The cytoskeleton and cell shape Flashcards
What 3 things defines cell shape?
How?
1) Adjoining cells
- Physically boxes in cells
2) Cell adhesions
- To the ECM and other cells
3) Extra-cellular matrix
- Confines the shape of the cell
What is the shape of the cell critical for?
Function
What processes is cell shape involved in?
- Migration
- Phagocytosis
- Transport
- Cytoskeletal dynamics
What is cortical actin and where is is present?
What is the function?
Cortex of actin around the surface of the cell
Important in providing support to the cell and giving it shape
What are stress fibres?
Aligned polymerised actin the in cell
With crosslinking
What are podosomes?
Where are they present?
Foot-like structures on the ventral part of the cell
Are adhesive - anchoring the cell to the ECM
What are the actin structures in a migrating cell?
1) Microvilli
2) Filopodia
3) Lamellipodia
4) Podosome
5) Stress fibres
6) Cortex
What are filopodia used for?
What are they extensions of?
For the cell to ‘sense’ the environment
Extensions of the lamellipodia
What are lamellipodia important in?
Movement of the cell
What gives strength to the migrating cell?
Many stress fibres in strips
Actin cortex
What are the dynamic changes in actin structure in the cell regulated by?
Phosphorylation
What happens to actin when the cell is inactive?
Low amounts of polymerised actin
When does actin spontaneously polymerise?
When there is an appropriate concentration of actin and enough salt in the solution
What is the only ‘bottle neck’ in actin polymerisation?
Why?
Initial oligomerisation (first 2 monomers coming together)
Very energetically unfavourable and therfore slow to occur
What occurs at the end of actin polymerisation?
Polymerisation reaches saturation
Where the rate of new monomers being added is equal to the rate of monomers coming off
Steady state of fibres
Describe the process of actin treadmilling
- Monomers are added to the barbed end (+)
- Monomers are bound to ATP when they bind
- Phosphate then released (ATP–>ADP)
- Monomers leave the pointed end (-)
- Monomers bound to ADP
What is treadmilling regulated by?
- Phosphorylation
- Many accessory proteins
What does the accessory protein profillin do?How?
What kind of protein is it?
Monomer binding protein:
- Increases the rate of monomer addition to the + end
- Binds to actin monomers and enables the monomer to be phosphorylated
- Then, delivers the monomers to the growing filament
What 2 things do the accessory proteins Arp2/3 do? How?
What kind of proteins are they?
Nucleators and branching proteins:
- Form the starting base of actin polymerisation
- By resembling actin and binding to the - end actin
- Interact with existing filaments allowing other actin branches to nucleate from them
What does the branching of actin provide in the cell?
Strength
What does the accessory protein Gelosin do? How?
What kind of protein is it?
Capping and severing protein:
- Severs filaments
- Regulates actin assembly and disassembly
- By binding to the + end of actin - preventing further growth
What do the accessory proteins apha-actin and filamin do? How?
What kind of protein is it?
Crosslinking proteins:
- Bind filaments together to give strength
What are the 2 classes of G protein and how are they different?
1) Small GTPases
- Monomeric
2) Heterotrimeric G proteins
- Membrane bound complex
What do small GTPases do?
Bind and hydrolyse GTP into GDP
What size are small GTPases?
21kDa
What occurs to GTPases after translation and why?
Post-translational lipid modifications
In order to target them to specific sites in the membrane
What superfamily do GTPases belong to?
What are the members of the family sometimes called? What does this mean?
Ras superfamily
Family members sometimes called ras-type proteins (meaning small monomeric GTPase)
How many different family members are there in the Ras family of GTPases?
36
Why is the superfamily of small monomeric GTPases called Ras?
Ras family is the founding GTPase
What are the proteins in the Ras branch important in?
Cell proliferation and cancer
What is Rho?
A family of Ras-like GTPases
What are Rho proteins involved in?
Cytoskeletal dynamics and migration
What the 3 main branches within the Rho family?
1) Rac
2) Rho
3) Cdc42
How is Rac activated?
By binding to GTP
What are the downstream targets of Rac?
PAK/WAVE2
What are GEFs?
What do they do?
