Lecture 2 - Actin Flashcards
List the higher order architectures of actin
Filopodia - parallel bundles located at leading edge of the cell
Stress fibres - antiparallel contractile stuctures
Lamellipodia and cortex - branched filaments
Describe how the remodelling of actin is regulated
remodelled in response to environmental cues that stimulate cell division, differentiation, or locomotion.
Central to this remodelling process, cells have regulatory mechanisms to assemble and disassemble actin filaments rapidly:
- Inhibition of spontaneous polymerisation of G-actin -> F-actin
- Nucleation of new actin filaments
- Control of actin filament length
- Elongation/shortening of prexisting actin filaments
=coordinated through actin-binding proteins
What are actin- binding proteins?
- regulate actin polymerisation and organisation
- associate with actin monomers or assembled filaments (f-actin)
What are actin monomer binding proteins?
G-actin binding proteins regulate polymerisation and nucleation Examples of these include thymosin and profilin, both of which regulate polymerisation of G-actin monomers into microfilaments, but do so in very different ways.
How is actin polymerisation regulated by thymosin and profilin?
Thymosin beta 4 (found in metazoan cells) is a small peptide that sequesters monomeric actin preventing polymerisation and also nucleotide exchange causing accumulation of monomeric ADP-actin.
Profilin, is present in most eukaryotic cells (including animals, plants and yeast) it also binds to G-actin, but increases the rate of nucleotide exchange and, unlike thymosin, only prevents polymerisation to the minus end of F-actin (because it binds opposite the nucleotide binding cleft which points towards the minus end). Thus, in the presence of profilin F-actin extends preferentially at the (+) end.
How is actin nucleation regulated?
Formins bring individual actin monomers together.
The Arp2/3 complex performs a similar function. This complex consists of two Actin Related Proteins and 5 other subunits. In addition its nucleation function the Arp2/3 complex also initiates branching of microfilaments (such as those found in the cell cortex) by binding to an F-actin filament (while still carrying out nucleation function) and initiating polymerization of a new branch. (as seen in lamellapodia and cortex)
What are the functions of actin filament binding proteins?
Severing and capping proteins regulate filament length
Cross linking proteins organise filaments and bundles
Severing proteins can break actin filaments into shorter fragments
Capping proteins can act to stabilise actin filaments
An actin filament capped at both ends will not gain or lose monomers and is therefore stabilised
Respond to different signals - multiple layers of regulation
What is gelsolin?
Gelsolin is a filament binding protein that has the ability to dissolve a mesh of actin filaments
Gelsolin disrupts the organisation by binding to F-actin, severing the filament and then capping the (+) end to prevent further growth.
Uncapped (-) ends can disassemble
Gelsolin’s severing and capping actions are stimulated in response to elevated calcium levels – translating responses to an extracellular signal that causes localised Ca2+ to cytoskeletal remodelling (Note that gelsolin activity is inhibited by PIP2 - example of how one signal can turn a function on and another turn it off).
What is cofilin?
Cofilin (also known as ADF-cofilin, for actin-depolymersing-factor), binds to ADP-bound actin, either within F-actin, or as monomeric G-actin.
-dissociates subunits
-prevents nucleotide exchange
-disassembles filaments
If it binds to ADP-actin at the end of a filament it increases depolymerisation (remember that this is found predominantly at the minus end), but if it binds deeper in the filament it causes severing. Another action of cofilin is to prevents nucleotide exchange – consequently inhibiting polymerisaion at the (+) end, thus promoting overall filament disassembly (opposite action to profillin).
What is fimbrin?
a monomeric protein with two actin binding domains each of which can bind an actin filament giving rise to the tight filament bundles found in filopodia
Describe features of myosin
•encoded by a multigene family (13 identified to date, different myosins perform different function – some of which are cell type specific; I, II and V are the most commonly studied)
•1 or 2 heavy chains in complex with light a light chain, or chains
Globular head region of myosin heavy chain binds to actin and has ATPase activity that generates force used to ‘walk’ along actin filaments.
-most abundant are myosins I and II
Myosin I - monomer (one heavy chain)
Myosins II and V = dimers (two heavy chains)
Describe myosins I and V
Tail domains on both myosin I and V bind membranes and thus link the cytoskeleton to membranes.
Myosin I binds the plasma membrane – anchoring actin filaments in filopodia to the cell surface and microvilli - functions as a monomer.
Myosin V binds to membrane of transport vesicles leaving the head domains to bind actin filaments to power transport- acts as a dimer.
Describe myosin II
Myosin II differs from myosins I and V in that it doesn’t link actin filaments to membrane, but rather generates contractile force, such as in striated muscle or during cytokinesis where it functions to tighten the contractile ring between the two daughter cells.
In striated muscle, individual myosin II dimers are assembled into higher order structures, with tails packed tightly together to form a thick filament from which the actin-binding globular heads protrude.
Describe myosin movement
ATP-binding causes
conformational change in myosin
Disruption actin binding site (releases)
ATP-hydrolysis causes
conformational change in myosin.
hydrolysis products trapped
Actin binding site restored (Head pivots and binds)
Conformational changes in head
& neck are transmitted & amplified to other parts of the molecule through the light chains bound to the neck. (power stroke)
