Lecture 20 & Term 2 Lecture 2 Actin: Organisation/Assembly/Binding/Movement

Question 1

Cytoskeleton

Answer 1

Network of filaments extending throughout eukaryotic cells
Highly dynamic
Responsible for cell movement/shape
For muscle contraction
Organelle movement
Vesicle transport
Secretion and uptake
Chromosome separation at mitosis
Separation of daughter cells at mitosis

Resistance of cells and tissues e.g. to mechanical stress

Question 2

Cytoskeleton has 3 independent protein networks

Answer 2

Microfilaments - 7-9nm wide - actin, stabilised by other proteins

Microtubules - 25nm - alpha and beta tubulin dimers rigid but dynamic can assemble and disassemble rapidly

Intermediate fibres - 10nm - various e.g. keratin in skin and hair, lamina that line nuclear envelope and provide strength

Question 3

Organisation of network depends on cell function and type

Answer 3

Moving cells like macrophages have very active actin microfilaments pushing cell forwards

Question 4

Actin cytoskeleton

Answer 4

Required for: cell movement/division, vesicle transport, phagocytosis and movement of organelles

Myosins are molecular motor proteins that work alongside actin

Most actin filaments are dynamic - length/organisation changeable

But some are stable e.g. microvilli

Various signalling pathways regulate actin organisation and dynamics

Actin binding proteins help organise the structure

Question 5

Different organisations of actin within cells

Answer 5

Microvilli - stable wafting profusions

Adhesion belt- around base of microvilli

Cell cortex - edges

Filopodia - probing actin extensions of cell - maybe to sense surroundings

Lamellipodia - for movement

Stress fibres - antiparallel contractile structures

Contractile ring - in dividing cells

Question 6

Actin can’t be crystallised

Answer 6

This is because it forms filaments. However it can be bound to one of its binding proteins to prevent filaments forming and then the complex can be crystallised - for X ray crystallography

Question 7

Structure of actin monomer (G-Actin)

Answer 7

Actin polypeptide folds into 4 subdomains with 2 lobes separated by a cleft

42kD
Several isoforms
Cytoplasmic beta actin
Cleft binds ATP

Question 8

Actin filament (F-actin)

Answer 8

2 strands of monomers
ATP binding site in same orientation as all other subunits
End of filament with the exposed cleft is called the - (minus) end
The strands are twisted with 14 monomers per turn

Question 9

Actin filament polarity +/- barbed/pointed

Answer 9

The monomers are all arranged in same orientation with ATP binding cleft facing towards minus end and opposite end positive.

Demonstrated by decorating the actin filament with head domains of myosin

Generates an arrowhead pattern pointing towards minus end

Hence minus end referred to as pointed end and plus end referred to as barbed end

Question 10

Actin polymerisation

Answer 10

F actin is assembled from G actin monomers and is dynamic

Addition or loss of subunits depends on conc. of available G actin

The initial start of filament formation is slow (nucleation)

Followed by a rapid elongation phase

Eventually as conc of free G-Actin drops a steady state is reached where addition=loss

Question 11

Critical concentration (Cc)

Answer 11

Cc is the conc. of free G actin at which addition at one end is balanced by loss at the same end

i.e. no net loss or gain at that end

Cc at the (+) end is lower than at the (-) end
Above Cc there is a net addition of subunits/ below Cc net loss

Each end has a different Cc

The lower Cc at (+) end indicates the faster binding constant

Therefore the (+) end will grow at a lower monomer conc. Than the (-) end

Question 12

Actin is an ATPase - helps to drive treadmilling

Answer 12

After ATP-Actin is bound at (+) end the ATP is hydrolysed to ADP+Pi

Pi is slowly released so that towards (-) end actin subunits contain ADP

This results in small confirmational changes in actin changing binding kinetics

ADP actin bonds less strongly to other actin monomers hence the diff in association and dissociation rates at opposite ends of the filament.

