L19 - Cell Migration Flashcards
What GTPases are involved in:
Channels and receptors -
Cell polarity -
Vesicular trafficking -
Migration -
Cell division -
Mechanobiology -
Channels and receptors - GPCRs
Cell polarity - Cdc42
Vesicular trafficking - Rabs and Arfs
Migration - Rho
Cell division - Ran
Mechanobiology - Rho
Which GTPase is the basis of cancer?
Ras
How does GTPase signalling work?
-GTPase signalling depends on the bound nucleotide
-Hydrolysis GTP
-Bound to the ribose to form a guanosine
-Guanosine Triphosphate (GTP)
-Get off gamma phosphate releases energy and causes a change in the molecule
-GTPases are unstable in reactive form as it will always want to be GTP
-Just bound triphosphate is not enough, it needs to cycle
-Ability to cycle the nucleotide becomes important
-Signalling bound to GTP and hydrolysis is switching off signalling and is GDP
-GTP bound Rac1
What do these nucleotide and effector-binding sites do:
Blue box -
P-loop -
Mg2+ -
Switch regions -
Blue box - this is all fixed so not interesting part of the molecule, most of this structure is the same with other GTPases and highly conserved
P-loop - phosphate binding loop, phosphates coordinated by P-loop and control shape of GTPase
Mg2+ - highly negative phosphate binds tightly
Switch regions - these do the worm and bind to downstream effectors
Why aren’t phosphorylation and antibodies used to measure GTPase activity? (2)
Phosphorylation - there is no phosphorylation event
Antibody - change is too small
What is used to measure GTPase activity?
Effector binding - evolved to be so precisely matched they can distinguished between active and inactive change
How does catalysis of GTP hydrolysis take place? (4)
-Bound nucleotide has energy barrier for hydrolysis
-Position the water to hydrolyse the bound for the best position for hydrolysis (Glu-61) catalytic glutamine
-Have Glysine 12 in P-loop
-Neutralises some negative change and lowers the energy barrier (P-loop, hydrogen bonds and lysine)
Describe the cyclic regulation of GTPases? (4)
-Load with GTP: remove bound GDP so it can be replaced with GTP. GEFs stabilise transition state and GEF accelerate exchange rate to switch on the GTPase
-GTP bound binds to downstream effectors
-GAP (GTPase activating protein) moves it away from signalling state and assists with hydrolysis
-GDI (guanine nucleotide dissociation inhibitor) bind to a form of GTPase and hold in cytosol to switch it off
What are GAPs?
GTPase activating proteins
What do GAPs do?
-GAP contributes to stabilisation and stabilised position of catalytic glutamine
What are GEFs?
Guanine Nucleotide Exchange Factors
What is the mechanism for actin-based motility? (5)
-Stimulate with growth factor, induces migration towards growth factor by formation of filopodia
-Filopodia activated because of Cdc42
-Lamellipodia protrudes forward and form new adhesions and is regulated by Rac1
-Formation of actin stress fibres bind contractility and contraction of actin myosin and is regulated by RhoA
-Rho kinase phosphorylated myosin light chain and results in activation of actomyosin contraction
What is used to guide migration?
Localised signals
