Prof. Thompson's lectures L6-L9 Flashcards
Cell steering
Compass model
Bifurcations and bias model
Dictyostelium
Small genome, amoebae come together.
Use cAMP as chemoattractant.
REMI
Insert foreign DNA in gene, gene won’t be expressed. So then you’ll knwo what it’s for. Forward genetics. For example come up with theory that if dictyostelium cant sense cAMP, wont aggregate
Cell autonomous/non-synergisable mutant
Can’t sense the signal. The gene mutated was needed to respond to signal. Even when mixed with WT cells, always shows phenotype -> can’t aggregate.
Non cell-autonomous/ synergisable mutant
Can’t make signal but can response when mixed with WT. Will aggregate when mixed with WT.
ACA mutants
synergisable. Can respond to signal but cant make adenylate cyclase which makes cAMP
cAR1 mutants
Non-synergisable. cAMP receptor 1. A GPCR. When activated. results in dissociation of G subunits which activates front of cell. So without receptor, Can’t respond to cAMP.
GPCR FRET
Can see when GPCR activated-> get dissociation of G subunits. Put CFP on alpha. YFP on beta. NO more energy transfer, no more FRET. So will see a drop in fluorescence when cAMP. Shows the dissociation.
CRAC
Recruited to leading edge. Member of family- PH domain containing proteins.
PH domain containing proteins
Recruited to leading edge (include CRAC). Bind phospholipids including PIP3.
Latrunculin A
Depolymerises actin. Still localises CRAC so CRAC is upstream of actin- doesnt need actin to be recruited.
PIP3
Phospholipid found at the PM. Important in regulating many processes so highly regulated. Made from PIP2 by PI3K. Can be broken back down by PTEN. PH domain proteins move to front because PIP3 is there.
PI3-kinase
phosphorylates PIP2->PIP3
PTEN
A phosphotase. Breaks down PIP3 to PIP2
PI3k-GFP
Can see that PI3K is localised to front within a few seconds. More active at front because more of it at front, (not because it’s everywhere and just activated at front)
LY
Removes PI3k from cell. So no PIP3 made. PI3k still recruited to front so shows PI3k doesnt need pip3 to get to front. Doesnt rely on pip3 or its own activity to be recruited to front… so what does it need?
Ras recruiting
Ras can bind to PH domain containing proteins. So as Ras is activated at edge of cell, binds PI3k at edge. To test, knock out RasC abd Ras G -> cells will not aggregate.
RasC and RasG mutants
Mutants fail to chemotx, supporting fact that Ras is important. Can measure amount of Ras when stimulated with cAMP. Have to knock out both C and G as they compensate each other if only one is mutated.
chemotaxis steps- summary
- cAMP/chemoattractant binds to GPCR
- Dissociation of G subunits.
- This leads to RasGEF activating Ras which binds to PI3K.
- PI3K binds to activated Ras at front of cell -> makes PIP2 into PIP3
- More PIP3 at front of cell so PH containing proteins come to front and bind to PIP3.
- These proteins (crac etc.) regulate F-actin assembly and cell polarity.
Hoeller, O and Kay R.R in 2007
Contraversial findings. Knocked out all 5 pi3k genes and even PTEN in dictyostelium. So no pip3. Put in chemotaxis assay and cells still moved!
Dont need pip3..
PI3K1-5/PTEN KO cells
Slightly slower movement = only defect.
IN weak gradient, PI3k very important.
IN strong gradient- cell finds other machinery, doesnt care about pi3k.
RIP3
Ras also binds to RIP3. (ras interacting protein). Component of TORC2 complex.
TORC2 complex
Highly conserved regulator of growth and cytoskeleton. Kinase related to PI3k.
consideration of Ca2+ as second messenger in cell migration
Considered because when cells get a signal they make IP3 which releases the stores of calcium. So when cAMP hits cell-> calcium released. But when calcium knocked out, made no difference
so not sure what calcium is up to.
GbpA and GbpB
Phosphodiesterases that break down cGMP
Primordial germ cells PGC
Give rise to sperm and eggs. add GFp to promoter, can watch them move across.
GbpC and GbpD
cGMP binding proteins
SDF-1
Chemokine. diffusable signalling molecule, provides gradietn for PGCs to move up
CXCR4
GPCR needed for primordial germ cell migration
Signal relay
When migration distance is big. cells leaving trails of guidance molecules.
