L25 - Cytoskeleton and Cell Shape Flashcards
What 3 things define cell shape?
Adjoining cells – physically boxes cells in
Cell adhesions
Extra-cellular matrix
What cells show the important of cell shape for function?
Nerves
Muscles
Red blood cells
Why is cell shape vital for cellular activity?
Migration – cell shape changes drive movement
Phagocytosis – cytoskeleton changes shape when engulfing
Transport – cytoskeleton movements used to move vesicles
Cytoskeletal dynamics
F-actin monomers role?
No structural role
F-actin polymers role?
Cortical actin - around the edge of the cell to maintain its shape
Filopodium - to sense surrounding environment
Lamellipodium – important for migration
Podosome – used to anchor the cell
Internally stress fibres – many stripes together to provide strength
The actin cytoskeleton in the inactive state is?
Very stable
Very little unpolymerized actin in cytoplasm
What regulated disassembly of F-actin?
Phosphorylation
Why do actin filament self-polymerise?
Actin in solution will polymerise by itself – because of their shape
Initial step is energetically unfavourable
Actin polymerisation steps
- Add salt to a group of monomers to allow polymerisation
- Olgiomers polymerise to form a growing actin filament
- A point where the rate of monomers added and falling off is equal – steady state of fibres
What is actin treadmilling?
This is the process of how we regulate actin polymerisation in cells
How are monomers added to actin filaments?
Added to the + end
Removed at the – end
What regulates the addition of monomers to actin filaments?
Phosphorylation regulates this - ATP ADP and Pi resulting in monomer release
What accessory protein is associated with actin filaments?
Profilin – binds to monomers enabling them to be phosphorylated so they can be added to the + end
They increase the rate of monomer addition
What are the 4 mechanism of actin binding proteins?
Branching
- Arp2/3 interacts with existing filaments to induce branching
Capping/severing
- Gelsolin binds to + end to block further growth
Nucleators
- Arp2/3 with profilin initiates fibre growth
Bundling/crosslinking
- Α-actin filamin reinforce the structure to give it strength
What are small GTPases?
Small monomeric 21kDa proteins
- Don’t confuse with heterodimeric G proteins
Have intrinsic GTPase activity that can bind to and hydrolyse GTP
What modifications do small GTPases have?
Many have post-translational lipid modifications to target them to specific membrane sites
What family do small GTPases belong to?
All small GTPases belong to a large family
Archetypcal member being Ras
What are some examples of the Ras superfamily of GTPases?
1 Ran - mitotic spindle organisation 27 Arf - membrane budding 61 Rab - endosomal trafficking 36 Ras - cell proliferation and a oncogene 20 Rho - cytoskeleton and migration
If RAC is bound to GTP it is?
Active
This is a timed process due to its enzymatic activity
GTP is broken down to GDP fairly quickly
If RAC is bound to GDP it is?
Inactive
What does active RAC activate?
Downstream targets - PAK and WAVE2
What helps catalyse the exchange of GDP for GTP?
Guanine nucleotide exchange factor (GEF)
What helps catalyse the breakdown of GTP to GDP?
GTPase activating protein (GAP)
What slows down the dissociation of GDP?
Guanine nucleotide dissociation inhibitor (GDI)
What causes a change in the structure of GTPase switch regions?
Binding of nucleotide causes a structural change in the switch regions
Bound nucleotide dictates signalling activity
GTP hydrolysis is an intrinsic property of the protein that turns signalling off
What do RhoA, Rac1 and Cdc42 coordinate?
Coordinate actin cytoskeletal organisation
In turn controls cell morphology, cell movement and cell polarity
What does Cdc42 control?
Controls polymerisation of actin filaments and formation of actin spikes or filopodia
What does Rac1 control?
Controls organisation of new actin filaments into dynamic ruffling structures or lamellipodia
What does RhoA control?
Stabilises actin filaments into a more rigid skeletal framework - stress fibres
What have dominant negative and constitutively active mutant GTPases shown?
Point mutations in the nucleotide-binding sites of small GTPases can make the protein
- Constitutively active - always on
- Dominant negative - always off and inhibitory
What does a constitutively active GTPase mutant cause?
Substitution of the catalytic glutamine in switch 2 stops GTP hydrolysis
Always GTP bound
Always signalling active
What does a dominant negative GTPase mutant cause?
Substitution of the P-loop stops nucleotide binding
Nucleotide-free
Mops up active GEFs
What part of the cell do Rho mutants affects?
The actin cytoskeleton
Constitutively active Rho activation leads to?
Stress fibre formation
Constitutively active Rac activation leads to?
Membrane ruffles
Constitutively active Cdc42 activation leads to?
Filopodia formation
What do activated Rho proteins bind to?
A specific 16 amino acid sequence in effector proteins
CRIB motif - Cdc42 Rac1 Interactive Binding
How does Rac stimulate actin filaments?
Activates WAVE proteins
These activate Arp2/3
These create actin filaments
How does Cdc42 stimulate actin filaments?
Activates WASP proteins
These activate Arp2/3
These create actin filaments
How does Rho activate increases in myosin contractility and stress fibre formation?
Activates Rho kinase
Activates myosin phosphorylation
