cell migration

Question 1

what are small GTPases

Answer 1

• Small - 21 kDa proteins
• One of the largest groups of signalling proteins: Ras superfamily
• Change conformation upon activation e.g gpcr
• Bind and activate downstream effectors

Question 2

effect of active ARf6

Answer 2

inhibitory

Question 3

what does signalling active mean

Answer 3

signalling = gtp active

Question 4

what does hydrolysis active mean

Answer 4

hydrolysis = chopping up enzyme = switching off

Question 5

GTPase structure

Answer 5

• Phosphate coordination by P-loop
  
  • phosphates are coordinated by p loop
  • controls shape of gtpase
• Mg2+ essential for nucleotide binding and activity
• Switch regions bind effectors

Question 6

how can gtpase activity be measured

Answer 6

measuring effector binding by measuring activity of switch proteins
staining doesn’t work bc too small

Question 7

why is catalysis of hydrolysis important

Answer 7

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes

Question 8

how does catalysis of GTP hydrolysis occur

Answer 8

1. attacking water -> catalytic glutamine
2. counteracting of negative charge at phosphates
   - glutamine 61 holds water in place best

Question 9

molecules involved in cyclic regulation of gtpases

Answer 9

GEFs - Accelerate exchange of GDP for GTP and stabilises nucleotide free form = GDP can fall off and GTP can come in
GAPs - accelerate hydrolysis of bound nucleotide

Question 10

how do GTPase activating proteins work

Answer 10

• less flexible structure to increase change of reaction
• have restricted freedom to lower entropy barrier and accelerate hydrolysis = stable catalytic glutamine
• argenine brings in a + charge to draw - charge away from phosphate

Question 11

how do GEFs work

Answer 11

stabilises nucleotide free form = GDP can fall off and GTP can come in = Accelerates exchange of GDP for GTP

Question 12

how does T17N mutation affect Rac in GEFs

Answer 12

• disrupts nucleotide binding at all
• turns off all gtpases in cell

Question 13

domain and family of GEFs

Answer 13

dbl-homology domain
dock-family
sec7 domain

Question 14

how do GTPases control actin-based motility

Answer 14

stimulate a cell with growth factor → activation of cdc42(rho gtpase) → formation of filopodia (arms) → activation of Rac1 → formation of lamellipodia (head moving forward) → RhoA activation → actin stress fibres are formed causing contraction of cell

Question 15

gtpase forming filopodia during actin-based motility

Answer 15

cdc42 (rho)

Question 16

gtpase forming lamellipodia during actin-based motility

Answer 16

activation of Rac1

Question 17

gtpase forming actin stress fibres and contraction of cell during actin-based motility

Answer 17

rhoA

Question 18

how does rhoA cause actomyosin contraction

Answer 18

GTP-RhoA activates Rho K → phosphorylates MLC → contraction of A-M contraction = pulls cell forward, muscle movement of organism

Question 19

features of cdc42, rac and rhoa signals

Answer 19

• Cdc42/Rac are protrusive signals (grow)
• RhoA is a contractile signal (shorten)

Question 20

effect of rac 1 localisation on migration

Answer 20

activity at front of cell = positive effect forward
spread out = less movement
without direction e.g skin fibroblasts they can’t move in right place to heal wound