The Relationship Between Intermediate Filaments and Microfilaments Before and During the Formation of Desmosomes and Adherens-Ty
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The Wiskott-Aldrich Syndrome: the Actin Cytoskeleton and Immune Cell Function
Disease Markers 29 (2010) 157–175 157 DOI 10.3233/DMA-2010-0735 IOS Press The Wiskott-Aldrich syndrome: The actin cytoskeleton and immune cell function Michael P. Blundella, Austen Wortha,b, Gerben Boumaa and Adrian J. Thrashera,b,∗ aMolecular Immunology Unit, UCL Institute of Child Health, London, UK bDepartment of Immunology, Great Ormond Street Hospital NHS Trust, Great Ormond Street, London, UK Abstract. Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by immune dysregulation, microthrombocytopaenia, eczema and lymphoid malignancies. Mutations in the WAS gene can lead to distinct syndrome variations which largely, although not exclusively, depend upon the mutation. Premature termination and deletions abrogate Wiskott-Aldrich syndrome protein (WASp) expression and lead to severe disease (WAS). Missense mutations usually result in reduced protein expression and the phenotypically milder X-linked thrombocytopenia (XLT) or attenuated WAS [1–3]. More recently however novel activating mutations have been described that give rise to X-linked neutropenia (XLN), a third syndrome defined by neutropenia with variable myelodysplasia [4–6]. WASP is key in transducing signals from the cell surface to the actin cytoskeleton, and a lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration. Keywords: Wiskott-Aldrich syndrome, actin polymerization, lymphocytes, -
The Desmoplakin Carboxyl Terminus Coaligns with and Specifically Disrupts Intermediate Filament Networks When Expressed in Cultured Cells Thaddeus S
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central The Desmoplakin Carboxyl Terminus Coaligns with and Specifically Disrupts Intermediate Filament Networks When Expressed in Cultured Cells Thaddeus S. Stappenbeck and Kathleen J. Green Department of Pathology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 Abstract. Specific interactions between desmoplakins tides including the 90-kD carboxy-terminal globular I and 11 (DP I and II) and other desmosomal or cyto- domain of DP I specifically colocalized with and ulti- skeletal molecules have been difficult to determine in mately resulted in the complete disruption of IF in part because of the complexity and insolubility of the both cell lines. This effect was specific for IF as micro- desmosome and its constituents . We have used a mo- tubule and microfilament networks were unaltered . lecular genetic approach to investigate the role that This effect was also specific for the carboxyl terminus DP I and 11 may play in the association of the desmo- of DP, as the expression of the 95-kD rod domain of somal plaque with cytoplasmic intermediate filaments DP I did not visibly alter IF networks. Immunogold (IF) . A series of mammalian expression vectors en- localization of COS-7 cells transfected with constructs coding specific predicted domains of DP I were tran- including the carboxyl terminus of DP demonstrated siently expressed in cultured cells that form (COS-7) an accumulation of mutant protein in perinuclear aggre- and do not form (NIH-3T3) desmosomes. Sequence gates within which IF subunits were sequestered. -
Vinculin Antibody Cat
Vinculin Antibody Cat. No.: 7809 Vinculin Antibody Figure 2 Western Blot Validation in Human, Mouse and Rat Cell Lines Figure 3 Western Blot Validation in PC3 Cell Lysate Loading: 15 μg of lysates per lane. Antibodies: Vinculin Loading: 15 μg of lysates per lane. Antibodies: Vinculin 7809 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. 7809 (Lane 1: 0.5 μg/mL and Lane 2: 1 μg/mL), 1h Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 incubation at RT in 5% NFDM/TBST. Secondary: Goat anti- dilution. rabbit IgG HRP conjugate at 1:10000 dilution. Figure 4 Immunocytochemistry Validation of Vinculin in Jurkat Cells Figure 5 Immunofluorescence Validation of Vinculin in Immunocytochemical analysis of Jurkat cells using anti- Jurkat Cells Vinculin antibody (7809) at 5 μg/ml. Cells was fixed with Immunofluorescent analysis of 4% paraformaldehyde- formaldehyde and blocked with 10% serum for 1 h at RT; fixed Jurkat cells labeling Vinculin with 7809 at 20 μg/mL, antigen retrieval was by heat mediation with a citrate followed by goat anti-rabbit IgG secondary antibody at buffer (pH6). Samples were incubated with primary 1/500 dilution (green) and DAPI staining (blue). antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin. Specifications September 23, 2021 1 https://www.prosci-inc.com/vinculin-antibody-7809.html HOST SPECIES: Rabbit SPECIES REACTIVITY: Human, Mouse, Rat HOMOLOGY: Predicted species reactivity based on immunogen sequence: Pig: (100%) Anti-Vinculin antibody (7809) was raised against a peptide corresponding to 23 amino IMMUNOGEN: acids near the center of human Vinculin. -
