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Lecture 8 Intermediate filaments Microfilaments (actin filaments): 5-9 nm Microtubules: 25 nm Intermediate filaments: 10 nm Intermediate filaments Ishikawa, H., Bischoff, R. & Holtzer, H. (1968). Mitosis and intermediate filamentsized filaments in developing skeletal muscle. J. Cell Biol. 38, 538–555. Intermediate filaments Intermediate filaments in the cytoplasm of the mammalian cell 20 µm IF in a glial cell Filaments of fibrillary acidic protein 100 µm Two types of intermediate filaments in cells of the nervous system Neurofilaments in a nerve cell axon Glial filaments in glial cells Cross-section of an axon showing both MT and IF IF anchored to desmosomes and hemidesmosomes Extracellular matrix Plectin cross-links IF to microfilaments and MT IF organization in metazoan cells Herrman et al. (2007). Nat. Rev. Mol. Cell. Biol. 8: 562-573 Demonstration of mechanical connections between extracellular matrix and nucleoplasm “Harpooning” of the cell by a micropipette Maniotis et al. (1997). Proc. Natl Acad. Sci. USA 94, 849–854. >65 genes encoding IF proteins in humans Tissue-specific expression of IF IFs in tumor diagnostics Components of IFs (in contrast to MF and MT) are not globular proteins Levels of organization and assembly of IF A model of intermediate filament construction A model of intermediate filament assembly ULF= unit-length filaments Krimse et al. (2007). A Quantitative Kinetic Model for the in Vitro Assembly of Intermediate Filaments from Tetrameric Vimentin. J. Biol. Chem. 282, 18563-18572. IF assembly in vivo Cell fusion A model of intermediate filament construction Coiled-coil structure is an ubiquitous feature of intermediate filaments Coiled-coil structure is an ubiquitous feature of intermediate filaments a,d: small apolar residues (Leu, Ile, Met, Val) Structural model of cytoplasmic and nuclear intermediate filament protein dimers Herrman et al. (2007). Nat. Rev. Mol. Cell. Biol. 8: 562-573 Francis Crick: Discoverer of the genetic code (Matt Ridley, 2006, HarperCollins) In the […] laboratory he took charge of making sure that everybody knew about everyone else’s scientific work. […] ‘Crick week’ was a week of seminars when the lab members told each other about their results. Sitting at the front, Crick was a terrifying presence, concentrating hard, interrupting frequently, and of course at the end giving a licid summary of not only what the speaker had just said but what they should have said and what it all meant. […] Even those asking questions were sometimes corrected: “The question you should have asked is … and the answer is …”. Graeme Mitchison recalls that, as a result, the seminars were both a terrifying ordeal, and an excellent spectator sport. Major types of IF in vertebrate cells TYPES OF IF COMPONENT POLYPEPTIDES LOCALIZATION Nuclear Lamins A, B, and C Nuclear lamina (inner lining of nuclear envelope) Vimentin-like Vimentin Many cells of mesenchymal origin Desmin Muscle Glial fibrillary acidic protein Glial cells Peripherin Some neurons Type I keratins (acidic) Epithelial cells and their derivatives (e.g., hair and nails) Epithelial Type II keratins (basic) Axonal Neurofilament proteins (NF-L, NF-M, and NF-H) Nuclear lamina (inner lining of nuclear envelope) The domain organization of intermediate filament protein monomers Heterooligomeric character of IFs is a basis of intra- and interfilament heterogeneity A strong filament formed from elongated fibrous subunits with strong lateral contacts Atomic Force Microscopy as a tool for biology Herrman et al. (2007). Nat. Rev. Mol. Cell. Biol. 8: 562-573 Mechanical properties of actin, tubulin, and vimentin polymers Dynamic nature of IF within a cell – FRAP technique http://www.ibiology.org/ibioseminars/cell-biology/robert-goldman-part-1.html Bob Goldman on IF (iBiology.org) http://www.ibiology.org/ibioseminars/cell-biology/robert-goldman-part-1.html Lev Sergejevič Teremin (1896-1993) What regulates a stability of intermediate filaments? The nuclear lamina is composed of a special class of IF Gerace, L., Blum, A. & Blobel, G. (1978). Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. J. Cell Biol. 79, 546–566 Gerace, L. & Blobel, G. (1980). The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell 19, 277–287. The nuclear lamina The nuclear lamina Nuclear pore complex CYTOSOL Nuclear envelope Nuclear lamina Chromatin NUCLEUS The nuclear lamina 1 µm nuclear lamina in a frog oocyte Disassembly of nuclear lamina in prophase is driven by phoshorylation of lamins by Cdk2p >230 mutations in lamin A cause a complex set of at least 13 different human diseases Hutchinson-Gilford progeria atypical Werner syndrome muscular dystrophies cardiomyopathy http://www.cell.com/abstract/S0092-8674(12)00401-1 Hutchinson-Gilford progeria is caused by deletion in lamin A gene leading to aberant processing of the protein Coutinho et al. (2009). Immunity & Ageing 6:4 Participation of lamin B in the organization of spindle pole Lamins A & C: present primarily in differentiated cells Lamin B: essential for cell survival RNAi-mediated reduction of lamin B in C. elegans and HeLa cells: spindle defects & chromosome mis-segregation Ma et al. (2009). Requirement for Nudel and dynein for assembly of the lamin B spindle matrix Nat. Cell Biol. 11: 247-256 Nuclear lamina is important for recruitment of defined chromosomal regions and participates in gene regulation Disturbance of association of chromosomes with nuclear lamina in cells from Down syndrome patients may cause a transcriptome-wide effects Pope & Gilbert (2014). Nature 508: 323-324. Letourneau et al. (2014). Nature 508: 345-350. Keratin filaments join cells together in cell sheets Keratin-green; Desmosomes-blue Different keratins are present in different skin layers Blistering of the skin caused by a mutant keratin gene basal cell of epidermis basal lamina defective keratin network hemidesmosome 40 µm Wild-type mouse Mouse expressing abnormal keratin J. Cell Biol. 115: 1661-1674 (1991) Epidermolysis bullosa hereditaria simplex Coulombe, P. A. et al. (1991). Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66, 1301–1311 Alexander disease: autosomal-dominant neurodegenerative disorder caused by mutations in the gene for glial fibrillary acidic protein (GFAP) Desmin filaments in muscle Destruction of muscle architecture in desminopathy (muscular dystrophies and/or cardiomyopathies) Desmin aggregates (red) in myofibres Skeletal muscle from a patient (left: massive accumulation of granulofilamentous material) Herrman et al. (2007). Nat. Rev. Mol. Cell. Biol. 8: 562-573 The assembly and decay of desmin mutants WT desmin mutant desmins Herrman et al. (2007). Nat. Rev. Mol. Cell. Biol. 8: 562-573