ANAT Cell Biology Lecture 11 School of Medical Sciences The University of New South Wales. UNSW Copyright Notice

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1 ANAT Cell Biology Lecture 11 School of Medical Sciences The University of New South Wales The actin cytoskeleton Prof Peter Gunning Oncology Research Unit Room 502A Wallace Wurth Building p.gunning@unsw.edu.au Microfilament Dr M.A. Hill, 2008 Sllide 1 UNSW Copyright Notice COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by or on behalf of the University of New South Wales pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further copying or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice. Microfilament Dr M.A. Hill, 2008 Sllide 2 Dr M. A. Hill, 2008 Cell Biology Laboratory School of Medical Sciences, Faculty of Medicine The University of New South Wales, Sydney, Australia Web: m.hill@unsw.edu.au Lecture Overview Microfilaments Structure, function and regulation Actin Motility Adhesion, focal adhesions Actin binding proteins, myosin motors Muscle contraction UNSW Cell Biology Text: Molecular Biology of the Cell. Chapter on Cytoskeleton Microfilament Dr M.A. Hill, 2008 Sllide 3 Image: Dr. Barber at Pikeville College, KY Dr M.A. Hill,

2 Cytoskeleton Microfilament Dr M.A. Hill, 2008 Sllide 4 Structural Systems Microfilaments shape motility contractility cytokinesis transport compartments Microtubules transport karyokinesis Intermediate Filaments compression resistance Microfilament Dr M.A. Hill, 2008 Sllide 5 Actin functional diversity Microfilament Dr M.A. Hill, 2008 Sllide 6 Dr M.A. Hill,

3 Twisted chain 7 nm diameter Compared to MT Thinner, more flexible, shorter Point in same direction Microfilaments Different organisation in different cellular regions How can actin filaments make different structures? Microfilament Dr M.A. Hill, 2008 Sllide 7 MBoC Figure16-49 Actin Binding Proteins influence Actin Structure Capping Capping protein Tropomodulin Nucleation Nucleation Arp2/3 Arp2/3 Formin Formin Monomer Monomer Binding Binding Profilin Profilin Thymosin Thymosin Branching Arp2/3 Arp2/3 F-actin G-actin Stabilisation Tropomyosin Tropomyosin >40 isoforms Microfilament Dr M.A. Hill, 2008 Sllide 8 Severing ADF/Cofilin Gelsolin Bundling and Crosslinking Fascin αactinin Roles of Actin Filaments Cell movement 1. Structures involved 2. Dynamics and coordination Cell Adhesions 1. Cell-substratum 2. Cell-cell Microfilament Dr M.A. Hill, 2008 Sllide 9 Dr M.A. Hill,

4 Whole or part of cell Amoeba, neutrophil, macrophages Neuron processes axon, dendrites Common structures Contraction Intracellular transport Cell Movement Microfilament Dr M.A. Hill, 2008 Sllide 10 Image: MBoC Figure Leading/Trailing Edge extension/retraction Actin nucleation Lamellipodia Sheet-like extensions Filopodia Thin protrusions Integrins anchor to ECM Motile Structures MBoC Figure Microfilament Dr M.A. Hill, 2008 Sllide 11 Cell Migration Microfilament Dr M.A. Hill, 2008 Sllide 12 Dr M.A. Hill,

5 Adhesion Junctions Adhesion (cell-matrix) Integrin Links to extracellular matrix Microfilament Dr M.A. Hill, 2008 Sllide 13 Image: MBoC Figure Focal Adhesions Microfilament Dr M.A. Hill, 2008 Sllide 14 Cell-Cell Junctions Adherens (cell-cell) cadherin (E-cadherin) Links to cadherin in neighboring cell microfilaments anchor the plaque that occurs under the membrane of each cell. Microfilament Dr M.A. Hill, 2008 Sllide 15 Dr M.A. Hill,

6 Adherens Junctions heart muscle, layers covering body organs, digestive tract. transmembrane proteins-cadherins Cadherins linked to actin microfilaments Microfilament Dr M.A. Hill, 2008 Sllide 16 Actin and Adhesion Actin filaments are the physical connections between the cell and its environment. Actin filaments can provide a direct link from the inside of the cell to adjoining cells or to the extracellular space. Actin filaments are involved in transmitting signals from the external environment to the cell interior Microfilament Dr M.A. Hill, 2008 Sllide 17 Actin functional challenge Diversify function dynamics organisation mechanics Spatial specialisation pool sizes function Evolution simple principle Microfilament Dr M.A. Hill, 2008 Sllide 18 Dr M.A. Hill,

7 Actin Microfilament Formation Globular actin monomer (g actin) polymerise to Filamentous actin (f actin) Cells approx 50:50 Monomer can add to either (+ or - ) end Faster at + end Actin-ATP hydrolysed (ADP) following addition Destabilises (like MT) Microfilament Dr M.A. Hill, 2008 Sllide 19 MBoC Figure16-50/51 Nucleation/Elongation Nucleation Two actin molecules bind weakly addition of a third (trimer) stabilizes the complex forms a "nucleation site Elongation Additional actin molecules form a long helical polymer Initial period of growth Then equilibrium phase reached Dynamic Equilibrium The relative rates of elongation and depolymerization controls filament length Microfilament Dr M.A. Hill, 2008 Sllide 20 Actin Types 6 Mammalian actin types (isoforms) All are 43 Kd Protein 2 cytoskeletal isoforms in all non-muscle cells Beta (β) 7p22-p12 Gamma (γ) 17q25 4 muscle isoforms in different muscle cells Alpha (α) skeletal Alpha (α) cardiac Alpha (α) smooth Gamma (γ) smooth Microfilament Dr M.A. Hill, 2008 Sllide 21 Dr M.A. Hill,