Conserved Microtubule–Actin Interactions in Cell Movement and Morphogenesis
REVIEW Conserved microtubule–actin interactions in cell movement and morphogenesis Olga C. Rodriguez, Andrew W. Schaefer, Craig A. Mandato, Paul Forscher, William M. Bement and Clare M. Waterman-Storer Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions — regulatory and structural. These interactions comprise at least three conserved ‘mechanochemical activity modules’ that perform similar roles in these diverse cell functions. Over the past 35 years, great progress has been made towards under- crosstalk occurs in processes that require dynamic cellular asymme- standing the roles of the microtubule and actin cytoskeletal filament tries to be established or maintained to allow rapid intracellular reor- systems in mechanical cellular processes such as dynamic shape ganization or changes in shape or direction in response to stimuli. change, shape maintenance and intracellular organelle movement. Furthermore, the widespread occurrence of these interactions under- These functions are attributed to the ability of polarized cytoskeletal scores their importance for life, as they occur in diverse cell types polymers to assemble and disassemble rapidly, and to interact with including epithelia, neurons, fibroblasts, oocytes and early embryos, binding proteins and molecular motors that mediate their regulated and across species from yeast to humans. Thus, defining the mecha- movement and/or assembly into higher order structures, such as radial nisms by which actin and microtubules interact is key to understand- arrays or bundles. -
Absence of NEFL in Patient-Specific Neurons in Early-Onset Charcot-Marie-Tooth Neuropathy Markus T
ARTICLE OPEN ACCESS Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy Markus T. Sainio, MSc, Emil Ylikallio, MD, PhD, Laura M¨aenp¨a¨a, MSc, Jenni Lahtela, PhD, Pirkko Mattila, PhD, Correspondence Mari Auranen, MD, PhD, Johanna Palmio, MD, PhD, and Henna Tyynismaa, PhD Dr. Tyynismaa [email protected] Neurol Genet 2018;4:e244. doi:10.1212/NXG.0000000000000244 Abstract Objective We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot- Marie-Tooth (CMT) disease. Methods Motor neurons were differentiated from induced pluripotent stem cells of a patient with early- onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient’s cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neuro- filaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most down- regulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions The strict neuronal specificity of neurofilament has hindered the mechanistic studies of re- cessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. -
Plakoglobin Is Required for Effective Intermediate Filament Anchorage to Desmosomes Devrim Acehan1, Christopher Petzold1, Iwona Gumper2, David D
ORIGINAL ARTICLE Plakoglobin Is Required for Effective Intermediate Filament Anchorage to Desmosomes Devrim Acehan1, Christopher Petzold1, Iwona Gumper2, David D. Sabatini2, Eliane J. Mu¨ller3, Pamela Cowin2,4 and David L. Stokes1,2,5 Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, b-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength. Journal of Investigative Dermatology (2008) 128, 2665–2675; doi:10.1038/jid.2008.141; published online 22 May 2008 INTRODUCTION dense plaque that is further from the membrane and that Desmosomes are large macromolecular complexes that mediates the binding of intermediate filaments. -
The Intrinsically Disordered Protein SPE-18 Promotes Localized Assembly of MSP in Caenorhabditis Elegans Spermatocytes Kari L
© 2021. Published by The Company of Biologists Ltd | Development (2021) 148, dev195875. doi:10.1242/dev.195875 RESEARCH ARTICLE The intrinsically disordered protein SPE-18 promotes localized assembly of MSP in Caenorhabditis elegans spermatocytes Kari L. Price*,¶, Marc Presler‡,¶, Christopher M. Uyehara§ and Diane C. Shakes ABSTRACT Buracco et al., 2019; Brouhard and Rice, 2018; Bodakuntla et al., Many specialized cells use unconventional strategies of cytoskeletal 2019; de Forges et al., 2012). However, a full understanding of control. Nematode spermatocytes discard their actin and tubulin cytoskeletal control requires consideration of less-studied proteins following meiosis, and instead employ the regulated assembly/ whose properties challenge our standard assumptions. disassembly of the Major Sperm Protein (MSP) to drive sperm One such protein is the nematode Major Sperm Protein (MSP), motility. However, prior to the meiotic divisions, MSP is sequestered assembly/disassembly dynamics of which power the crawling through its assembly into paracrystalline