cell migration Flashcards
what are small GTPases
- 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
effect of active ARf6
inhibitory
what does signalling active mean
signalling = gtp active
what does hydrolysis active mean
hydrolysis = chopping up enzyme = switching off
GTPase structure
- 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
how can gtpase activity be measured
measuring effector binding by measuring activity of switch proteins
staining doesn’t work bc too small
why is catalysis of hydrolysis important
GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes
how does catalysis of GTP hydrolysis occur
- attacking water -> catalytic glutamine
- counteracting of negative charge at phosphates
- glutamine 61 holds water in place best
molecules involved in cyclic regulation of gtpases
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
how do GTPase activating proteins work
- 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
how do GEFs work
stabilises nucleotide free form = GDP can fall off and GTP can come in = Accelerates exchange of GDP for GTP
how does T17N mutation affect Rac in GEFs
- disrupts nucleotide binding at all
- turns off all gtpases in cell
domain and family of GEFs
dbl-homology domain
dock-family
sec7 domain
how do GTPases control actin-based motility
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
gtpase forming filopodia during actin-based motility
cdc42 (rho)
gtpase forming lamellipodia during actin-based motility
activation of Rac1
gtpase forming actin stress fibres and contraction of cell during actin-based motility
rhoA
how does rhoA cause actomyosin contraction
GTP-RhoA activates Rho K → phosphorylates MLC → contraction of A-M contraction = pulls cell forward, muscle movement of organism
features of cdc42, rac and rhoa signals
- Cdc42/Rac are protrusive signals (grow)
- RhoA is a contractile signal (shorten)
effect of rac 1 localisation on migration
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
