Actin and actin-based motility Flashcards
BIO1339 ReCap
You learnt that F actin is formed from G-actin monomers, and that the actin undergoes “treadmilling”
What does actin do?
Actin is one of the most abundant proteins in most cell types
Essential for: maintaining cell shape, motility, and cell division
Dynamic
Can form stable structures by associating with other proteins.
Interacts with around 60 other proteins to perform various functions
Describe the structure of actin
An asymmetric protein that can self-assemble to form polarised actin filaments.
- Formed from monomers of G actin
- Each filament may be thought of as a two-stranded helix with a twist repeating every 37 nm
- Multiple, lateral interactions between the two strand prevent separation
- 6nm wide
Formation and polarity
Plus end, actin Bound with ATP joins
Minus end, actin bound with ADP leaves
Describe treadmilling of actin filaments
Rate of addition at barbed end = rate of loss at pointed end
Length is constant but the polymer moves
“Treadmilling” vs dynamic instability of MTs
Describe actin as an ATPase
- Actin is an ATPase (an enzyme)
- Actin subunits added at both ends, at higher rate at the + end
- Hydrolysis of ATP to ADP occurs within the filament
- Most actin in F-actin is ADP-bound
- ADP/ATP-free actin can also polymerise (but rate of nucleotide binding regulates dynamics/stability)
Name the three drugs that affect actin filaments
Slide 10
Phalloidin, cytochalasin, latrunculin
Descrbe cytochalasin-D and Latrunculin-A
Prevent filment growth
- Latrunculin-A binds to G-actin and prevents filament growth
- Cytochalasin-D binds F-actin at barbed end and prevents filament growth
- Filaments will depolymerise in the presence of these agents
Describe F actin organisation in the cell
Filopodia
- Uniform polarity: also seen in the microvilli and in the dendritic meshwork
Dendritic meshwork:
- Graded polarity bundles (lamellipodium). Meshwork is key to pushing the plasma membrane forward
Stress fibres
- Filaments within the bundle have alternating polarity, like muscle fibres - alternating polarity is key to use actin in conjunction with myosin II to contract things inside the cell. Myosin walks towards + end
Describe force generation in actin
Uses myosin
Large superfamily of related molecular motors. Walk along or propel the sliding of actin filaments. ATP hydrolysis is essential for movement
Describe the dendritic nucleation paradigm
A model to explain how extracellular signals are able to trigger actin assembly in the right place and in the correct organisation
What are the key actin binding proteins?
Nucleating protein
Severing protein
Capping protein
Myosin motor protein
Describe actin cytoskeletal assembly
Extracellular signals trigger actin cytoskeletal assembly facilitated by actin binding proteins
GTPases are important
Describe steps 1 and 2.
The Rho GTPases are molecular switches
There are lots of different types of small GTPases within the cell.
They all act as molecular switches that act to amplify a given signal at a particular spatial location within the cell.
Rho GTPases are important for cell migration
Describe molecular switches
There are lots of different types of small GTPases within the cell.
They all act as molecular switches that act to amplify a given signal at a particular spatial location within the cell.
Rho GTPases are important for cell migration
Three Rho family GTPases to consider when discussing actin organisation
Describe the different effects of the Rho family GTPases on actin structure
Slide 23
Cdc42 is a regulator of cell polarity
What does cross talk between the Rho GTPases enable?
Cell to fine tune actin organisation
Describe Rho family GTPases activation
Activated at specific locations
- Not evenly and equally distributed in the cytoplasm
- Targeted to specific membrane and subcellular compartments that are essential for their function by:
(1) post-translational modification by isoprenoid or fatty acid lipids:
Farnesylation
Geranylgeranylation
Palmitoylation
(2) carboxyl-terminal sequences
Describe mutations in CDC42
- Small GTPase
- Homozygous nonsense mutations are embryonically lethal
- Missense variants underlie a clinically heterogenous group of phenotypes characterised by:
Postnatal – growth deficiency
Intellectual disability
Seizures
MRI brain anomalies
Tone anomalies
Endocrine anomalies
Facial dysmorphism
Cardiac anomalies
Recurrent infections
Mutations perturb CDC42 function by altering the switch between active and inactive states
Which actin binding proteins are essential for migration – What experiments investigated this?
