Microfilament © Dr M.A. Hill, 2008 Sllide 1
ANAT3231 - 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 Email: [email protected]
Microfilament © Dr M.A. Hill, 2008 Sllide 2
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© Dr M. A. Hill, 2008 Cell Biology Laboratory
School of Medical Sciences, Faculty of Medicine The University of New South Wales, Sydney, Australia
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Microfilament © Dr M.A. Hill, 2008 Sllide 3
Lecture Overview • Microfilaments
– Structure, function and regulation
• Actin – Motility – Adhesion, focal adhesions – Actin binding proteins, myosin motors – Muscle contraction
• UNSW Cell Biology • http://cellbiology.med.unsw.edu.au/units/science/lecture07.htm
• Text: Molecular Biology of the Cell. Chapter on ‘Cytoskeleton’
Image: Dr. Barber at Pikeville College, KY
Microfilament © Dr M.A. Hill, 2008 Sllide 4
Cytoskeleton
Microfilament © Dr M.A. Hill, 2008 Sllide 5
Structural Systems Microfilaments
• shape • motility • contractility • cytokinesis • transport • compartments
Microtubules
• transport • karyokinesis
Intermediate Filaments
• compression resistance
Microfilament © Dr M.A. Hill, 2008 Sllide 6
Actin functional diversity
Microfilament © Dr M.A. Hill, 2008 Sllide 7
Microfilaments • Twisted chain 7 nm
diameter • Compared to MT
– Thinner, more flexible, shorter
• Point in same direction • Different organisation in
different cellular regions • How can actin filaments
make different structures?
MBoC Figure16-49
Microfilament © Dr M.A. Hill, 2008 Sllide 8
F-actin
Nucleation Arp2/3 Formin
Monomer Binding Profilin Thymosin
Branching Arp2/3
Capping Capping protein Tropomodulin
Stabilisation Tropomyosin
G-actin
Severing ADF/Cofilin Gelsolin
Bundling and Crosslinking Fascin αActinin
Actin Binding Proteins influence Actin Structure
Branching Arp2/3
Stabilisation Tropomyosin >40 isoforms
Capping Capping protein Tropomodulin
Nucleation Arp2/3 Formin
Monomer Binding Profilin Thymosin
Microfilament © Dr M.A. Hill, 2008 Sllide 9
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 10
Cell Movement • Whole or part of cell
– Amoeba, neutrophil, macrophages
– Neuron processes • axon, dendrites
– Common structures – Contraction
• Intracellular transport
Image: MBoC Figure 16-54
Microfilament © Dr M.A. Hill, 2008 Sllide 11
Motile Structures • Leading/Trailing Edge
– extension/retraction – Actin nucleation
• Lamellipodia – Sheet-like
extensions • Filopodia
– Thin protrusions • Integrins anchor to
ECM
MBoC Figure 16-55
Microfilament © Dr M.A. Hill, 2008 Sllide 12
Cell Migration
Microfilament © Dr M.A. Hill, 2008 Sllide 13
Adhesion Junctions • Adhesion (cell-matrix)
– Integrin – Links to extracellular matrix
Image: MBoC Figure 16-75
Microfilament © Dr M.A. Hill, 2008 Sllide 14
Focal Adhesions
Microfilament © Dr M.A. Hill, 2008 Sllide 15
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 16
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 17
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 18
Actin functional challenge Diversify function
• dynamics • organisation • mechanics
Spatial specialisation
• pool sizes • function
Evolution
• simple principle
Microfilament © Dr M.A. Hill, 2008 Sllide 19
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)
MBoC Figure16-50/51
Microfilament © Dr M.A. Hill, 2008 Sllide 20
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 21
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 22
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 23
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 24
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 25
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 26
Actin Binding Proteins
Image: MBoC Figure 16-79
Microfilament © Dr M.A. Hill, 2008 Sllide 27
Actin Motors - Myosin
Microfilament © Dr M.A. Hill, 2008 Sllide 28
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 29
Actin Motors- Myosin
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
Actin (red), Myosin II (green) Late Philip Presley, MBL: Fluorescence filter tuning of Zeiss Photomicroscope- III, allowing precise registration for the dual channel exposures.
Image Source: http://faculty-web.at.northwestern.edu/med/fukui/04-Cytoskeleton.html
Microfilament © Dr M.A. Hill, 2008 Sllide 30
Myosin Movement
MBoC Figure 16-71
Microfilament © Dr M.A. Hill, 2008 Sllide 31
Muscle Types • Skeletal, cardiac
– Striated – sarcomeres
• Smooth – non-striated
Image: MBoC Figure 16-82
Microfilament © Dr M.A. Hill, 2008 Sllide 32
Skeletal Muscle
MBoC Fugure 16-83/85
http://www.lab.anhb.uwa.edu.au/mb140/
Microfilament © Dr M.A. Hill, 2008 Sllide 33
Muscle Contraction
• sliding of filaments actin against myosin – troponin and tropomyosin
• contraction of skeletal and cardiac muscle regulated by Ca2+ flux
• smooth muscle cells and non-muscle cells – contraction same mechanism – contractile units smaller less highly ordered
• activity and state of assembly controlled by Ca2+ -regulated phosphorylation of a myosin
Text modifird from MBoC Muscle Summary
Microfilament © Dr M.A. Hill, 2008 Sllide 34
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 35
Actin functional challenge
Diversify function • dynamics • organisation • mechanics
Spatial specialisation
• pool sizes • function
Evolution
• simple principle
Microfilament © Dr M.A. Hill, 2008 Sllide 36
F-actin
Nucleation Arp2/3 Formin
Monomer Binding Profilin Thymosin
Branching Arp2/3
Capping Capping protein Tropomodulin
Stabilisation Tropomyosin
G-actin
Severing ADF/Cofilin Gelsolin
Bundling and Crosslinking Fascin αActinin
Actin Binding Proteins influence Actin Structure
Branching Arp2/3
Stabilisation Tropomyosin >40 isoforms
Capping Capping protein Tropomodulin
Nucleation Arp2/3 Formin
Monomer Binding Profilin Thymosin
Microfilament © Dr M.A. Hill, 2008 Sllide 37
Actin Filaments Tropomyosins Binds Along the Sides of ActinMicrofilaments
Actin: 2 isoforms
Tropomyosin: >40 isoforms
Microfilament © Dr M.A. Hill, 2008 Sllide 38
Distinct subcellular sorting of cytoskeleton Tm isoforms
Tm1,2,3,5a,5ab,6
Tm1,2,3
Tm1,2,3
Tm5a,b
Tm1,2,3
Tm5NM2
Tm5a,b
Tm5NM1,2
Tm1,2,3
Tm5
Tm5NM1 actin
Microfilament © Dr M.A. Hill, 2008 Sllide 39
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 40
Hall, A. (1998) Science 279:509-514
Rho activation
Rac activation
Cdc42 activation
mimics
mimics
mimics
Tm5NM1 over-expression
Tm3 over-expression
TmBr3 over-expression
Stress Fibres
Lamellipodia
Filopodia
Microfilament © Dr M.A. Hill, 2008 Sllide 41
Tm5NM1
Tm3
Shorter Filaments
fascin
Arp2/3
ADF
+ Inactive LimK
pADF
p p
Myosin Motors
p
Filopodia Stress Fibers
α-actinin1
α-actinin4
Proposed mechanism
+
Active LimK
pADF
ADF
p p
Longer Filaments
Gunning et al (2008) Physiological Reviews
Microfilament © Dr M.A. Hill, 2008 Sllide 42
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?