Actin Filaments Flashcards
What are some important roles of actin filaments in the cytoskeleton?
- cell motility
- contractility
- shape changes
- cytokinesis
- cell polarity
- phagocytosis
- macropinocytosis (engulfment and uptake process of large amounts of fluids and membranes.)
How abundant is actin?
Very abundant:
- 20% of mass in skeletal muscle
- 5% of total protein in non-muscle
Actin Proteins (Classes)
Globular monomer with two domains (ATP binding site is between the 2 domains).
3 Classes of actin:
Class 1 - Non-muscle beta, gamma, and smooth muscle gamma-actin.
Class 2 - Skeletal, cardiac and vascular alpha-actin
Class 3 - Actin-RPV, centractin, lower eukaryote actins.
Actin Isoform Localisation in non-muscle Cells:
Alpha-actin - in stress fibres.
Beta-actin - in membrane ruffles; involved in cell movement which requires distortion of the plasma membrane.
Gamma-actin - at cell periphery (sub-plasmalemmal array)
Upregulation of [Alpha/Beta/Gamma]-actin accompanies increased cell movement.
Upregulation of Beta-actin accompanies increased cell movement.
Actin Structures in Cells:
Transient/labile surface structures on cells that are due to assembly of actin:
- Lamellipodia
- Filopodia
- Ruffles
Stable actin structures:
- Microvilli (contain bundles of 20-30 actin filaments
stabilised by cross-linking proteins [villin, fimbrin, and espin])
Actin associated junctions:
- Focal Complexes
- Focal Adhesions
Control and Regulation of Actin Filaments:
- Actin filaments are polarised (have a plus end and a minus end)
- Actin assembly requires energy from ATP hydrolysis (Actin tightly binds to ATP and Mg2+, ATP then hydrolysed to ADP after polymerisation)
- Actin binding proteins regulate monomer to filament ratio by exploiting the difference in the assembly characteristics at either end
- Cell signalling alters actin assembly: via small GTPases:
Rac/Rho/Cdc42
Actin assembly into filaments, and their polarity:
Actin filament assembly is facilitated by formins.
Pure actin nucleates as trimers and elongates by addition of monomers, forming actin filaments.
Actin filament polar-ends show different kinetics:
Minus-end = Slow growing (aka pointed-end)
Plus-end = Fast growing (aka barbed-end)
Subunits add and come off both ends, but different critical concentrations operating at either end:
Plus end: Low critical concentration, only need ~0.1μM to start adding subunits
Minus end: Higher critical concentration, need over 0.8μM to start adding subunits
So between 0.1-0.8 μM, subunits will add at plus end and drop off at the minus end, giving “treadmilling”.
Formation of New Actin Filaments Can Come From 3 Sources:
- De novo nucleation
- Uncapping
- Severing Existing Filaments
Pure actin assembles spontaneously into filaments:
-Assembles from ATP bound G-Actin (globular momomeric actin)
-ATP hydrolysed to ADP after polymerisation,
lags behind = ATP cap
-ATP hydrolysis = ADP-actin = depolymerisation state
Filament Elongation and Treadmilling:
Elongation favoured at barbed-end (+ end).
Between 0.1-0.8 μM (The critical concentration), there is a net addition of subunits to the barbed-end and a net loss of subunits at the pointed-end.
This is a flux of subunits through the filament known as “Treadmilling”.
Acting Binding Proteins: Sequestering proteins (Names and roles of 3 main ones to know)
Regulatory proteins control assembly and keep
Globular-actin concentration high:
- Profilin (Binds G-actin/ATP, lowering the critical concentration, thus limiting elongation to the barbed-end by suppressing spontaneous nucleation. Note: Too much or too little profilin is detrimental to filament formation).
- Capping protein (Caps the barbed-end, stops elongation)
- Thymosin-beta4 (Sequesters G-actin making it polymerisation-incapable, 100 x greater affinity for ATP-actin than ADP-actin. Means that when the barbed-end becomes uncapped, revealing the high affinity actin binding site, there is a pool of actin ready to assemble immediately at these sites.)
Capping Proteins examples
CapZ: Binds barbed-end
Tropomodulin: Binds pointed-end
What proteins link the actin filaments to the plasma membrane?
ERM:
- Ezrin
- Radixin
- Moesin
How is the state of actin in cells regulated?
Changes in actin configuration are mediated locally by signalling. This signalling is mediated by small GTPases:
- Rac –> Forms lamellipodia (Thin, sheet-like membrane protrusions found at the leading edge of motile cells)
- Rho –> Forms stress fibres/focal adhesions
- Cdc42 –> Forms filopodia (Thin cytoplasmic projections)
Models to explain how actin assembly drives formation of lamellipodia at the leading edge of the cell:
- Treadmilling Model: Depolymerisation only at the pointed ends. Growth of filaments at barbed ends concentrated at leading edge.
- Dendritic Brush Model:
- New actin filaments nucleated ON existing filaments
- Capping of pointed ends of actin filaments
- Requires actin depolymerisation