To figure this out, researchers used bacteria.
Some species,for example, listeria, rickettsia and shigella, actually hijack the host actin cytoskeleton in order to be able to maintain a productive infection.
They make use of the host cells actin and the various actin binding proteins to form something called an actin rocket which they use to jet propel themselves around inside the host cell.
Describe the reconstitution of actin-based motility using only 4 proteins
- Take listeria and purified actin and various combinations of actin binding proteins and put them in a test tube.
- See which combinations allowed a listeria rocket to form.
- Cytoskeletal proteins are recruited to the bacterial surface
- This results in actin polymerisation at one pole of the bacterium, propelling the bacterium along
- The proteins required are used for normal mammalian cell motility; therefore understanding how bacteria move gives insights into how other cells move
What is needed for reconstructing bacterial motility?
Actin
Arp2/3 complex
Capping protein
ADF/cofilin
DOES NOT require myosin, as is a motor independent process
Describe the Arp2/3 complex
The Arp2/3 complex is a nucleation factor
Helps the cell to regulate where new actin filaments are nucleated
Nucleation is the rate-limiting step in forming an actin filament
Why are nucleation factors needed?
Actin monomers bind together – dimer, then trimer- this is slow if just passive diffusion
The trimer is the first stable state of an actin filament
Once the trimer is formed, polymerisation/elongation from the barbed end can happen really fast
Arp2/3 complex helps the formation of the stable actin trimer by mimicking the structure of actin dimer
Describe nucleation of branched filaments
Arp2/3 complex is also used. Binds in such a way to create a 70 degree angle. Binds to the side of a pre-existing filament
Most actin filaments in the lamellipodial meshwork are oriented with their growing (+) ends toward the plasma membrane
Branches produce force only while they are relatively short and stiff, pushing the membrane forward as new monomers are added into the gap between membrane and meshwork
An angled orientation also makes it easier to insert an actin monomer between the + end and the plasma membrane – a branch is able to produce more force without falling apart.
Describe the leading edge of a migrating cell
Branched array of actin filaments
Arp2/3 complex localised to branch sites
Describe Arp2 knockdown cells
Do not have lamellipodia
By short haipin RNA
No sheet-like protrusions, but linear protrusions are still there
Why are capping proteins needed?
Longer filaments are more prone to buckling. Some filaments are not oriented in a productive way
Describe ADF cofilins
Third family of proteins needed to reconstitute rocketing motility in listeria. ADF cofilins don’t regulate actin filament assembly directly
They are actin severing proteins, so regulate disassembly
Describe the lamellipodium during migration
Same width. ADF cofilins maintain the width and stop filaments getting too long. Actin is in a steady dynamic state at the leading edge
ADF/cofilin is only active at the rear. Trimming off the actin filaments at the right time to ensure lamellipodium stays productive. Regulated by spatial localisation and phosphorylation
Describe actin monomer supply at the leading edge
Rate-limiting
Ratio level of G- to F-actin in chick fibroblasts.
Migrating fibroblasts were fixed in formaldehyde and the ratio image determined by dividing the G-actin image by the F-actin image.
The G/F actin ratio ranges from:
low (G low=blue)
Intermediate (equal = green)
high (G high = red)
There is a problem:
Problem:
Actin monomer supply is rate limiting
It needs to be immediately recycled and used for polymerisation
Actin only polymerises in the ATP-bound form
BUT actin is disassembled in the ADP-bound form
What does profilin do?
Catalyses the regeneration of ATP-actin from ADP-actin
