Cytoskeleton 2 (1) PDF

Cellular Biology & Homeostasis CYTOSKELETON Part 2 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES 1. Describe the structure and function of:  Microtubules (dynamic instability, MT-organizing center, MT-associated proteins, examples of cilia and flagella)  Intermediate filaments (give som...

Cellular Biology & Homeostasis CYTOSKELETON Part 2 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES 1. Describe the structure and function of:  Microtubules (dynamic instability, MT-organizing center, MT-associated proteins, examples of cilia and flagella)  Intermediate filaments (give some examples) MICROTUBULES Microtubules are polymers of the protein tubulin The tubulin subunit is itself a heterodimer formed from two closely related globular proteins called alphatubulin and beta-tubulin (each 445-450 amino acids length) • Each tubulin has a binding site for one GTP molecule:  the GTP that is bound to alpha-tubulin is trapped and is never hydrolyzed nor exchanged;  the GTP in beta-tubulin may be in either the GTP or the GDP form and is exchangeable MICROTUBULES Microtubules are hollow cylindrical structures built from protofilaments. Each composed of alphabeta tubulin heterodimers stacked head to tail and then folded into a tube MICROTUBULES How can microtubules grow and shrink?  By a process called dynamic instability Dynamic instability refers to the coexistence of assembly and disassembly at the ends of a microtubule.  The microtubule can dynamically switch between growing and shrinking phases MICROTUBULES- DYNAMIC INSTABILITY Dynamics are influenced by the binding and hydrolysis of GTP which occurs only within the betasubunit of the tubulin dimer: • The addition of GTP-containing tubulin to the end of a protofilament causes the end to grow • If GTP hydrolysis proceeds more rapidly than the addition of new subunits, the microtubule begins to shrink MICROTUBULES – DYNAMIC INSTABILITY https://www.youtube.com/watch?v=XqTyD8P67kM https://www.youtube.com/watch?v=AoPRd4AZEAY&t=3s VIDEO: Dynamic stability https://upload.wikimedia.org/wikipedia/commons/transcoded/a/a0/Microtubul eDynamicInstability.ogv/MicrotubuleDynamicInstability.ogv.480p.vp9.webm MICROTUBULES- MAPs Microtubule-associated proteins (MAPs) move along microtubules bringing transport vesicles to target organelles in the cell: • Kinesin, travels (normally) towards (+) end • Dynein, travels towards (-) end Transport of cargo MICROTUBULES - MAPs Types of kinesin ATP hydrolysis occurs in the globular head domains  Generates movement along the microtubule via the microtubule-binding domains Dynein is composed of two identical heavy chains, which make up two large globular head domains, and a variable number of intermediate and light chains.  Transport of intracellular cargos towards the (-) end of the microtubule Kinesin has a similar structure to dynein.  Transport of a variety of intracellular cargoes, including vesicles, organelles, protein complexes, and mRNAs toward the microtubule's (+) end MICROTUBULES -MAPs The selective stabilization of the microtubules can polarize a cell  MAPs can move organelles and vesicles within the cell Cell polarity refers to spatial differences in shape, structure, and function within a cell.​ Microtubule motors read MAPs https://bioscope.ucdavis.edu/2018/09/06/microtubule-motors-read-maps/ MICROTUBULES - TRANSPORT OF CARGO https://www.youtube.com/watch?v=UGc6pkpU8qM https://www.youtube.com/watch?v=7sRZy9PgPvg Molecular motor https://www.youtube.com/watch?v=-7AQVbrmzFw https://www.youtube.com/watch?v=y-uuk4Pr2i8 MICROTUBULES MAPs can move vesicles with pigments (melanosomes) in the skin https://www.youtube.com/watch?v=hNZ8Ui5Txbc https://www.youtube.com/watch?v=nMtVb5Hsi3Y MICROTUBULES - MTOC Microtubules originate from a specific cellular location known as microtubule organizing center (MTOC) • In animal cells, the centrosome is the major MTOC MICROTUBULES - MTOC • When a cell divides, the MT rearrange to form a bipolar mitotic spindle, which is responsible for aligning and segregating the chromosomes  Astral microtubuli • After division is complete, both daughter cells reorganize their MT and actin filaments Mitosis https://www.youtube.com/watch?v=C6hn3sA0ip0 MICROTUBULES – Cilia and Flagella Microtubules have major structural role in eukaryotic cilia and flagella (highly specialized and efficient motility structures)  Always extend directly from a MTOC (= basal body 9:2 structure) Action of dynein motor proteins (along MT strands along cilium/flagellum) allow for bending and generate force for movement.  