L1: cytoskeleton Flashcards
What is actin?
Actin: A highly conserved cytoskeletal protein that is present in all eykaryotes. At the molecular level it is a somewhat flattened globular protein consisting of ~375 residues. It can be divided into 4 subdomains which surround the medial cleft that binds to a nucleotide (ATP, ADP) 7nm
describe actin polymerisation
actin monomers (globular (G actin) polymerise to filamentous actin. The actin filaments grow faster from the barbed (young) (+) end compared to the pointed end (old). (-). and filament elongation is predominantly driven by atp-actin monomers. atp comes in at the plus end bound to g actin. adp out at the minus end bound to g actin. G-ADP is less stable and is more likely to disassociate. As actin monomers assemble into filaments they undergo a conformational change to a flatter structure and this triggers the proteins atpase activitty.
what is actin treadmilling?
In the steady state, the actin filament will demonstrate treadmilling by the addition of atp-actin monomers to its barbed end while adp actin monomers dissociate from its pointed end at the same rate, resulting in a constant filament length.
what is actin needed for?
in cells, actin produces force for motile and morphogenetic processes such as migration, morphogenesis, endocytosis, organelle dynamics and cell division.
what are associated proteins?
proteins involved in filament formation and breakdown.
GTP binding proteins e.g: Rac (a gtpase).
GEFs (GTP/GDP exchange factors): GDP-rac (inactive) vs GTP-rac (active)
GEFs activate rac where rac-gtp forms lamellipodia
GAPs: gtpase activating proteins. Rac-gtp to rac-gdp by GTP–> GDP +Pi (off switch) stop/reduce actin filament formation.
other GTP binding proteins:
cdc42- filopodia
rho- stress fibres
Non GTP binding proteins: Arp2/3- regulates actin filament formation. promotes formation of branched actin filaments. binds to actin filaments and nucleates growth at a 70 degree andlge. requires nucleation ptromoting factors like WASP and VCA.
what is arp2/3?
the arp2/3 complex is a heptameric complex composed of two actin related proteins (arp2 and arp3) and 5 scaffolding subunits (arpC1 to acrpC5). arp2/3 is mediated by NFPs where among these WASP family proteins and the WAVE family protein complexes are most important for endocytosis and cell migration. These NFPs are anchored to lipid membranes and activated by gtpases like rac1 and cdc42.
describe arp2/3 activity
Actin-loaded NPFs (e.g., WASP, WAVE) are localized at the membrane.
The Acidic (A) domain of NPFs binds to the ArpC1 subunit of Arp2/3 (high-affinity interaction).
This brings the Arp2/3 complex to the membrane.
The WH2 domain of NPFs delivers G-actin to Arp2.
The second NPF binds to Arp3.
This delivers a second actin monomer to Arp3, completing the nucleation complex.
This primes the Arp2/3 complex for nucleation.
when two NFPs bind to the arp2/3 complex and two actin monomers, the arp2/3 complex undergoes a conformational change allowing scaffolding subunits to bind to the side of a pre-existing ‘mother’ filament. the arp2 and arp3 subunits are repositioned to adopt a filament barbed end like conformation.
following the detachment of the WCA domains a new filament can grow from the arp2 and arp3 subunits taking the form of a branch that forms a 70 degree angle with the mother filament.
what happens in the innactive form?
arp2 blocks the barbed end of arp3. nfps induce a conformational change when inducing nucleation.
what are formins?
formins are a family of homodimeric proteins that control the elongation of actin filament barbed ends in many cellular actin networks. they have a formin homology 2 (FH2) and FH1. FH2 dimerises forming a ring-like structure anchored to the growing filament that encircles actin for nucleation (filament barbed ends and controls actin monomer incorporation i.e delivers actin for polymerisation). It can bind and stabilise actin dimers and contributes to actin nucleation. FH1 is composed of polyproline sequences that bind to profilin and delivers G actin. Helps exchange its bound nucleotide from adp to atp so more g-atp is available for polymerisation at the barbed end. so forms linear actin filaments.
when is the ring/donut-like structure accessible?
only accessible when rho or rac bind. induce a conformational change so opens it uo and is accessible for actin to enter the ring.
