actin and cytoskeleton

cytoskeleton

• 3 independent protein networks:

1. microfilaments - 6-9 nm, cell shape and muscle contraction, separation of daughter cells in mitosis

2. microtubules - larger than microfilaments, form hollow tubes, involved in chromosome separation in mitosis, organelle movement, vesicle transport

3. intermediate filaments - more stable than microtubule and actin filaments, create physical properties of the cell, responsible for resistance of cells and tissues to mechanical stress

• microfilaments and microtubules are very dynamic - used for moving things around the cell

• cytoskeleton is responsible for cell movement, shape, muscle contraction

actin cytoskeleton

• required for movement, cell division, vesicle transport, phagocytosis, movement of organelles

• myosin motor proteins work with actin filaments - move along actin filaments, can be attached to transport vesicles or other actin filaments

• actin filaments are dynamic - length and organisation rapidly changeable

• signaling pathways regulate actin organisation and dynamics - cell can change organisation of actin in response to external signals

• actin binding proteins - help to organise structures

actin monomer - G actin

• Actin polypeptide folds into 4 subdomains that generate 2 lobes separated by ATP binding cleft (Mg²⁺ ion complexed with ATP/ADP)

• base of cleft - ATPase, site where Mg²⁺ and ATP are bound

• floor of cleft acts as a hinge that allows lobes to flex relative to eachother

• when ATP/ADP is bound, this affects the conformation of G-actin - without bound nucleotide it will denature

• relatively small - 42 kD

• globular protein, ATPase

• assembles into polymer F-actin - reversible reaction

actin filament - F-actin

• 2 strands of monomers - has polarity, helical arrangement

• actin monomers spontaneously form into filaments, ATP binding cleft oriented in same direction in all subunits - points towards minus end

• minus end - pointed end, plus end - barbed end - due to myosin binding with slight tilt pointing towards minus end since ATP binding cleft points towards minus end

• plus end grows faster than minus end and minus end shrinks faster than plus end

• strands are twisted with 14 monomers per turn - repeat structure of 36nm

polymerisation of G-actin monomers into F-actin filaments

• addition or loss of subunits depends on concentration of G-actin available - below certain concentration filaments cannot assemble

• 3 key phases:

1. nucleation phase - G-actin subunits combine into an oligomer with 2-3 subunits, when 3 subunits in length can act as nucleus for next phase

2. elongation phase - short oligomer increases rapidly in length, addition of actin monomers to both ends

3. steady-state phase - G-actin monomers exchange subunits with filament ends as concentration of available free G-actin drops, no net change in total length of filaments since growing and shrinking at the same rate

• initial start of filament formation is slow - individual monomers make 4 interactions with surrounding actin monomers, there are 4 attachments for each monomer with another monomer and each attachment has a role in stabilising the monomer so polymer is more likely to come apart again when less monomers are joined

critical concentration

• minimum concentration of G-actin monomers present such that F-actin filaments form

• C_c = rate of addition/rate of dissociation

• binding constant depends on concentration

• C_c at (+) end is lower than at the (-) end - faster binding constant at (+) end so it grows at a lower monomer concentration than the (-) end

• above C_c - net addition of subunits, filaments form

• below C_c - net loss of subunits, filaments do not form

• higher concentration of G-actin = more likely monomer will collide with end and bind

• dissociation constant does not depend on concentration - due to dynamic interaction of monomers within the filaments

• at steady state - concentration of G-actin remains at C_c

actin treadmilling

• particular subunits appear to move through the filament

• actin is an ATPase - hydrolyses ATP to ADP

• after ATP-G-actin is added to (+) end, ATP is hydrolysed

• Pi is slowly released so that towards the (-) end, the actin subunits contain ADP - this results in small conformational changes in actin which changes binding kinetics

• ADP actin binds less strongly to other actin monomers - actin dissociates more readily at (-) end

• rate of addition at (+) end quicker than at (-) end and rate of dissociation at (-) end quicker than at (+) end - in comparison to rates of addition rates of dissociation at both ends relatively similar

• at steady state - ATP-actin monomers preferentially added to (+) end whilst ADP-actin subunits dissociate at (-) end

control of actin treadmilling

• controlled by actin binding proteins

• cofilin - binds to ADP-F-actin at (-) end, this destabilises the filament between regions with and without cofilin so that it breaks into short pieces - accelerates dissociation and shrinkage at (-) end since more free (-) ends generated, net shrinkage of filament at (-) end

• profilin - binds to ADP-G-actin and opens nucleotide-binding cleft so that ADP is released - allows actin to be recharged with ATP

• profilin also helps to destabilise the structure at (-) end by blocking binding sites for G-actin - ATP-G-actin added to (+) end, once new actin subunit is bound to filament proflin dissociates

• thymosin β₄ - binds to ATP-G-actin and inhibits its addition so controls concentration of free monomer, can bind to or release ATP-actin to decrease/increase actin dynamic

• when some ATP-G-actin is incorporated into actin filaments, more thymosin β₄ will dissociate so that sufficient actin subunits are available for polymerization

capping proteins

•     Bind to filament ends

•   CapZ – binds to (+) end of filament, inhibits subunit addition/loss

•   Activity of CapZ inhibited by regulatory phospholipid phosphatidylinositol 4,5-bisphosphate found in cell membrane

•    Certain regulatory proteins can bind to (+) end to prevent CapZ binding while still allowing assembly

•     Tropomodulin – binds to (-) end of filament, found mostly in cells in which actin filaments need to be stabilised for long periods of time, works with tropomyosin to stabilize actin filaments

•       Another class of proteins can cap and sever (+) end of actin filaments – eg gelsolin