Prokaryotes possess tubulin-like proteins, BUT prokaryotic flagella are totally different in structure from eukaryotic flagella, do not contain MT-based structures MICROTUBULES – Cilia and Flagella Flagella: are used to move cells in an aqueous environment (e.g. spermatozoids) Cilia: can move fluid around a cell (e.g. mucus in the respiratory epithelium) Flagella Cilia Sperm under the microscope https://www.youtube.com/watch?v=JQ5RvbjWFtQ Mucociliary movement https://www.youtube.com/watch?v=HMB6flEaZwI MICROTUBULES MICROTUBULES TAXOL (PACLITAXEL) • The principle of anticancer chemotherapy: Blocking microtubules to kill cancer cells • Blocks depolymerization • Cytostatic drug used in cancer therapy (breast/ovarian cancer) Pacific yew MICROTUBULES VINCA ALKALOIDS Vinca rosae, vinca minor MICROTUBULES COLCHICINE Alkaloid of the autumn crocus (Colchicum autumnale) INTERMEDIATE FILAMENTS INTERMEDIATE FILAMENTS • No polarity → (+) or (-) end • Subunits don’t contain ATP or GTP • Not involved in cell movement • No motor proteins associated But… intermediate filaments are associated with cell-cell junctions, strengthening cells Keratin (red) and nuclear lamin (blue) and epithelia, tissue mechanical stability INTERMEDIATE FILAMENTS • Line the inner face of the nuclear envelope, forming a protective cage for the cell’s DNA. • In the cytosol, they are twisted into strong cables that can hold epithelial cells sheets together • Help nerve cells o extend long and robust axons • Part of hair and fingernail structure Keratin intermediate network in rat kangaroo epithelial cell. Photo: Molecular expressions™ Cytoplasmic and nuclear intermediate filaments in a rat kangaroo kidney epithelia cell. Photo: Nikon’s small world competition INTERMEDIATE FILAMENTS Model of intermediate filament construction Central building block:  1 pair of 2 intertwined proteins = ‘coiled-coil’ structure  Bound together by hydrophobic interactions In a final IF there are 16 dimers of IF monomers (=32 coiled coils) INTERMEDIATE FILAMENTS INTERMEDIATE FILAMENTS KERATIN FILAMENTS: • Most diverse intermediate filament family • Produced by keratinocytes in the epidermis • Formation of horns, nails, hair, scales • Anchoring of epithelial cells via desmosomes/hemidesmosomes INTERMEDIATE FILAMENTS The diversity of keratins is clinically useful in the diagnosis of epithelial cancers (carcinoma)  Particular set of keratin filaments expressed gives an indication of the epithelial tissue in which the cancer originated and thus can help guide the choice of treatment Mutation in keratin genes cause several genetic diseases INTERMEDIATE FILAMENTS In diseases where the gene coding for keratin is mutated, the final protein disrupts the normal keratin network in the basal cells of the skin and the epidermis can easily be detached (blistering) • E.g. epidermolysis bullosa simplex (EBS) INTERMEDIATE FILAMENTS - EBS FYI KRT5 missense variant in a Cardigan Welsh Corgi with epidermolysis bullosa simplex Epidermolysis bullosa simplex in sibling Eurasier dogs is caused by a PLEC non-sense variant INTERMEDIATE FILAMENTS Other intermediate filaments Neurofilaments: high concentrations along the axons of vertebrate neurons • Participate in axonal growth (length and diameter) • Provide strength and stability to the axon Lamin: mechanical stability of cell nucleus Desmin: scaffold function for sarcomere (skeletal and cardiac muscle) CYTOSKELETON - summary Microfilaments • two helical crossed actin Structure strands Diameter • 7-8 nm Subunit • Actin • Maintenance of the cell Functions shape • Changes of the cell shape • Muscle contraction • Cell movement • In the periphery of the cell, Localization sometimes running parallel Microtubules Intermediate filaments • Tubes of 11-18 strands per MT • Long molecule polymers • 24-25 nm with 15 nm Lumen • 8-12 nm • Tubulin • α- und β-Tubulin • Keratin, Lamin, Vimentin • Maintenance of the cell shape • Maintenance of the cell • Cell movement shape • Chromosome movement during • Mechanical strength cell division • Formation of the nuclear • Movement of organelles lamina • Coming from an organizing center (example: centrosomes) and spreading towards the cell periphery • Distributed along the whole cytoplasm of the cell, building a network of filaments in the cytosol SMALL WORLD Photo competition https://www.nikonsmallworld. com/subjects/actin Clara Camargo, DVM [email protected]