L2 Intermediate Filaments PDF

BS31004 The Cell Shape dynamic and Movement cytoskeleton L2 : Intermediate filaments Dr Alan Prescott [email protected] Immunofluorescence microscopy Electron microscopy actin intermediate filament...

BS31004 The Cell Shape dynamic and Movement cytoskeleton L2 : Intermediate filaments Dr Alan Prescott [email protected] Immunofluorescence microscopy Electron microscopy actin intermediate filaments filaments microtubules  Intermediate filaments Most resilient structures in the cell Can withstand large stresses and strains Cell type-specific expression patterns Large multigene family All similar secondary structure - -helical, coiled-coils Some cytoplasmic and others nuclear Network through cell from plasma membrane to nucleus The Intermediate Filament Multigene Family TYPE V TYPE VI lamins: filensin A-type=A,C CP49 TYPE I B-type=B1,B2,B3 keratins 9-20 trichocyte keratins TYPE IV Ha1-4, Hax, others neurofilaments: NF-L, NF-M NF-H -internexin nestin TYPE III vimentin desmin TYPE II GFAP keratins 1-8 peripherin trichocyte keratins Hb1-4, Hbx, others  Intermediate filament proteins Robust proteins - need denaturants to dissolve them – Removal of denaturant (eg. Urea) by dialysis initiates protein assembly No energy source required – In-vitro: Di- and monovalent cations, physiological pH, reducing agents at room temperature Assembly = dimer - tetramer - higher oligomers – Heptad repeats in -helices: form coiled coils – Sequence motifs at either end of rod domain important in assembly Small soluble pool Filaments are apolar – Subunit exchange along whole filament  Structure of intermediate filaments assembles in parallel dimers then antiparallel tetramers alpha-helical rod domain L1 L12 L2 1A 1B 2A 2B E1 V1 H1 H2 V2 E2 head domain tail domain “stutter” - another linker? essential for filament contributes assembly to filament assembly  Heptad repeats visualised as a helical wheel Heptad repeats are the basis e g of coiled-coil b c formation in a d intermediate filament f f protein dimers c d a g e b The -helix is the most common protein structure in nature The intermediate filament multigene family Type I keratin epithelia Type II keratin epithelia Type III vimentin-like mesenchyme Type IV neurofilaments neurones Type V lamins all nuclei Type VI (variable) At least 65 genes in the human genome Tissue-specific expression Usefulness in diagnostic pathology  Visualisation of intermediate filament protein tetramers by electron microscopy Intermediate filaments polymerise spontaneously and rapidly in vitro (no ATP/GTP, cofactors or associated proteins required)  Intermediate filaments resist stretching but are easily bent Figure 16-9 Molecular Biology of the Cell (© Garland Science 2008) Physical properties of the major cytoskeletal filaments Structure and assembly of intermediate filaments. Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008) Figure 16-8 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008) Keratin intermediate filament proteins Type I Proteins – Acidic isoelectric points / Lower Mr Type II proteins – Neutral or basic isolelectric points / Higher Mr Obligate heteropolymers Equimolar association of Type I and II required for assembly Specific paired expression in tissues Position of keratins on 2D gels Type I Type II Types I & II: Two families of keratin filament protein Type II Type I K2p K9 K1 K10 cornified, dry K2e stratified, barrier K3 K12 corneal K4 K13 mucosal, wet K6a K16 fast turnover K6b K17 K5 K14 K15 basal K19 K8 K18 simple K7 K20 Mirjana Liovic Keratin intermediate filaments are dynamic, as soluble keratin is incorporated into filaments.  Immunoperoxidase staining of keratin K5 in epidermis (basal cells) in skin Immunoperoxidase staining of K13 in buccal epithelium K10 fluorescence staining in epidermis (skin): suprabasal cells positive, basal cells are negative Purification and in vitro assembly of bovine muzzle keratins In vitro assembled keratin filaments  Intermediate filament attachment sites KERATINS – Desmosomes – Hemidesomosomes Type III IF proteins – Desmosomes - GFAP/Desmin – Ankyrin - plasma membrane Keratins and desmosomes seen by immunofluorescence Desmosomes and Intermediate filaments Intermediate filament - desmosome networks link cell to cell in tissues Epidermolysis bullosa simplex Blistering skin Dominant inheritance Basal keratinocytes are fragile Faults in keratin filament formation caused by mutations in basal cell keratins K5 or K14 When intermediate skin blistering filaments fail... disorders arise Transgenic mice carrying a mutant keratin gene exhibit blistering similar to that in the human disease epidermolysis bullosa simplex. The intermediate filament multigene family Type I keratin epithelia Type II keratin epithelia Type III vimentin-like mesenchyme Type IV neurofilaments neurones Type V lamins all nuclei Type VI (variable) Different filament networks can coexist in the same cell keratin vimentin Muscle: distinct locations for different intermediate filaments  Desmin surrounds the sarcomere in skeletal muscle The intermediate filament multigene family Type I keratin epithelia Type II keratin epithelia Type III vimentin-like mesenchyme Type IV neurofilaments neurones Type V lamins all nuclei Type VI (variable) Lamin intermediate filaments are all intranuclear extra piece in helix 1B CaaX Box (B lamins) nuclear localization signal Long c-term Tail domain  Intermediate filaments in the nucleus A- and B-type lamins,form framework of nuclear lamina  -helical/coiled coils ~ 55nm long (~350 amino acids): longer than cytoplasmic rod domains B-lamins in all nuclei; A-type lamins in differentiated cells Lamins cannot co-assemble with cytoplasmic filaments (due to length and sequence characteristics of -helix) Disassembly/reassembly of the nuclear lamina by phosphorylation at mitosis Interconnect nuclear pores; docking sites on nuclear membrane via LAPs Lamin meshwork in oocyte nuclei  GFP-lamin B in live cells Frans Ramaekers Nuclear lamina is linked to the cytoplasmic cytoskeleton The intermediate filament multigene family Type I keratin epithelia Type II keratin epithelia Type III vimentin-like mesenchyme Type IV neurofilaments neurones Type V lamins all nuclei Type VI (variable)  Cross-linked IFs Less cross-links in axon (NF-H non- in Glial cells helical extension) Figure 16-22 Molecular Biology of the Cell (© Garland Science 2008)  Neurofilament protein structure NF-L NF-H.. KSP KSP KSP KSP.. Quick-freeze deep etch preparation for EM, showing cross bridges between neurofilaments consisting of extended tail domains (Chen et al., J Cell Sci. 113, 2000)  Type VI ? Lens specific IFproteins:CP49,filensin Filensin+ CP49+crystallin Human lens 5 weeks CP49 Filensin The intermediate filament multigene family Type I keratin epithelia Type II keratin epithelia Type III vimentin-like mesenchyme Type IV neurofilaments neurones Type V lamins all nuclei Type VI (variable)