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UNSW ANAT3231 Cell Biology Lecture 11- Microfilaments

Tuesday, April 22, 2008

© Dr M.A. Hill, 2008 1

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]


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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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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



© Dr M.A. Hill, 2008 2


Cytoskeleton


Structural Systems Microfilaments

•  shape •  motility •  contractility •  cytokinesis •  transport •  compartments

Microtubules

•  transport •  karyokinesis

Intermediate Filaments

•  compression resistance


Actin functional diversity



© Dr M.A. Hill, 2008 3


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


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





Roles of Actin Filaments

•  Cell movement 1. Structures involved 2. Dynamics and coordination

•  Cell Adhesions 1. Cell-substratum 2. Cell-cell



© Dr M.A. Hill, 2008 4


Cell Movement •  Whole or part of cell

–  Amoeba, neutrophil, macrophages

–  Neuron processes •  axon, dendrites

–  Common structures –  Contraction

•  Intracellular transport

Image: MBoC Figure 16-54


Motile Structures •  Leading/Trailing Edge

–  extension/retraction –  Actin nucleation

•  Lamellipodia –  Sheet-like

extensions •  Filopodia

–  Thin protrusions •  Integrins anchor to

ECM

MBoC Figure 16-55


Cell Migration



© Dr M.A. Hill, 2008 5


Adhesion Junctions •  Adhesion (cell-matrix)

–  Integrin –  Links to extracellular matrix



Focal Adhesions


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.



© Dr M.A. Hill, 2008 6


Adherens Junctions •  heart muscle, layers

covering body organs, digestive tract.

•  transmembrane proteins-Cadherins

•  Cadherins linked to actin microfilaments


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


Actin functional challenge Diversify function

•  dynamics •  organisation •  mechanics

Spatial specialisation

•  pool sizes •  function

Evolution

•  simple principle



© Dr M.A. Hill, 2008 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)

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


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



© Dr M.A. Hill, 2008 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


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?


Small GTPase Regulate the Actin Cytoskeleton

Rho Stress Fibres Rac Lamellapodia Cdc 42 Filipodia How do signals turn into structure?



© Dr M.A. Hill, 2008 9


Small GTPases regulate polymerisation and create different structures

– Activate monomer binding protein

•  Sequester •  Release

– Activate polymer binding proteins •  Bundling •  cross-linking •  Severing •  contracting


Actin Binding Proteins



Actin Motors - Myosin



© Dr M.A. Hill, 2008 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


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


Myosin Movement

MBoC Figure 16-71



© Dr M.A. Hill, 2008 11


Muscle Types •  Skeletal, cardiac

–  Striated –  sarcomeres

•  Smooth –  non-striated



Skeletal Muscle

MBoC Fugure 16-83/85

http://www.lab.anhb.uwa.edu.au/mb140/


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



© Dr M.A. Hill, 2008 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?


Actin functional challenge

Diversify function •  dynamics •  organisation •  mechanics

Spatial specialisation

•  pool sizes •  function

Evolution

•  simple principle


F-actin



Branching Arp2/3


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






© Dr M.A. Hill, 2008 13


Actin Filaments Tropomyosins Binds Along the Sides of ActinMicrofilaments

Actin: 2 isoforms

Tropomyosin: >40 isoforms


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


Isoforms Define Specific Functional Properties of Actin Filaments

•  Spatially segregated filaments contain

different tropomyosins. •  Spatially segregated filaments have

different functional roles in the cell.



© Dr M.A. Hill, 2008 14


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


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


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?

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