slices from a PTEN/p53 deleted, PDGF-B driven mouse model of high-grade glioma. Electrode locations were co-registered to the underlying histology to ascertain the influence of the varying histologic landscape on the observed electrophysiologic changes. Peritumoral, infiltrated, and tumor sites were sampled in tumor-bearing slices. Following the addition of zero Mg solution, all three histologic regions in tumor-bearing slices showed significantly greater increases in firing rates when compared to the control sites. Tumor-bearing slices demonstrated increased proclivity for SLEs, with 40 events in tumor-bearing slices and 5 events in control slices (p-value = .0105). Observed SLEs were characterized by either low voltage fast (LVF) onset patterns or short bursts of repetitive widespread, high amplitude low frequency discharges. Seizure foci comprised areas from all three histologic regions. The onset electrode was found to be at the infiltrated margin in 50% of cases and in the peritumoral region in 36.9% of cases. These findings reveal a landscape of histopathologic and electrophysiologic alterations associated with ictogenesis and spread of tumor-associated seizures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2019.104676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147009PMC
February 2020

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

Proc Natl Acad Sci U S A 2019 07 24;116(31):15550-15559. Epub 2019 Jun 24.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Jacksonville, FL 32224;

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1902847116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681735PMC
July 2019

Single-cell transcriptome analysis of lineage diversity in high-grade glioma.

Genome Med 2018 07 24;10(1):57. Epub 2018 Jul 24.

Department of Systems Biology, Columbia University Medical Center, New York, NY, 10032, USA.

Background: Despite extensive molecular characterization, we lack a comprehensive understanding of lineage identity, differentiation, and proliferation in high-grade gliomas (HGGs).

Methods: We sampled the cellular milieu of HGGs by profiling dissociated human surgical specimens with a high-density microwell system for massively parallel single-cell RNA-Seq. We analyzed the resulting profiles to identify subpopulations of both HGG and microenvironmental cells and applied graph-based methods to infer structural features of the malignantly transformed populations.

Results: While HGG cells can resemble glia or even immature neurons and form branched lineage structures, mesenchymal transformation results in unstructured populations. Glioma cells in a subset of mesenchymal tumors lose their neural lineage identity, express inflammatory genes, and co-exist with marked myeloid infiltration, reminiscent of molecular interactions between glioma and immune cells established in animal models. Additionally, we discovered a tight coupling between lineage resemblance and proliferation among malignantly transformed cells. Glioma cells that resemble oligodendrocyte progenitors, which proliferate in the brain, are often found in the cell cycle. Conversely, glioma cells that resemble astrocytes, neuroblasts, and oligodendrocytes, which are non-proliferative in the brain, are generally non-cycling in tumors.

Conclusions: These studies reveal a relationship between cellular identity and proliferation in HGG and distinct population structures that reflects the extent of neural and non-neural lineage resemblance among malignantly transformed cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-018-0567-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058390PMC
July 2018

MRI-localized biopsies reveal subtype-specific differences in molecular and cellular composition at the margins of glioblastoma.

Proc Natl Acad Sci U S A 2014 Aug 11;111(34):12550-5. Epub 2014 Aug 11.

Pathology and Cell Biology,

Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and nonneoplastic cells that remain after surgery form the biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-sequencing (RNA-seq) and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared with tissue from the nonenhancing (NE) margins of tumors. Comparisons with the The Cancer Genome Atlas dataset showed that the samples from CE regions resembled the proneural, classical, or mesenchymal subtypes of GBM, whereas the samples from the NE regions predominantly resembled the neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from nonneoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology analysis showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-seq data from the GBM samples to that of nonneoplastic brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes: the NE regions of proneural tumors were enriched in oligodendrocyte progenitor genes, whereas the NE regions of mesenchymal GBM were enriched in astrocytic and microglial genes. These subtype-specific patterns provide new insights into molecular and cellular composition of the infiltrative margins of GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1405839111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151734PMC
August 2014

Tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) by Hck regulates macrophage function.

J Biol Chem 2014 Mar 30;289(11):7897-906. Epub 2014 Jan 30.

