Cytoskeleton 2 2024.pdf

Cellular Biology & Homeostasis CYTOSKELETON Part 2 VP 2024 Clara Camargo, DVM LEARNING OBJECTIVES 1. Describe the structure and function of:  Microtubules (dynamic instability, MT-organizing center, MT-associated proteins, cilia and flagella)  Intermediate filaments (cell junction, mechanical stab...

Cellular Biology & Homeostasis CYTOSKELETON Part 2 VP 2024 Clara Camargo, DVM LEARNING OBJECTIVES 1. Describe the structure and function of:  Microtubules (dynamic instability, MT-organizing center, MT-associated proteins, cilia and flagella)  Intermediate filaments (cell junction, mechanical stability, mutations affecting this filament) 2. Understand how drugs can affect the functioning of microtubules (give examples) MICROTUBULES Microtubules are polymers of the protein tubulin The tubulin subunit is itself a heterodimer formed from two closely related globular proteins called alpha-tubulin 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 hydrolysed nor exchanged;  the GTP in beta-tubulin hydrolyses during polymerization (growing) → GDP. Upon microtubule depolymerization (shrinking), GDP is exchanged to GTP, and beta-tubulin can polymerize once more. MICROTUBULES Microtubules are hollow cylindrical structures built from heterodimeric protofilaments Each composed of: alpha-beta tubulin heterodimers stacked head to tail and folded into a tube MICROTUBULES How can microtubules grow (polymerize) and shrink (depolymerize)?  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  The structural plasticity and dynamics of microtubules depend on the activities of the GTP-binding domain of α-, β-tubulin MICROTUBULES- DYNAMIC INSTABILITY GTP hydrolysis occurs only within the beta-subunit of the tubulin dimer The addition of GTP-containing tubulin to the end of a protofilament causes the end to polymerize (grow) If GTP hydrolysis proceeds more rapidly than the addition of new subunits, the microtubule begins to depolymerize (shrink) MICROTUBULES- DYNAMIC INSTABILITY Clinical relevance of microtubules dynamic instability The microtubule network is recognized for its role in regulating: Cell growth and movement Cell signaling events → which modulate fundamental cellular processes Dynamic Microtubule Arrays in Leukocytes and Their Role in Cell Migration and Immune Synapse Formation FYI https://www.frontiersin.org/articles/10.3389/fcell.2021.635511/full 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 (+ end or – end)  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 FYI https://bioscope.ucdavis.edu/2018/09/06/microtubule-motors-read-maps/ 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 Centrosome: made up of 2 cylinders called centrioles Centrioles - very small cylindrical organelle near the nucleus in animal cells, occurring in pairs and involved in the development of spindle fibers in cell division. 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 highly specialized and efficient motility structures  flagella Basal body microtubule-base 9:2 structure How do cilia and flagella move? https://www.youtube.com/watch?v=9nZYlyFGm50 Action of dynein motor proteins on 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 upwards in the respiratory epithelium) Cilia Flagella Sperm under the microscope https://www.youtube.com/watch?v=JQ5RvbjWFtQ Mucociliary movement https://www.youtube.com/watch?v=HMB6flEaZwI MICROTUBULES MICROTUBULES PACLITAXEL (TAXOL) Anticancer chemotherapy: prevents depolymerization of MT, decreasing the dynamic nature of these cytoskeletal structures Cytostatic drug used in cancer therapy (breast/ovarian cancer)  inhibits progression of mitotic cells to G1 phase by interference with spindle formation without affecting other microtubule functions during anaphase and telophase Pacific yew MICROTUBULES VINCA ALKALOIDS Vinca rosae, vinca minor Vincristine binds irreversibly to microtubules and spindle proteins → interferes with the formation of the mitotic spindle → arresting tumor cells in metaphase MICROTUBULES Colchicine can aid in familial Shar-pei fever treatment FYI  Prevents polymerization of MT and consequently secretion of SAA (serum amyloid A – reactive amyloidosis) COLCHICINE https://files.brief.vet/migration/article/15736/familial-shar-pei-fever-15736-article.pdf 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 However… intermediate filaments are associated with cell-cell junctions, strengthening cells and epithelia, tissue Keratin (red) and nuclear lamin (blue) 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:  2 intertwined proteins = ‘coiled-coil’ dimer  Bound together by hydrophobic interactions In a final IF there are 16 dimers of IF monomers (=32 coiled coils) INTERMEDIATE FILAMENTS EPITHELIAL 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 American Academy of Dermatology Association https://www.aad.org/public/diseases/az/epidermolysis-bullosa-overview 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 → the epidermis can easily be detached (blistering) E.g., epidermolysis bullosa simplex (EBS) 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 Chromosome movement during shape cell division Mechanical strength Movement of organelles Formation of the nuclear Cell movement 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