CBS Cytoskeleton KEATS 23_24 Tagged 2 PDF

Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Cytoskeleton Teaching Objectives To be able to describe the thr...

Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Cytoskeleton Teaching Objectives To be able to describe the three major structural components of the cytoskeleton, their assembly and disassembly and their associated proteins To understand the importance of the cytoskeleton in various aspects of cell structure, intracellular motility and cell movement Be aware of the diseases caused by problems of cytoskeleton 2  Cytoskeleton - outline Network of protein filaments with in a cell Range of functions – Connection with Extracellular Matrix (ECM) – Maintaining cell shape – Intracellular transport – Cytokinesis – Chromosome separation – Cell movement  Composition of Cytoskeleton Actin microfilaments – Polymers of actin – 7-9 nm diameter Intermediate filaments – Tissue specific proteins – 10 nm Microtubules – Polymers of tubulin – 25 nm diameter  Actin microfilaments Monomer is globular protein (G-actin) contains ATP-binding domain G-actin polymerises to microfilament (F-actin), process involves ATP hydrolysis Polymerisation via non-covalent interactions, monomers in Head to Tail orientation, each microfilament has a (+) and (-) end Monomers can be added at both (+) and (-) end Dynamic structure: length depends of relative rate of loss and gain of G- actin monomers Actin microfilaments are usually present as two tightly wound chains Comprises ~5% of cellular protein  Actin microfilament structure Biochemistry and Molecular Biology Fig8.4  Function of actin Muscle contraction – covered in “Skeletal Muscle” lecture Mechanical support – e.g. in microvilli Biochemistry and Molecular Biology Fig8.14 Maintaining cell shape Cell movement Biochemistry and Molecular Biology Fig8.13  Actin binding proteins G actin binding proteins – thymosin β4 : inhibits polymerisation Cross-linking proteins – villin : parallel bundles in microvilli – filamin : joining at angles to create a mesh Severing – gelsolin : cuts and binds (+) end; the other part depolmerises – “gel to sol” Biochemistry and Molecular Biology Fig8.15  Contraction in non-muscle cells Muscle myosin is also present in non-muscle cells Interaction between myosin and actin microfilaments allow movement , requires ATP hydrolysis Movement within cells – Cytokinesis : ring of actin forms in the centre of cell anchors to the plasma membrane, myosin contraction constricts the cell Movement of cells – Lamellipodia mediated cell movement across extracellular matrix (ECM)  Lamellipodia formation Extensions of cells contain actin network Generated by rapid growth of actin filaments at the cell membrane Tip of lamellipodia interacts with ECM via integrins Contraction involving myosin allows cell movement time  Intermediate Filaments (IF) Polymers of individual IF proteins - 10nm in diameter Different IF proteins in different cell types – Epithelia cells : keratin(s) Physical support and external structures – Axons : neurofilamin(s) Structural arrangement of axons – Universal (nuclear) : lamins A, B, C Supporting nuclear structure Usually stable and not dynamic – Lamins are exception as nuclear membrane reforms during mitosis  Formation of IF polymer 1 1. Intermediate filament protein (monomer) 2. Helical dimer 2 3. Two dimers combine to form a tetramer (the fundamental unit of the IF) 3 4. Tetramers link in a staggered formation and end-to-end to form the filament 4 5. Subunit exchange is slow but occurs throughout the length of the filament 5  Interaction between IFs and cytoskeleton IFs can link actin mircofilaments and also microtubules e.g. plectin  IF: clinical considerations Mutations in keratins can result in the skin blistering disease epidermolysis bullosa simplex (EBS) Abnormal expression of neurofilamins can result in the neurodegenerative disease amyotrophic lateral sclerosis (ALS)  Microtubules : protofilament Tubulin monomer is a heterodimer : α-tubulin and β-tubulin Protofilament has a (-) and (+) end Monomers can be rapidly added and removed from both (+) and (-) ends 13 parallel protofilaments Biochemistry and Molecular Biology Fig8.17 arranged in a hollow tube Microtubules approx. 25nm diameter Microtubule-Organising Centre Usually have one end attached to a Microtubule-Organising Centre (MTOC) One MTOC associated with nucleus Microtubules “grow out” from MTOC until reach destination and then are stabilised Assembly and disassembly of microtubule Pi + end - end GTP GDP GTP bound monomers GDP bound monomers assemble onto filament dissociate rapidly Pi  Function of microtubules Dynamic scaffold – Spindle for chromatid separation during mitosis Movement of cargo to specific locations in cell Central internal support of cilia Stabilise structure of cells – e.g. platelets Organise the structure of organelles – e.g. endoplasmic reticulum  Spindle formation Spindle consists of microtubules Spindle formation initiated from the centrosome (type of Microtubule- Organising Centre) Centrosomes contain a pair of centrioles Centrioles contain stable fused microtubules Centrosomes form at two poles of cell Kinetochore microtubules attached to the centromere of chromatid Aster microtubules attach centrosome to cell membrane Mitotic spindle Biochemistry and Molecular Biology fig8.21  Mitotic spindle Mitotic spindle – images of cells  Movement of cargo within cell Two motor proteins associated with microtubules ATP hydrolysed to move cargo along the microtubule Kinesin moves towards (+) end, towards the cell periphery Dynein moves towards (–) end, near nucleus Biochemistry and Molecular Biology fig8.19  “Tramways” within cell Molecular Biology of the cell Figure 16-98 Vesicles move 10 cm per day so can take more than a week to move down long axons  Cilia Membrane bound hair-like extensions Microtubules is central support MTOC is called Basal Body located close to membrane Microtubules facilitate the movement of components up and down within cilia All cells have single primary cilia Disassembled during mitosis Specialised types: – Stereocilia in the inner ear – sound detection – Motile cilia in respiratory / lung ciliated epithelia – beat and move fluid around  Stereocilia in the inner ear The cells are depolarised or hyperpolarised by deflections caused by sound Actin filaments keep the stereocilia rigid Primary versus motile cilia Ainsworth, C. Cilia: Tails of the unexpected. Nature 448, 638–641 (2007)  Ciliopathies Situs inversus – Congenital condition, organs are in mirrored position – Defect in cilia-mediated movement of growth factors in the embryo Autosomal Dominate Polycystic Kidney Disease (ADPKD) – Formation of kidney cystic that expand and disrupt normal tissue – Mutated proteins (polycystin-1 and -2) associated with abnormal function of primary cilia  Microtubules : clinical considerations Disruption of mitotic spindle can interfere with cell division Anti-cancer chemotherapeutic agents – Colchicine : binds tubulin monomers prevents microtubule formation – Taxol : binds and stabilises microtubules https://odanlab.com/product/colchicine/ https://taxol.weebly.com/description-what-is-taxol.html  Role of cytoskeleton in cell-cell adherence actin microfilaments Junctions between cells are connected to the cell cytoskeleton – desmosome – gap junctions Attachments to the ECM are connected to the cell IF cytoskeleton – hemidesmosomes – focal adhesions ECM  Summary Cell biology and function of actin filaments Cell biology and function of IFs Cell biology and function of microtubules Nature and function of cilia