Lecture 20 & Term 2 Lecture 2 Actin: Organisation/Assembly/Binding/Movement Flashcards
Cytoskeleton
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
Cytoskeleton has 3 independent protein networks
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
Organisation of network depends on cell function and type
Moving cells like macrophages have very active actin microfilaments pushing cell forwards
Actin cytoskeleton
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
Different organisations of actin within cells
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
Actin can’t be crystallised
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
Structure of actin monomer (G-Actin)
Actin polypeptide folds into 4 subdomains with 2 lobes separated by a cleft
42kD
Several isoforms
Cytoplasmic beta actin
Cleft binds ATP
Actin filament (F-actin)
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
Actin filament polarity +/- barbed/pointed
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
Actin polymerisation
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
Critical concentration (Cc)
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
Actin is an ATPase - helps to drive treadmilling
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.
Actin treadmilling
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
Actin monomer binders
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)
Treadmilling is controlled by actin binding proteins
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
Cell movement by actin assembly
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)
Actin filament treadmilling summary
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
Myosin - partners of actin
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)
Myosin structure
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
3 common classes
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
Myosin power stroke summary
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)
Accessory proteins required for maintenance of integrity of a sarcomere
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
Actin microfilament summary
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