Guanine nucleotide exchange factors:
- Catalyse the exchange of GDP –> GTP, to regulate the activity of the GTPase
When is the only time a GTPase can activate its downstream targets?
When bound to GTP
What happens quickly after the GTPase is bound to GTP?
What is this mechanism?
GTP is broken down into GDP, causing the GTPase to go into an inactive state
Timed mechanism
What are GAPs and what do they do?
GTPase activating protein:
- Increase the enzymatic activity of a small GTPase by increasing the inactive form of the GTPase
- Causes GTP –> GDP
What do guanine nucleotide dissociation inhibitors do?
Binds to the GDP-bound form an prevents exchange, maintaining the small GTPase in an inactive state
How do GTPases signal?
By activating downstream pathways when they are in the active state (bound to GTP)
What function does the hydrolysis of GTP by a GTPase serve?
Timing function - to bring the enzyme back into its inactive state
What does the binding of GTP to Rac1 cause?
What does this allow?
A structural change in the ‘switch region’
Allowing the GTPase to interact with their downstream targets
What are the switch regions in a GTPase?
2 loops: switch 1 and switch 2, which bind either side of the nucleotide
What are the principle Rho family GTPases and what do they do?
What do these features do?
RhoA
Rac1
Cdc42
Coordinate actin cytoskeletal organisation, which ultimately controls:
- Cell morphology
- Cell movement
- Cell motility
These features organise cells to form multicellular organisms
How does Cdc42 control actin cytoskeletal organisation?
Controls the polymerisation of actin filaments and the formation of actin spikes/fillopodia
How does Rac1 control actin cytoskeletal organisation?
Controls the organisation of new actin filaments into dynamic ruffling/lamellipodia
How does RhoA control actin cytoskeletal organisation?
Stabilises and consolidates actin filaments into stress fibres (more rigid skeletal framework)
How can the function of GTPases be studied?
Where are these mutations present and what do they result in?
By identifying mutations that change their ability to be turned on/off
Point mutations in the nucleotide-binding sites, can make the protein:
- Constitutively active
- Dominant negative
Describe the constitutively active GTPase mutant
Substitution of the catalytic glutamine in the switch, stopping GTP hydrolysis, by inactivating the enzymatic activity
What is special about the enzymatic regions of GTPases?
The enzymatic region in ALL small monomeric GTPases is conserved
Describe the dominant negative GTPase mutant
Substitution of the P-loop stops nucleotide binding - nucleotide free
Still capable of binding to GEFs - to mop them up and prevent them from activating the wild type GTPase
What does constitutively active Rho mutants lead to?
What does this cause in the cell?
Stress fibre formation
Causes the cell to become very rigid
What does constitutively active Rac mutants lead to?
What does this cause in the cell?
Many membrane ruffles (lamellipodia in all directions)
Cell is immobile, as tries to move in all directions
What does constitutively active Cdc42 mutants lead to?
What does this cause in the cell?
Lots of filopodia formation
What is the downstream pathway of Rac?
Activation of WAVE proteins:
- Leading to the activation of Arp2/3
- Leading to the polymerisation of actin filaments
What is the downstream pathway of Cdc42?
How is this different to Rac?
Activation of WASP proteins:
- Leading to the activation of Arp2/3
- Leading to the polymerisation of actin filaments in a DIFFERENT WAY to Rac - causing a different response in the cytoskeleton
What is the downstream pathway of Rho?
Activation of Rho kinase:
- Leading to Myosin activation
- Leading to increases myosin contractility and stress fibre formation
What do activated Rho family proteins bind? What is this?
A CRIB motif in effector proteins:
- A specific 16 amino acid sequence
- Cdc42/Rac1 Interactive Binding
What things activate GEFs and GAPs?
Where do these occur?
What outputs do these ultimately lead to how?
- Integrin interaction with the ECM
- RTK
- GPCRs
- Cadherins
Ultimately leads to:
- Changes in cell adhesion
- Actin polymerisation
- Focal adhesions
- Cell migration
By controlling the small GTPases that organise the cytoskeleton (Rac, Rho, Cdc42)
What are the accessory proteins which aid treadmilling?
- Profillin
- Arp2/3
- Gelosin
- Alpha-actin
- Filamin
