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Dynamic Cells > Prof. Thompson's lectures L6-L9 > Flashcards

Prof. Thompson's lectures L6-L9 Flashcards

1
Q

Cell steering

A

Compass model

Bifurcations and bias model

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2
Q

Dictyostelium

A

Small genome, amoebae come together.

Use cAMP as chemoattractant.

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3
Q

REMI

A

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

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4
Q

Cell autonomous/non-synergisable mutant

A

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.

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5
Q

Non cell-autonomous/ synergisable mutant

A

Can’t make signal but can response when mixed with WT. Will aggregate when mixed with WT.

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6
Q

ACA mutants

A

synergisable. Can respond to signal but cant make adenylate cyclase which makes cAMP

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7
Q

cAR1 mutants

A

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.

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8
Q

GPCR FRET

A

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.

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9
Q

CRAC

A

Recruited to leading edge. Member of family- PH domain containing proteins.

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10
Q

PH domain containing proteins

A

Recruited to leading edge (include CRAC). Bind phospholipids including PIP3.

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11
Q

Latrunculin A

A

Depolymerises actin. Still localises CRAC so CRAC is upstream of actin- doesnt need actin to be recruited.

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12
Q

PIP3

A

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.

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13
Q

PI3-kinase

A

phosphorylates PIP2->PIP3

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14
Q

PTEN

A

A phosphotase. Breaks down PIP3 to PIP2

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15
Q

PI3k-GFP

A

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)

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16
Q

LY

A

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?

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17
Q

Ras recruiting

A

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.

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18
Q

RasC and RasG mutants

A

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.

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19
Q

chemotaxis steps- summary

A
  1. cAMP/chemoattractant binds to GPCR
  2. Dissociation of G subunits.
  3. This leads to RasGEF activating Ras which binds to PI3K.
  4. PI3K binds to activated Ras at front of cell -> makes PIP2 into PIP3
  5. More PIP3 at front of cell so PH containing proteins come to front and bind to PIP3.
  6. These proteins (crac etc.) regulate F-actin assembly and cell polarity.
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20
Q

Hoeller, O and Kay R.R in 2007

A

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..

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21
Q

PI3K1-5/PTEN KO cells

A

Slightly slower movement = only defect.
IN weak gradient, PI3k very important.
IN strong gradient- cell finds other machinery, doesnt care about pi3k.

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22
Q

RIP3

A

Ras also binds to RIP3. (ras interacting protein). Component of TORC2 complex.

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23
Q

TORC2 complex

A

Highly conserved regulator of growth and cytoskeleton. Kinase related to PI3k.

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24
Q

consideration of Ca2+ as second messenger in cell migration

A

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.

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25
Q

GbpA and GbpB

A

Phosphodiesterases that break down cGMP

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26
Q

Primordial germ cells PGC

A

Give rise to sperm and eggs. add GFp to promoter, can watch them move across.

27
Q

GbpC and GbpD

A

cGMP binding proteins

28
Q

SDF-1

A

Chemokine. diffusable signalling molecule, provides gradietn for PGCs to move up

29
Q

CXCR4

A

GPCR needed for primordial germ cell migration

30
Q

Signal relay

A

When migration distance is big. cells leaving trails of guidance molecules.

31
Q

Collective cell migration examples

A
  1. Border cells in drosophila
  2. Lateral line in zebrafish
  3. dictyostelium development
  4. dorsal closure in drosophila
32
Q

Border cells

A

Undergo partial EMT-? delaminate from epithelium so can move as 10-cell unit.

33
Q

UAS

A

Upstream activation sequence. Put UAS upstream of a genee and gene will be inactive until add GAL4.

34
Q

GAL4

A

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.

35
Q

EGF

A

Epiderman growth factor. found in gradient along length of oocyte. remove and border cells wont move.

36
Q

PVF

A

PDGF and VEDF related factor. found in gradient along length of oocyte. If you remove, border cells wont move.

37
Q

Dominant negative

A

Has extracellular portion that binds growth factor, but won’t communicate. Soaks up all growth factor.

38
Q

Cadherin

A

HIgh levels in polar cells in middle. Glues 10 cells together in border cell migration

39
Q

Hidsight

A

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

40
Q

RREB

A

Homologue of hindsight. Remove RREB by iRNAs. Wounds dont heal, and cells wont come together.

41
Q

Lateral line in zebrafish

A

Group of 100 cells moves across embryo and deposits rosets of cells along way that will beome mechanosensory organs.

42
Q

sdf-1

A

chemokine that lateral line follows along path.

sdf-1 morpholino KO - lateral line wont follow

43
Q

CXCR4

A

SDF-1 receptor. Only expressed at front of lateral line.

44
Q

CXCR7

A

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.

45
Q

FGF

A

High FGF at back of lateral line. Represses CXCR4 so only found at back where you want CXCr7

46
Q

Wnt

A

High Wnt conc. at front of lateral line. represses cxcr7 becuase at fron want cxcr4.

47
Q

Dark field imaging

A

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.

48
Q

cAMP

A

Cells amplify camp in chain.

cAMP-> GPCR-> G-> PI3K-> PIP2 TO PIP3-> CRAC-> stimulates ACA and recruits machinery

49
Q

ACA

A

Adenylate cyclase makes more cAMP at back of cells to relay

50
Q

Adenyate cyclase null cell

A

Put this in chain of cells, signal not passed on, chains stops working.

51
Q

Phosphodiesterase in cells

A

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)

52
Q

tip cells

A

must stay at front of slug. They make more cAMP (express more ACA).
They also have more myosin- greater motile force.

53
Q

Slug adhesion

A

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.

54
Q

Drosophila dorsal closure

A

2 epithelial sheets meet. Segmented so must be coordinated.

55
Q

Basket/kayak/slipper

A

Drosophila dorsal closure mutants. Mutations in JNK cascade and Ena KOs.

56
Q

JNK pathway

A

Makes AP-1. Needs Jun and Fos to make ap1. Activates expression of the growth factor- Dpp.

57
Q

AP-1

A

A transcription factor made by jnk pathway. Activates transcription of some genes. e.g. Dpp which is a growth factor

58
Q

Decapentaplegic (Dpp)

A

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.

59
Q

Rac and cdc42 dorsal hole

A

If inactivated, hole cannot close. showing filopodia=vital

60
Q

Ena at dorsal hole

A

promotes filopodia formation to close hole. Helps filopodia meet and match to correct segments in epithelial sheet. important

61
Q

TGF-beta

A

Secreted at wound edge in humans. coordinates cell behaviour. IN embryo- would be good in wound healing £££

62
Q

PVR

A

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.

63
Q

Hemocytes

A

Drosophila equivalent to macrophages. Rush to wound. need rhoGTPases. cdc42 senses the wound to keep hemocytes on track.

64
Q

Myosin purse string

A

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.

Dynamic Cells (5 decks)

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