structures called fibrous motility of nematode spermatozoa (Klass and Hirsh, 1981; bodies (FBs). The accessory proteins that direct this sequestration Sepsenwol et al., 1989; Italiano et al., 1996; reviewed by Roberts process have remained mysterious. This study reveals SPE-18 as an and Stewart, 2012; Smith, 2014). Although MSP-based motility intrinsically disordered protein that is essential for MSP assembly appears superficially similar to its actin-based counterpart, the within FBs. In spe-18 mutant spermatocytes, MSP forms disorganized molecular mechanisms are distinct. Much of what we know about cortical fibers, and the cells arrest in meiosis without forming haploid MSP dynamics was gleaned from the parasitic nematode Ascaris, sperm. -
Transiently Structured Head Domains Control Intermediate Filament Assembly
Transiently structured head domains control intermediate filament assembly Xiaoming Zhoua, Yi Lina,1, Masato Katoa,b,c, Eiichiro Morid, Glen Liszczaka, Lillian Sutherlanda, Vasiliy O. Sysoeva, Dylan T. Murraye, Robert Tyckoc, and Steven L. McKnighta,2 aDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390; bInstitute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology, 263-8555 Chiba, Japan; cLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520; dDepartment of Future Basic Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan; and eDepartment of Chemistry, University of California, Davis, CA 95616 Contributed by Steven L. McKnight, January 2, 2021 (sent for review October 30, 2020; reviewed by Lynette Cegelski, Tatyana Polenova, and Natasha Snider) Low complexity (LC) head domains 92 and 108 residues in length are, IF head domains might facilitate filament assembly in a manner respectively, required for assembly of neurofilament light (NFL) and analogous to LC domain function by RNA-binding proteins in the desmin intermediate filaments (IFs). As studied in isolation, these IF assembly of RNA granules. head domains interconvert between states of conformational disor- IFs are defined by centrally located α-helical segments 300 to der and labile, β-strand–enriched polymers. Solid-state NMR (ss-NMR) 350 residues in length. These central, α-helical segments are spectroscopic studies of NFL and desmin head domain polymers re- flanked on either end by head and tail domains thought to be veal spectral patterns consistent with structural order. -
Microtubule-Associated Protein Tau (Molecular Pathology/Neurodegenerative Disease/Neurofibriliary Tangles) M
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4051-4055, June 1988 Medical Sciences Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: Identification as the microtubule-associated protein tau (molecular pathology/neurodegenerative disease/neurofibriliary tangles) M. GOEDERT*, C. M. WISCHIK*t, R. A. CROWTHER*, J. E. WALKER*, AND A. KLUG* *Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; and tDepartment of Psychiatry, University of Cambridge Clinical School, Hills Road, Cambridge CB2 2QQ, United Kingdom Contributed by A. Klug, March 1, 1988 ABSTRACT Screening of cDNA libraries prepared from (21). This task is made all the more difficult because there is the frontal cortex ofan zheimer disease patient and from fetal no functional or physiological assay for the protein(s) of the human brain has led to isolation of the cDNA for a core protein PHF. The only identification so far possible is the morphol- of the paired helical fiament of Alzheimer disease. The partial ogy of the PHFs at the electron microscope level, and here amino acid sequence of this core protein was used to design we would accept only experiments on isolated individual synthetic oligonucleotide probes. The cDNA encodes a protein of filaments, not on neurofibrillary tangles (in which other 352 amino acids that contains a characteristic amino acid repeat material might be occluded). One thus needs a label or marker in its carboxyl-terminal half. This protein is highly homologous for the PHF itself, which can at the same time be used to to the sequence ofthe mouse microtubule-assoiated protein tau follow the steps of the biochemical purification. -
A Unique and Specific Interaction Between Αt-Catenin and Plakophilin
