Regulation of cell migration

Question 1

How big are GTPases?

Answer 1

Small 21kDa proteins

Question 2

What is the structure of GTP? (2)

Answer 2

• Guanine nucleotide binds to a ribose molecule to form guanosine
• Guanosine is joined to 3 phosphate groups to form GTP

Question 3

What is the difference between a ‘signalling active’ and ‘hydrolysis active’ GTPase? (2)

Answer 3

• Signalling active is bound to GTP because it is active and able to bind to downstream effectors
• Hydrolysis active is bound to GDP because it is hydrolysing GTP and switching off the signalling activity

Question 4

What is the structure of a GTPase? (3)

Answer 4

• P loop: binds to phosphate groups
• Mg2+: essential for nucleotide binding
• Switch regions 1 and 2: bind to downstream effectors

Question 5

How do you detect an active GTPase? (2)

Answer 5

• Effector binding
• The conformational change between the active and inactive form of the GTPase is very subtle so an antibody wouldn’t be able to discriminate between the 2 forms but an effector can

Question 6

How is GTP hydrolysis catalysed? (2)

Answer 6

• Glutamine residue positions the attacking water molecule in the optimal position to hydrolyse the phosphate bond
• P loop contains lysine residues which counteracts the negative charge of the phosphates in the GTP

Question 7

What are active mutants of Rac? (2)

Answer 7

• Q61L: substituting the glutamine gets rid of its catalytic activity so the GTPase is always GTP-bound
• G12V: pushes Q61 out of position and disturbs the P loop

Question 8

How are GTPases regulated? (4)

Answer 8

• GEF removes the bound GDP and replaces it with GTP to switch on the GTPase
• GTP-bound GTPase binds to downstream effectors
• GAP assists GTP hydrolysis by the GTPase
• GDI binds to the GDP-bound form and keeps it switched off

Question 9

What is a GDI?

Answer 9

Guanine nucleotide dissociation inhibitor

Question 10

What is a GEF?

Answer 10

Guanine nucleotide exchange factor

Question 11

What is a GAP?

Answer 11

GTPase activating protein

Question 12

How does p50 GAP assist GTP hydrolysis by Rac1? (3)

Answer 12

• GTPase stabilises the water and bound Mg2+, causes restricted freedom of the water molecule, results in a reduced entropy barrier for GTP hydrolysis
• p50 GAP stabilises the position of the catalytic glutamine
• p50 GAP brings positively charged arginine which counteracts the negative charge of the phosphate which destabilises the bond for hydrolysis

Question 13

How do GEFs accelerate exchange of GDP for GTP?

Answer 13

Stabilises nucleotide free, Mg2+ free GTPase so more GTP can bind

Question 14

What is an inactive mutant of Rac?

Answer 14

T17N mutation inhibits GTP binding and is dominant negative because it binds up all the GEFs

Question 15

What kinds of GEFs are there? (3)

Answer 15

• Double-homology
• DOCK family
• Sec7

Question 16

How are GTPases involved in actin-based motility? (3)

Answer 16

• Cdc42 responsible for production of filopodia
• Rac1 responsible for lamellipodia
• RhoA responsible for contraction of stress fibres to move the cell

Question 17

What is the role of RhoA in actin-based motility? (3)

Answer 17

• GTP-RhoA activates Rho kinase
• Rho kinase phosphorylates the myosin light chain
• Results in actomyosin contraction

Question 18

How are antagonistic signals timed in actin-based motility? (4)

Answer 18

• Cdc42 and rac are protrusive signals so they are switched on to start migration
• RhoA is a contractile signal so is suppressed at the start
• Then cdc42/rac are switched off and rhoA is switched on
• Cycles backwards and forwards

Question 19

How do cells migrate on a 2D matrix? (3)

Answer 19

• Random
• Flat
• Fast

Question 20

How do cells migrate on a 3D matrix? (3)

Answer 20

• Directional
• Integrated with the matrix
• Often slower but more efficiently