Question 13

Actin treadmilling

Answer 13

The rate of addition of ATP actin is much faster at the (+) end than at the (-) end whereas rate of dissociation is similar

After ATP actin is added to (+) end ATP is slowly hydrolysed to ADP thus (-) end of F actin will contain ADP Actin

At steady state ATP actin monomers are preferentially added to the (+) end whereas ADP actin units disassemble at (-) end

Question 14

Actin monomer binders

Answer 14

Monomer binders: involved in dynamics of actin growth/disassembly

Nucleotide exchange e.g. profilin
Monomer capping/sequestration e.g. thymosins
Monomer delivery and polymerisation e.g.twinfillin
Nucleation e.g. Arp2/3, WASP and formins

Bundlers and crosslinkers

Microvilli (fimbrin, scruin,villin,espin)
Filopodia and stress fibres (fascin and alpha actinin)

Cytoskeletal linkers
Actin to intermediate filaments (spectrin)
Actin to intermediate filaments & microtubules (e.g. plectin,MACF,MAP2)
Actin to microtubule (tau)

Myosins - conventional/non >17 classes

Rulers and stabilisers ( e.g adducin,caldesmon and nebulins)

Anchors to membranes and membrane proteins (e.g. alpha actinin and ERM proteins)

Sidebinders and signallers e.g. contractin coronin and drebrin

Capping and severing
(+) End (capping protein capz, formin, tensin)
(-) End (Arp2/3 and tropomodulin)
Severing and capping (e.g. gelsolin, villin)
Depolymerization and severing (e.g. ADF/ cofililin and AlP1)

Branch formation
(E.g. Arp 2/3 and WASP/SCAR/WAVE)

Question 15

Treadmilling is controlled by actin binding proteins

Answer 15

Cofilin - binds ADP-actin at (-) end and destabilises filament to release actin monomers bound to cofilin (which then dissociates)

Profilin binds ADP-actin and released ADP (confirmational change) allows recharge with ATP

Thymosin beta 4 sequesters ATP-actin controlling conc. Of free monomer

Question 16

Cell movement by actin assembly

Answer 16

Cells are attached to ECM via focal adhesions aka integrins

1) extension - movement starts by extension of membrane driven by actin assembly (from (+) end of actin)

2) adhesion - extension attaches forming a new adhesion

3) translocation - cell internal content shifted forward by actin/myosin

4) de-adhesion - old focal adhesion is detached integrins and membrane recycled by endocytosis

3)

Question 17

Actin filament treadmilling summary

Answer 17

Actin assembly at (+) end located at membrane.
Cofilin destabilises (-) controlling filament length providing space for (+) growth
Profilin removes ADP from ADP-actin allowing it to bind to ATP and bind to (+k end

Question 18

Myosin - partners of actin

Answer 18

Large family of proteins - human genome encodes about 40 kinds
Diff myosins for diff jobs
all use ATP energy
Myosin head domains convert ATP hydrolysis to mechanical work (movement)

Question 19

Myosin structure

Answer 19

Actin binding site & ATP binding site on head regions

Essential light chain for stability

Regulatory light chain for control of myosin via Ca2+ and phosphorylation

Heavy chain tail - variable - determines binding

Question 20

3 common classes

Answer 20

Class 1: single head, 10-14 nm , for membrane association and endocytosis

Class 2: 2 headed, 5-10nm for contraction of muscle - pulls actin filaments together

Class 5: 2 headed, 36 nm for organelle transport - connects vesicle to actin

~14 types, some are one headed but most have 2.
Almost all move towards (+) end of actin

Myosins are motor proteins that move along actin filaments

Question 21

Myosin power stroke summary

Answer 21

1) myosin binds ATP, head released from actin
2) hydrolysis of ATP > ADP+Pi, myosin head rotates to cocked position
3) myosin head binds actin filament
4)”power stroke” release of Pi, elastic energy straightens myosin, moves actin filament left
5) ADP released, ATP bound, head released from actin (1)

Question 22

Accessory proteins required for maintenance of integrity of a sarcomere

Answer 22

Cap z - stabilises actin filament at (+) end
Tropomodulin - same at (-) end

Nebulin - giant protein with repeating actin binding domains, extends from z disc to tropomodulin and determines length of filament in muscle

Titi - another giant protein is an elastic spring like molecule. A pair of titins extend the whole sarcomere

Question 23

Actin microfilament summary

Answer 23

Polymers of G-Actin monomers
Polarised growth of filaments treadmilling
Filament polarity and stability controlled by ATP
Actin structures regulated by many associated proteins
Many actin structures enable mobility in conjunction with myosin
Muscle and non muscle cells rely on sliding of myosin along actin filaments for movement