YR1 Lecture 1H - The Cytoskeleton - Dr Morven Cameron 2021 PDF

Dr Morven Cameron [email protected] Learning outcomes Recognise that the cytoskeleton is the fundamental basis for cell movement and for intracellular transport, and that the actin cytoskeleton is also the basis of skeletal muscle function List examples of diseases that are a result of...

Dr Morven Cameron [email protected] Learning outcomes Recognise that the cytoskeleton is the fundamental basis for cell movement and for intracellular transport, and that the actin cytoskeleton is also the basis of skeletal muscle function List examples of diseases that are a result of mutations or ‘poisoning’ of the cytoskeleton, and recognise that it is also a significant drug target (i.e. vincristine, taxol). Why should I care? Basis of cellular structure and intracellular transport. Many diseases/dysfunctional states arise due to alterations in cytoskeletal elements: – Myopathies/dystophies; cancer; heart failure; neurodegeneration; deafness. Target of several natural toxins: – Clostridial toxins; enterotoxin; E-coli necrotic factor; phalloidin (death cap mushrooms). Target of several important drugs: – Vincristine, vinblastine, taxol, colchicine, nocodazole. Functions Provides support and maintains cell shape. Protects the cell. Allows muscle contraction. Enables cellular motion. Intracellular transport. Critical role in cell division. Cytoskeletal elements Microfilaments: – 3-6 nm in diameter. – Mainly form the cortex beneath the plasma membrane. Intermediate filaments: – 10 nm diameter. – Extend from perinucleus to cell periphery; enriched at sites of cell contact. Microtubules: – 25 nm in diameter. – Extend from perinucleus to cell periphery. Cytoskeletal elements Microfilaments: – 3-6 nm in diameter. – Mainly form the cortex beneath the plasma membrane. Intermediate filaments: – 10 nm diameter. – Extend from perinucleus to cell periphery; enriched at sites of cell contact. Microtubules: – 25 nm in diameter. – Extend from perinucleus to cell periphery. Microfilaments Made up of actin monomers (G-actin) globular protein. Contains a nucleotide binding site (ATP or ADP) which regulates shape of protein. Actin-ATP à polymerisation à ActinADP. Microfilaments G-actin (monomer) becomes F-actin (actin filament) – twisted helical chains, has polarity: barbed (+) and pointed end (-). Modulation: – Polymerisation is aided by profilin occurs at both ends but favours the barbed end at critical concentrations of G-actin molecules. – Capping proteins prevent further polymerisation. – Severing proteins: gelsolin, ADF/cofilin. – Microfilaments can be cross-linked or bundled together to form many different structures. + - Microfilaments Inner life of a cell: http://www.xvivo.net/animation/the-inner-life-of-the-cell/ Functions of microfilaments in cells? Maintenance of cell shape Actin – myosin interaction Myosin is from a large family of motor proteins that have varying roles in different cell types. The most well known actin – myosin interaction occurs in muscle cells to produce contraction. However, actin – myosin interactions are also involved in cell crawling. Utilises the energy from ATP hydrolysis to move along the microfilament. Muscle contraction Microfilaments – cell crawling Actin filaments polymerize towards the direction of crawling. Lamellipodium protrude from the cell and attach to the substratum (basement membrane). Myosin II contracts pulling the cell in the direction of the newly formed actin filaments. Cell crawling Retinal pigment epithelial (RPE) cell crawling – in my lab Microfilaments primary regulators of cell movement – good or bad? Good: – – – – Muscle cells. Immune cells. Wound healing. Axon guidance – neurons. Bad – Cancer – metastasis. – Angiogenesis in diabetic retinopathy – epithelial cell migration. – Immune infiltration into the CNS – multiple sclerosis. – Neuronal sprouting – neuropathic pain. Cytoskeletal elements Microfilaments: – 3-6 nm in diameter. – Mainly form the cortex beneath the plasma membrane. Intermediate filaments: – 10 nm diameter. – Extend from perinucleus to cell periphery; enriched at sites of cell contact. Microtubules: – 25 nm in diameter. – Extend from perinucleus to cell periphery. Intermediate filaments >70 distinct intermediate filament proteins. Can be divided into six major classes: I Acidic Keratins II Basic Keratins III Desmin GFAP Peripherin Vimentin IV Neurofilaments V Lamins VI Nestin Epithelia Epithelia Muscle Glial cells Peripheral neurons Mesenchyme Neurons Nuclear envelope Neural stem cells Intermediate filaments Tend to