From the Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 and.

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck(-/-) bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck(-/-) BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP(-/-) BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.509497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953300PMC
March 2014

Wiskott-Aldrich syndrome protein regulates leukocyte-dependent breast cancer metastasis.

Cell Rep 2013 Aug 1;4(3):429-36. Epub 2013 Aug 1.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp(-/-) macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777703PMC
August 2013

The chemotactic defect in wiskott-Aldrich syndrome macrophages is due to the reduced persistence of directional protrusions.

PLoS One 2012 18;7(1):e30033. Epub 2012 Jan 18.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp⁻/⁻ bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 - elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030033PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261183PMC
July 2012

Signaling networks regulating leukocyte podosome dynamics and function.

Cell Signal 2011 Aug 20;23(8):1225-34. Epub 2011 Feb 20.

Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.

Podosomes are ventral adhesion structures prominent in cells of the myeloid lineage. A common aspect of these cells is that they are highly motile and must to traverse multiple tissue barriers in order to perform their functions. Recently podosomes have gathered attention from researchers as important cellular structures that can influence cell adhesion, motility and matrix remodeling. Adhesive and soluble ligands act via transmembrane receptors and propagate signals to the leukocyte cytoskeleton via small G proteins of the Rho family, tyrosine kinases and scaffold proteins and are able to induce podosome formation and rearrangements. Manipulation of the signals that regulate podosome formation and dynamics can therefore be a strategy to interfere with leukocyte functions in a multitude of pathological settings, such as infections, atherosclerosis and arthritis. Here, we review the major signaling molecules that act in the formation and regulation of podosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellsig.2011.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095719PMC
August 2011

Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins.

PLoS One 2011 Feb 14;6(2):e16485. Epub 2011 Feb 14.

Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016485PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038862PMC
February 2011

Nck1 and Grb2 localization patterns can distinguish invadopodia from podosomes.

Eur J Cell Biol 2011 Feb-Mar;90(2-3):181-8. Epub 2010 Sep 17.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.

Invadopodia are matrix-degrading ventral cell surface structures formed in invasive carcinoma cells. Podosomes are matrix-degrading structures formed in normal cell types including macrophages, endothelial cells, and smooth muscle cells that are believed to be related to invadopodia in function. Both invadopodia and podosomes are enriched in proteins that regulate actin polymerization including proteins involved in N-WASp/WASp-dependent Arp2/3-complex activation. However, it is unclear whether invadopodia and podosomes use distinct mediators for N-WASp/WASp-dependent Arp2/3-complex activation. We investigated the localization patterns of the upstream N-WASp/WASp activators Nck1 and Grb2 in invadopodia of metastatic mammary carcinoma cells, podosomes formed in macrophages, and degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. We provide evidence that Nck1 specifically localizes to invadopodia, but not to podosomes formed in macrophages or degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. In contrast, Grb2 specifically localizes to degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells, but not invadopodia or podosomes formed in macrophages. These findings suggest that distinct upstream activators are responsible for N-WASp/WASp activation in invadopodia and podosomes, and that all these ventral cell surface degradative structures have distinguishing molecular as well as structural characteristics. These patterns of Nck1 and Grb2 localization, identified in our study, can be used to sub-classify ventral cell surface degradative structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2010.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017226PMC
July 2011

The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages.

Eur J Cell Biol 2011 Feb-Mar;90(2-3):205-12. Epub 2010 Aug 21.

Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Wiskott-Aldrich Syndrome Protein (WASP) is a hematopoietic cell-specific regulator of Arp2/3-dependent actin polymerization. Despite the presence of the highly homologous N-WASP (neural-WASP), macrophages from WAS patients are devoid of podosomes, adhesion structures in cells of the monocytic lineage capable of matrix degradation via matrix metalloproteases (MMPs), suggesting that WASP and N-WASP play unique roles in macrophages. To determine whether N-WASP also plays a unique role in macrophage function, N-WASP expression was reduced using silencing RNA in a sub-line of RAW 264.7 macrophages (RAW/LR5). Similar to reduction in WASP levels, cells with reduced N-WASP levels were rounder and less polarized. Interestingly, podosomes still formed when N-WASP was reduced but they were unable to perform matrix degradation. This defect was rescued by re-expression of N-WASP, but not by over-expression of WASP, indicating that these proteins play distinct roles in podosome function. Additionally, reducing N-WASP levels mistargets the metalloprotease MT1-MMP and it no longer localizes to podosomes. However, N-WASP was only found to co-localize with MT1-MMP positive vesicles at podosomes, suggesting that N-WASP may play a role on the targeting or fusion of MMP-containing vesicles to podosomes in macrophage-like cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2010.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012153PMC
July 2011

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis.

Exp Cell Res 2010 Dec 27;316(20):3406-16. Epub 2010 Jun 27.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2010.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976787PMC
December 2010

Regulation of WASp by phosphorylation: Activation or other functions?

Commun Integr Biol 2010 Mar;3(2):101-5

Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor that regulates actin polymerisation via the Arp2/3 complex. Its mutation in human syndromes has led to extensive studies on the regulation and activities of this molecule. Several mechanisms for the regulation of WASp activity have been proposed, however, the role of tyrosine phosphorylation remains controversial, particularly due to inconsistencies between results obtained through biochemical and cell biological approaches. In this mini-review, we are addressing the major aspects of WASp regulation with an emphasis on the role of tyrosine phosphorylation on WASp activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cib.3.2.10759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889963PMC
March 2010

Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion.

J Biol Chem 2010 Jul 15;285(30):23296-308. Epub 2010 May 15.

Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCalpha can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIalpha that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIalpha on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIalpha phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCalpha in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.098129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906322PMC
July 2010

Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages.

J Cell Sci 2009 Nov 6;122(Pt 21):3873-82. Epub 2009 Oct 6.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.051755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773189PMC
November 2009

The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo: a role for phosphatidylinositol 3-kinase and Cdc42.

J Biol Chem 2009 Aug 26;284(35):23302-11. Epub 2009 Jun 26.

Department of Anatomy, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA.

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.036384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749104PMC
August 2009

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells.

J Cell Biol 2008 Mar;180(6):1245-60

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, USA.

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200708123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2290849PMC
March 2008

PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation.

J Cell Sci 2006 Jul;119(Pt 13):2837-46

Division of Biomedical Sciences, Imperial College London, London, SW7 2AZ, UK.

Syndecan-4 is a ubiquitously expressed transmembrane heparan sulphate proteoglycan acting in concert with integrins in the formation of focal adhesions and stress fibres. Signalling events studied thus far suggest the formation of a ternary complex between syndecan-4, phosphatidylinositol 4,5-bisphosphate and protein kinase C alpha (PKCalpha). Syndecan-4 clustering at the cell surface has also been associated with RhoA-dependent signalling, but the relationship between PKCalpha and RhoA has not been resolved. Here we present evidence that syndecan-4, PKCalpha and RhoA are in a linear pathway necessary for the formation and maintenance of stress fibres in primary rat embryo fibroblasts. Inhibition of PKCalpha activity through the use of specific pharmacological inhibitors, a dominant-negative construct, or siRNA downregulation of protein levels, attenuated focal adhesion formation and the maintenance of stress fibres. However, these effects could be bypassed through independent activation of RhoA with lysophosphatidic acid, but not by clustering of syndecan-4 with ligand. Furthermore, inhibition of PKCalpha could block the increase in the GTP levels of RhoA induced by clustering of syndecan-4 at the cell surface. All these data point to a mechanism whereby syndecan-4 signals to RhoA in a PKCalpha-dependent manner and PKCalpha directly influences RhoA activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.03020DOI Listing
July 2006

RhoGDI: multiple functions in the regulation of Rho family GTPase activities.

Biochem J 2005 Aug;390(Pt 1):1-9

Division of Biomedical Sciences, Imperial College London, Exhibition Road, London SW7 2AZ, UK.

RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase-RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20050104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1184558PMC
August 2005
-->