anat3231 - cell biology lecture 11 · anat3231 - cell biology lecture 11 school of medical sciences...

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


Cytoskeleton


Structural Systems Microfilaments

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

Microtubules

•  transport •  karyokinesis

Intermediate Filaments

•  compression resistance


Actin functional diversity


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


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


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.


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


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


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?


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


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


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


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





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.


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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