2126 Research Article A unique and specific interaction between ␣T-catenin and plakophilin-2 in the area composita, the mixed-type junctional structure of cardiac intercalated discs Steven Goossens1,2,*, Barbara Janssens1,2,*,‡, Stefan Bonné1,2,§, Riet De Rycke1,2, Filip Braet1,2,¶, Jolanda van Hengel1,2 and Frans van Roy1,2,** 1Department for Molecular Biomedical Research, VIB and 2Department of Molecular Biology, Ghent University, B-9052 Ghent, Belgium *These authors contributed equally to this work ‡Present address: Wiley-VCH, Boschstrasse 12, D-69469 Weinheim, Germany §Present address: Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium ¶Present address: Australian Key Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006, Australia **Author for correspondence (e-mail: [email protected]) Accepted 24 April 2007 Journal of Cell Science 120, 2126-2136 Published by The Company of Biologists 2007 doi:10.1242/jcs.004713 Summary Alpha-catenins play key functional roles in cadherin- desmosomal proteins in the heart localize not only to the catenin cell-cell adhesion complexes. We previously desmosomes in the intercalated discs but also at adhering reported on ␣T-catenin, a novel member of the ␣-catenin junctions with hybrid composition. We found that in the protein family. ␣T-catenin is expressed predominantly in latter junctions, endogenous plakophilin-2 colocalizes with cardiomyocytes, where it colocalizes with ␣E-catenin at the ␣T-catenin. By providing an extra link between the intercalated discs. Whether ␣T- and ␣E-catenin have cadherin-catenin complex and intermediate filaments, the specific or synergistic functions remains unknown. In this binding of ␣T-catenin to plakophilin-2 is proposed to be a study we used the yeast two-hybrid approach to identify means of modulating and strengthening cell-cell adhesion specific functions of ␣T-catenin. -
Neurofilaments: Neurobiological Foundations for Biomarker Applications
Neurofilaments: neurobiological foundations for biomarker applications Arie R. Gafson1, Nicolas R. Barthelmy2*, Pascale Bomont3*, Roxana O. Carare4*, Heather D. Durham5*, Jean-Pierre Julien6,7*, Jens Kuhle8*, David Leppert8*, Ralph A. Nixon9,10,11,12*, Roy Weller4*, Henrik Zetterberg13,14,15,16*, Paul M. Matthews1,17 1 Department of Brain Sciences, Imperial College, London, UK 2 Department of Neurology, Washington University School of Medicine, St Louis, MO, USA 3 a ATIP-Avenir team, INM, INSERM , Montpellier university , Montpellier , France. 4 Clinical Neurosciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom 5 Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Québec, Canada 6 Department of Psychiatry and Neuroscience, Laval University, Quebec, Canada. 7 CERVO Brain Research Center, 2601 Chemin de la Canardière, Québec, QC, G1J 2G3, Canada 8 Neurologic Clinic and Policlinic, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland. 9 Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY, 10962, USA. 10Departments of Psychiatry, New York University School of Medicine, New York, NY, 10016, 11 Neuroscience Institute, New York University School of Medicine, New York, NY, 10016, USA. 12Department of Cell Biology, New York University School of Medicine, New York, NY, 10016, USA 13 University College London Queen Square Institute of Neurology, London, UK 14 UK Dementia Research Institute at University College London 15 Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden 16 Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden 17 UK Dementia Research Institute at Imperial College, London * Co-authors ordered alphabetically Address for correspondence: Prof. -
The Role of the Interaction of the Vinculin Proline-Rich Linker Region
ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1875–1886 doi:10.1242/jcs.133645 RESEARCH ARTICLE The role of the interaction of the vinculin proline-rich linker region with vinexin a in sensing the stiffness of the extracellular matrix Hiroshi Yamashita1,*, Takafumi Ichikawa1,*, Daisuke Matsuyama1, Yasuhisa Kimura1, Kazumitsu Ueda1,2, Susan W. Craig3, Ichiro Harada4 and Noriyuki Kioka1,` ABSTRACT information into biochemical signals. At FAs, transmembrane ECM receptors, integrins, are connected to the ends of actin stress Although extracellular matrix (ECM) stiffness is an important aspect fibers through various cytoplasmic proteins, including talin and of the extracellular microenvironment and is known to direct the vinculin. Cells sense ECM stiffness by ‘pulling’ on the ECM lineage specification of stem cells and affect cancer progression, using actomyosin-generated intracellular tension through FAs and the molecular mechanisms that sense ECM stiffness have not yet adjusting the tension that they exert accordingly (Geiger et al., been elucidated. In this study, we show that the proline-rich linker 2009; Parsons et al., 2010); cells on rigid ECM exert higher (PRL) region of vinculin and the PRL-region-binding protein vinexin intracellular tension than cells on soft ECM. Although some FA are involved in sensing the stiffness of ECM substrates. A rigid proteins have been suggested to engage in sensing intracellular substrate increases the level of cytoskeleton-associated vinculin, tension (del Rio et al., 2009; Friedland et al., 2009; Sawada et al., and the fraction of vinculin stably localizing at focal adhesions (FAs) 2006), the signaling mechanisms by which cells sense ECM is larger on rigid ECM than on soft ECM.