8 Actin Isoforms are Functionally Distict β- vs γ-actin in myoblasts β-actin promotes cell spreading and stress fibres γ-actin inhibits cell spreading and stress fibre formation Microfilament Dr M.A. Hill, 2008 Sllide 22 Actin Filament Function Actin filaments have to be nucleated The filaments are dynamic There are multiple types of actin Different actins have different roles The actins are functionally distinct But what signal controls polymerisation? Microfilament Dr M.A. Hill, 2008 Sllide 23 Small GTPase Regulate the Actin Cytoskeleton Rho Stress Fibres Rac Lamellapodia Cdc 42 Filipodia How do signals turn into structure? Microfilament Dr M.A. Hill, 2008 Sllide 24 Dr M.A. Hill,

9 Small GTPases regulate polymerisation and create different structures Activate monomer binding protein Sequester Release Activate polymer binding proteins Bundling cross-linking Severing contracting Microfilament Dr M.A. Hill, 2008 Sllide 25 Actin Binding Proteins Microfilament Dr M.A. Hill, 2008 Sllide 26 Image: MBoC Figure Actin Motors - Myosin Microfilament Dr M.A. Hill, 2008 Sllide 27 Dr M.A. Hill,

10 Actin Motors - Myosin Myosins Myosin I All cells One head domain Binds actin Myosin II Muscle myosin Also other cells Dimer, 2 heads Bind to each other to form myosin filament Thick filament Microfilament Dr M.A. Hill, 2008 Sllide 28 Actin Motors- Myosin Actin (red), Myosin II (green) Late Philip Presley, MBL: Fluorescence filter tuning of Zeiss Photomicroscope- III, allowing precise registration for the dual channel exposures. Myosin I (green), Myosin II (red) Dr. Edward Korn, Dr. Thomas Lynch, NIH: Polyclonal anti-acanthamoeba myosin-i antibody, revealed a unique localization to myosin isoforms Microfilament Dr M.A. Hill, 2008 Sllide 29 Image Source: Myosin Movement Microfilament Dr M.A. Hill, 2008 Sllide 30 MBoC Figure Dr M.A. Hill,

11 Skeletal, cardiac Striated sarcomeres Smooth non-striated Muscle Types Microfilament Dr M.A. Hill, 2008 Sllide 31 Image: MBoC Figure Skeletal Muscle MBoC Fugure 16-83/85 Microfilament Dr M.A. Hill, 2008 Sllide 32 Muscle Contraction sliding of filaments actin against myosin troponin and tropomyosin contraction of skeletal and cardiac muscle regulated by Ca 2+ flux smooth muscle cells and non-muscle cells contraction same mechanism contractile units smaller less highly ordered activity and state of assembly controlled by Ca 2+ - regulated phosphorylation of a myosin Microfilament Dr M.A. Hill, 2008 Sllide 33 Text modifird from MBoC Muscle Summary Dr M.A. Hill,

12 Questions A single filament can be µm in length. How can you ensure the filament has the same function along its length? How can you make functionally different filaments in different parts of the cell? Microfilament Dr M.A. Hill, 2008 Sllide 34 Actin functional challenge Diversify function dynamics organisation mechanics Spatial specialisation pool sizes function Evolution simple principle Microfilament Dr M.A. Hill, 2008 Sllide 35 Actin Binding Proteins influence Actin Structure Capping Capping protein Tropomodulin Nucleation Nucleation Arp2/3 Arp2/3 Formin Formin Monomer Monomer Binding Binding Profilin Profilin Thymosin Thymosin Branching Arp2/3 Arp2/3 F-actin G-actin Stabilisation Tropomyosin Tropomyosin >40 isoforms Microfilament Dr M.A. Hill, 2008 Sllide 36 Severing ADF/Cofilin Gelsolin Bundling and Crosslinking Fascin αactinin Dr M.A. Hill,

13 UNSW ANAT3231 Cell Biology Actin Tropomyosins BindsFilaments Along the Sides of Actin Microfilaments Tropomyosin: >40 isoforms Actin: 2 isoforms Microfilament Dr M.A. Hill, 2008 Sllide 37 Distinct subcellular sorting of cytoskeleton Tm isoforms Tm5a,b Tm1,2,3,5a,5ab,6 Tm1,2,3 Tm5a,b Tm5 Tm1,2,3 Tm1,2,3 Tm5NM1 actin Tm5NM1,2 Tm1,2,3 Tm5NM2 Microfilament Dr M.A. Hill, 2008 Sllide 38 Isoforms Define Specific Functional Properties of Actin Filaments Spatially segregated filaments contain different tropomyosins. Spatially segregated filaments have different functional roles in the cell. Microfilament Dr M.A. Hill, 2008 Sllide 39 Dr M.A. Hill,

14 Tm5 NM1 over-expression Rho activation mimics Stress Fibres TmBr3 over-expression Rac activation mimics Lamellipodia Tm3 over-expression Cdc42 activation mimics Filopodia Microfilament Dr M.A. Hill, 2008 Sllide 40 Hall, A. (1998) Science 279: Proposed mechanism Filopodia Stress Fibers α-actinin1 α-actinin4 fascin Myosin Motors p p p padf Arp2/3 Shorter Filaments Longer Filaments + Active LimK ADF Inactive LimK ADF + Tm5 NM1 Microfilament Dr M.A. Hill, 2008 Sllide 41 p p padf Tm3 Gunning et al (2008) Physiological Reviews Future How do you make specific filaments at specific sites in the cell? How do you make homopolymers? How do the tropomyosins control filament function with such precision? How do tropomyosins control cell signalling? Can we make anti-tropomyosin drugs? Microfilament Dr M.A. Hill, 2008 Sllide 42 Dr M.A. Hill,