Actin Flashcards
What are the roles of actin?
cell motility contractility shape changes cytokinesis cell polarity phagocytosis macropinocytosis
Overall what is Actins distrubution in the cell?
20% in skeletal
5% of all the proteins in non muscle
In non-muscle cells, where are the different actin isoforms, alpha, beta, and gamma found?
gamma- at he periphery
alpha- in STRESS fibers
beta- in ruffles, where cell distorts to move. More UPREGULATION of beta actin means more movement
What are some properties of actin?
375 amino acids
1) 43 kDa
2) globular monomer, with two domains, G-actin
3) At least 6 isoforms in mammals, encoded by separate genes
What are the three classes of actin?
Class 1 - Non-muscle beta, gamma and smooth muscle gamma-actin.
Class 2 - Skeletal, cardiac and vascular a-actin
Class 3 - Actin-RPV, centractin, lower eukaryote actins
Name all of the actin structures in the cell (labile, stable, and the junctions.)
Transient/labile surface:
Lamellipodia
Filopodia
Ruffles
Stable actin structures:
Microvilli (contain bundles of 20 actin filaments
stabilised by cross-linking proteins (the bundling proteins
villin, fimbrin and espin))
Actin associated junctions:
- focal complexes
- focal adhesions
How is actin assembled?
Process requires energy from nucleotide (ATP-ADP) hydrolysis in cells.
First spontaneously assembles from G-actin subunits around a nucleus (forming F-actin). When it is polmerized it binds to ATP (in its binding site). It is elongated. When it depolymerised ATP is hydrolysed to ADP.
Actin binding proteins regulate monomer to filament ratio
by exploiting the difference in the assembly characteristics at either end (the polarised positive and negative ends)
Here different kinetics are shown; slow growing-end is at the minus-end and the fast growing-end is at the barbed plus-end. As a result it grows in a arrowhead pattern.
Cell signalling alters actin assembly such as small GTPases.
What is the structure of actin?
Trimers and elongates by addition of monomers which forms either filaments or networks/bundles.
6-8nm wide
When do actin filaments form?
As a result of:
1) Uncapping
2) De novo nucleation
3) severing existing filaments
Describe actin formation tread-milling.
At the positive end less actin subunits needed to grow the filament, but a higher subunit concentration is needed if the negative end (pointed) is to grow and so growth mainly at positive barbed end.
Subunits are added at positive end and LOST AT NEGATIVE END and the two ends grow UNEQUALLY (tread-milling)
Describe the three stages of actin filament growth
1) Lag phase
2) polymerisation phase
3) steady state with treadmilling
How is free G-actin (used as subunits) maintained?
Regulatory proteins control assembly and keep
G-actin concentration high (50% of all actin in this state)
eg, 1)Profilin
2) Capping protein- caps the positive barbed-end of he F-ACTIN , stops elongation as free G-actins cannot bind
Tropomodulin binds to minus end.
3) Thymosin-b4- binds to G-actin making it polymerisation-incapable as there isnt a high enough concentration of free nucleotides
What is Profilin?
Regulatory protein of actin that binds to the G-ACTIN, stops it being added, limits elongation.
It is a Ubiquitous protein
It also associates with the barbed end (+) of actin filaments. This effectively reduces the critical concentration.
Too much and too little profilin is detrimental to filament formation.
Knockouts in mice and yeast show it to be an essential protein.
What is beta-thymosin
Regulatory protein of actin, found at the cell periphery that binds to the ATP-ACTIN (the free G-actin) moreso than the ADP-ACTIN, so when positive end is free actin is ready to assemble.
What are the two concentrations needed at either end of the actin filament
0.1 µM @ positive versus 0.6µM @negative
Give some examples of ACTIN binding proteins.
50 different types
a-actinin used for bundling
tropomyosin used for side binding
Cofilin used for severing filament
What is a formin?
Protein that helps actin assembly through the reloading of its whiskers with actin subunits
What role does the binding protein filamin have?
Filamin forms a cross-linked actin network at the cell cortex
What is a ERM protein and what does it do?
Ezrin Radixin Moesin: ERM proteins link the actin filaments to the plasma membrane by binding to the filament and a membrane protein.
There are many actin crosslinking proteins
1) fimbrin
2) a- actinin
2) spectrin
4) dystrophin
used for different fibers and functions; depending on their size and shape, they have differing interactions with the actin
What mediates the signalling that regulates actin state?
Ras superfamily of proteins
1) GTPase Rac (in meshwork lamellipodium)
2) Rho (in contractile stress fibers)
3) cdc42 (in parallel filopodium)
stimulated by growth factors, ECM and other hormones
Describe two models of proposed Actin assembly
1)Treadmilling model :
depolymerisation only at the pointed ends, occurs throughout
the entire filament array and is necessary to allow
treadmilling
growth of filaments from existing actin filament population
barbed ends concentrated at leading edge, pointed ends
throughout the cytoplasm (depolarising at the rear)
2)Dendritic brush model :
new actin filaments nucleated ON existing filaments at the leading edge,
capping of pointed ends of actin filaments
requires actin depolymerisation, releasing filaments.
What is the Arp2/3 complex?
consists of 7 polypeptides (Arp2 and Arp3, p40, p35, p21, p18 and p14(ARPC1-5) sharing similar structure/genes/homology to actin
It binds to and caps the pointed-end (negative) and can act as a start point for assembly (its nucleation point)
This enables assembly at barbed-end, binds to side of pre-existing filaments to produce BRANCHING (forms branched webs in lamellipodia)
It is in high concentration at leading edge.
ARP2/3 nucleates new filaments, caps pointed-ends and anchors them to pre-existing filaments (at y junctions)
It can be activated by signalling and regulatory proteins (NPFs-nucleation, Rho-family GTPases (cdc42, Rac, Rho) and actin polymerisation promoting factors like WASP,WAVE)
Rho-GTP stimulates actin growth and stress fibers
What effect can pathogens have on actin?
Listeria, Shigella, Helicobacter and several other pathogens hi-jack the actin machinery to promote infection such as bacterial escape from phagocytosis.
What is the role of the Rho-family GTPases (cdc42, Rac, Rho) in cell movement?
If RHO is activated, myosin 2 is activated at the back of the moving cell
If RAC is activated, APR is activated at he front of the moving cell
cdc42 is activated at the front of the moving cell.
Treadmilling of the actin filaments in the leading edge will help cell migrate.
In summary what are the factors that control actin and its protrusion of the cell?
1) cell surface receptors
2) Rho-family GTPases, Cdc42, Rac
3) WASP and Scar related proteins
4) Arp2/3 complex
5) capping protein
6) profilin
7) cofilin (ADF)