Collective cell migration examples
- Border cells in drosophila
- Lateral line in zebrafish
- dictyostelium development
- dorsal closure in drosophila
Border cells
Undergo partial EMT-? delaminate from epithelium so can move as 10-cell unit.
UAS
Upstream activation sequence. Put UAS upstream of a genee and gene will be inactive until add GAL4.
GAL4
Binds to UAS-> activates transcription. So if GFP-Moesin has UAS added- allows us to see the border cell migration. add gal4-> see moesin-rich protrusions.
EGF
Epiderman growth factor. found in gradient along length of oocyte. remove and border cells wont move.
PVF
PDGF and VEDF related factor. found in gradient along length of oocyte. If you remove, border cells wont move.
Dominant negative
Has extracellular portion that binds growth factor, but won’t communicate. Soaks up all growth factor.
Cadherin
HIgh levels in polar cells in middle. Glues 10 cells together in border cell migration
Hidsight
Hindsight = gene needed to turn cadherin on or off when appropriate. Hindsight mutant where all cells have cadherin so can’t move properly, all stuck.
Has zinc fingers so binds to dna like a TF
RREB
Homologue of hindsight. Remove RREB by iRNAs. Wounds dont heal, and cells wont come together.
Lateral line in zebrafish
Group of 100 cells moves across embryo and deposits rosets of cells along way that will beome mechanosensory organs.
sdf-1
chemokine that lateral line follows along path.
sdf-1 morpholino KO - lateral line wont follow
CXCR4
SDF-1 receptor. Only expressed at front of lateral line.
CXCR7
Present in cells at back of lateral line. Not true chemokine receptor, It acts as a dominant negative.
Soaks up the SDF-1 to maintain the gradient.
FGF
High FGF at back of lateral line. Represses CXCR4 so only found at back where you want CXCr7
Wnt
High Wnt conc. at front of lateral line. represses cxcr7 becuase at fron want cxcr4.
Dark field imaging
measures light changes only. Can see waves where mounds of dictyosteliu form. Pulses. And ripples when slug moves later on. Because cells cring to round and then extend psuedopods.
cAMP
Cells amplify camp in chain.
cAMP-> GPCR-> G-> PI3K-> PIP2 TO PIP3-> CRAC-> stimulates ACA and recruits machinery
ACA
Adenylate cyclase makes more cAMP at back of cells to relay
Adenyate cyclase null cell
Put this in chain of cells, signal not passed on, chains stops working.
Phosphodiesterase in cells
IN chain cells make phosphodiesterase to break down cAMP so that the signal doesnt get louder and louder. Keep it a wave.
(every 9 minute wave so receptors desensitized after for 9 mins)
tip cells
must stay at front of slug. They make more cAMP (express more ACA).
They also have more myosin- greater motile force.
Slug adhesion
cells at front constantly make ECM (slime sheath). Next cells use it to move along. Do this by using intergrin-like molecules and focal adhesions containing talin and paxillin b.
Drosophila dorsal closure
2 epithelial sheets meet. Segmented so must be coordinated.
Basket/kayak/slipper
Drosophila dorsal closure mutants. Mutations in JNK cascade and Ena KOs.
JNK pathway
Makes AP-1. Needs Jun and Fos to make ap1. Activates expression of the growth factor- Dpp.
AP-1
A transcription factor made by jnk pathway. Activates transcription of some genes. e.g. Dpp which is a growth factor
Decapentaplegic (Dpp)
Homologue of human GF TGF-beta. Present in leading cells at drosophila dorsal hole. Actvated contraction of amnoserosa cells below hole and changes behaviour of surrounding cells.
Rac and cdc42 dorsal hole
If inactivated, hole cannot close. showing filopodia=vital
Ena at dorsal hole
promotes filopodia formation to close hole. Helps filopodia meet and match to correct segments in epithelial sheet. important
TGF-beta
Secreted at wound edge in humans. coordinates cell behaviour. IN embryo- would be good in wound healing £££
PVR
Growth factor receptor at leading edge of wounds. When epidermis is wounded, PVR comes into contact with its ligand- PVF. PVF let out. Promotes formation of protrusions to heal.
Hemocytes
Drosophila equivalent to macrophages. Rush to wound. need rhoGTPases. cdc42 senses the wound to keep hemocytes on track.
Myosin purse string
Rho will bind to Rho kinase-> phosphorylates myosin. Contract cable. Knockout rho-> get huge lameelipodia around wound to compensate because wound healing very important.
Can do without purse string but cant do without filopodia.
