L25 - Cytoskeleton and Cell Shape

Question 1

What 3 things define cell shape?

Answer 1

Adjoining cells – physically boxes cells in
Cell adhesions
Extra-cellular matrix

Question 2

What cells show the important of cell shape for function?

Answer 2

Nerves
Muscles
Red blood cells

Question 3

Why is cell shape vital for cellular activity?

Answer 3

Migration – cell shape changes drive movement
Phagocytosis – cytoskeleton changes shape when engulfing
Transport – cytoskeleton movements used to move vesicles
Cytoskeletal dynamics

Question 4

F-actin monomers role?

Answer 4

No structural role

Question 5

F-actin polymers role?

Answer 5

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

Question 6

The actin cytoskeleton in the inactive state is?

Answer 6

Very stable

Very little unpolymerized actin in cytoplasm

Question 7

What regulated disassembly of F-actin?

Answer 7

Phosphorylation

Question 8

Why do actin filament self-polymerise?

Answer 8

Actin in solution will polymerise by itself – because of their shape
Initial step is energetically unfavourable

Question 9

Actin polymerisation steps

Answer 9

1. Add salt to a group of monomers to allow polymerisation
2. Olgiomers polymerise to form a growing actin filament
3. A point where the rate of monomers added and falling off is equal – steady state of fibres

Question 10

What is actin treadmilling?

Answer 10

This is the process of how we regulate actin polymerisation in cells

Question 11

How are monomers added to actin filaments?

Answer 11

Added to the + end

Removed at the – end

Question 12

What regulates the addition of monomers to actin filaments?

Answer 12

Phosphorylation regulates this - ATP  ADP and Pi resulting in monomer release

Question 13

What accessory protein is associated with actin filaments?

Answer 13

Profilin – binds to monomers enabling them to be phosphorylated so they can be added to the + end
They increase the rate of monomer addition

Question 14

What are the 4 mechanism of actin binding proteins?

Answer 14

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

Question 15

What are small GTPases?

Answer 15

Small monomeric 21kDa proteins
- Don’t confuse with heterodimeric G proteins
Have intrinsic GTPase activity that can bind to and hydrolyse GTP

Question 16

What modifications do small GTPases have?

Answer 16

Many have post-translational lipid modifications to target them to specific membrane sites

Question 17

What family do small GTPases belong to?

Answer 17

All small GTPases belong to a large family

Archetypcal member being Ras

Question 18

What are some examples of the Ras superfamily of GTPases?

Answer 18

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

Question 19

If RAC is bound to GTP it is?

Answer 19

Active
This is a timed process due to its enzymatic activity
GTP is broken down to GDP fairly quickly

Question 20

If RAC is bound to GDP it is?

Answer 20

Inactive

Question 21

What does active RAC activate?

Answer 21

Downstream targets - PAK and WAVE2

Question 22

What helps catalyse the exchange of GDP for GTP?

Answer 22

Guanine nucleotide exchange factor (GEF)

Question 23

What helps catalyse the breakdown of GTP to GDP?

Answer 23

GTPase activating protein (GAP)

Question 24

What slows down the dissociation of GDP?

Answer 24

Guanine nucleotide dissociation inhibitor (GDI)

Question 25

What causes a change in the structure of GTPase switch regions?

Answer 25

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

Question 26

What do RhoA, Rac1 and Cdc42 coordinate?

Answer 26

Coordinate actin cytoskeletal organisation | In turn controls cell morphology, cell movement and cell polarity

Question 27

What does Cdc42 control?

Answer 27

Controls polymerisation of actin filaments and formation of actin spikes or filopodia

Question 28

What does Rac1 control?

Answer 28

Controls organisation of new actin filaments into dynamic ruffling structures or lamellipodia

Question 29

What does RhoA control?

Answer 29

Stabilises actin filaments into a more rigid skeletal framework - stress fibres

Question 30

What have dominant negative and constitutively active mutant GTPases shown?

Answer 30

Point mutations in the nucleotide-binding sites of small GTPases can make the protein - Constitutively active - always on - Dominant negative - always off and inhibitory

Question 31

What does a constitutively active GTPase mutant cause?

Answer 31

Substitution of the catalytic glutamine in switch 2 stops GTP hydrolysis Always GTP bound Always signalling active

Question 32

What does a dominant negative GTPase mutant cause?

Answer 32

Substitution of the P-loop stops nucleotide binding Nucleotide-free Mops up active GEFs

Question 33

What part of the cell do Rho mutants affects?

Answer 33

The actin cytoskeleton

Question 34

Constitutively active Rho activation leads to?

Answer 34

Stress fibre formation

Question 35

Constitutively active Rac activation leads to?

Answer 35

Membrane ruffles

Question 36

Constitutively active Cdc42 activation leads to?

Answer 36

Filopodia formation

Question 37

What do activated Rho proteins bind to?

Answer 37

A specific 16 amino acid sequence in effector proteins | CRIB motif - Cdc42 Rac1 Interactive Binding

Question 38

How does Rac stimulate actin filaments?

Answer 38

Activates WAVE proteins These activate Arp2/3 These create actin filaments

Question 39

How does Cdc42 stimulate actin filaments?

Answer 39

Activates WASP proteins These activate Arp2/3 These create actin filaments

Question 40

How does Rho activate increases in myosin contractility and stress fibre formation?

Answer 40

Activates Rho kinase | Activates myosin phosphorylation