form more or less permanent structures in tissues such as skin and hair. Are phosphorlylated if dissasembly is required (i.e. cell division). Involved in cell-cell (desmosomes) and cellmatrix (hemidesmosomes) adhesion. https://micro.magnet.fsu.edu Intermediate filaments Intermediate filaments Intermediate fibers in hair Type I (acidic) filaments associate with type II (basic) filaments in the dimer. Cell–cell adhesion Desmosomes are found in epithelia, and cardiac muscle. Cell–matrix adhesion Intermediate fibers attach (indirectly) to adhesion proteins (integrins), which anchor the cell (mainly epithelial cells) to the basement membrane. Intermediate filament disorders - examples Type I & II disorders Epidermolysis bullosa simplex of Weber–Cockayne type Type III disorders Amyotrophic lateral sclerosis (ALS) Type IV disorders Parkinson’s disease. Alzheimers disease Type V disorders - Very varied due to nuclear localisation of type V intermediate filaments: - Lipodystrophy - Muscle laminopathies - Neurological disorders - Systemic laminopathies Meesman corneal dystrophy Type VI disorders - cateract http://www.interfil.org/ Cytoskeletal elements Microfilaments: – 3-6 nm in diameter. – Mainly form the cortex beneath the plasma membrane. Intermediate filaments: – 10 nm diameter. – Extend from perinucleus to cell periphery; enriched at sites of cell contact. Microtubules: – 25 nm in diameter. – Extend from perinucleus to cell periphery. Microtubules Subunits are dimers of the protein tubulin. – One monomer each of α-tubulin and βtubulin; bound GTP is hydrolysed during or immediately following polymerisation. – Polar: α-tubulin at minus end and βtubulin at the plus end – more tubulin is always added to the + end. – 13 tubulin polymers form the circumference; small tubes with internal diameter ~ 14 nm. All microtubules attach to a centrioles in the centrosome. Display dynamic instability. Microtubule Associated Proteins (MAPs) MAPs stabilise microtubules with protein interactions – e.g. Tau Functions tightly regulated by phosphorylation. Gives rise to specialised microtubule structures/functions: – Cilia – e.g. respiratory cilia – Flagella – Sperm – Centrioles during cell division Microtubules in cell division Microtubules in cell division Polymerisation De-polymerisation Microtubules in cell division Nikon Imaging Center at UCSF: https://www.youtube.com/watch?v=2J65DoinDKU Interaction with motor proteins Motor proteins, dynein and kinesin “walk” along microtubules. Dynein moves towards the negative pole (-), in a retrograde motion. – Retrograde = towards cell body. Kinesin moves towards the positive pole (+), in an anterograde motion. – Anterograde = away from the cell body Process is dependent on ATP hydrolysis. Involved in many roles, including vesicular transport, flagellar/cilia motion (dynein), transport of organelles. Vesicle transport Vesicle transport Cilia and flagella Both have a similar structure, but cilia are short and many, flagella are long and few. Cilia are expressed on a number of epithelial cells where they move particles. – e.g. Epithelial cells of the respiratory passages (nose, pharynx, trachea) contain huge numbers of cilia (107/mm2) where they dislodge and expel particles that collect in the mucous secretions. Sperm cells notably have a long and active flagella which they use to navigate to the egg. – Fallopian tubes also have cilia to guide the egg down the fallopian tubes. Dynein is involved in powering the beating motion of both flagella and cilia. Cilia and flagella Microtubule pathologies -examples Neurodegeneration – Parkinson’s disease, Alzheimer’s Disease, frontotemporal dementia and other tauopathies. – Tau forms a stabilising protein for microtubules. Ciliopathies – – – – Kartagener Syndrome Joubert Syndrome Meckel Syndrome Polycystic kidney disease Muscular dystrophy – Duchenne muscular dystrophy – one of Prof Morley’s research areas. Alzheimer's disease/tauopathies https://www.nia.nih.gov/alzheimers/publication/part-2-what-happens-brain-ad/hallmarks-ad Drugs that target microtubules Vincristine, vinblastine: Hodgkin's and nonHodgkin's lymphomas; acute lymphocytic leukemia and Wilm's tumor. Colchicine: Gout and Behçet's disease. Taxol (taxanes): Ovarian cancer, breast cancer, bronchogenic carcinoma (non-small cell type). Cytoskeleton Microfilaments: – Maintains cell shape. – Cell crawling. – Muscle contraction (in association with myosin). Intermediate filaments: – Semi-permanent. – Cell adhesion. – Hair and nails. Microtubules: – Cell division. – Vesicular and organelle transport. – Cilia/flagella.