14th Nov - Signalling Microdomains

Question 1

What are the two ‘phases’ of the plasma membrane?

Answer 1

The Liquid disordered (Phospholipids) phase

The Liquid ordered (sphingolipids) phase

Question 2

Describe the liquid disordered phase

Answer 2

Made up of loosely packed phospholipids - rapid lateral diffusion

Question 3

Describe the liquid ordered phase

Answer 3

Made up of cholesterol and sphingolipids, making the bilayer assembly more rigid and there is less lateral diffusion

Question 4

What are lipid rafts?

Answer 4

dynamic lipid microdomains in the cell membrane

Question 5

What are the two main types of lipid rafts?

Answer 5

Planar and caveolae

Question 6

What are caveolae?

Answer 6

Flask shaped pits in the plasma membrane

Question 7

What is caveolin?

Answer 7

A cholesterol binding protein, marker for caveloae anchored to the membrane by palmitoylation.

Question 8

What are the genes that encode for caveolin?

Answer 8

Cav-1
Cav-2
Cav-3

Question 9

Which tissues is Cav-1 localised to?

Answer 9

It is ubiquitous, with high levels in endothelia and smooth muscle cells

Question 10

Which tissues is Cav-2 localised to?

Answer 10

It is ubiquitous, with high levels in endothelial and smooth muscle cells

Question 11

Which tissues is Cav-3 localised to?

Answer 11

Muscle specific, expressed in skeletal, cardiac and smooth muscle

Question 12

Approximately how many caveolin molecules are present per caveloae?

Answer 12

150

Question 13

What is EDH2?

Answer 13

A dynamic ATPase that associates with the caveolar neck

Question 14

What is cavin?

Answer 14

A protein which coats the caveolar surface

Question 15

What are the distinctive characteristics of lipid rafts/caveolae which can be used to identify them?

Answer 15

Resistant to solubilsation by detergetns at 4C

Have a low buoyant density and float to the top of density gradients

Question 16

How did Lisanti (1994) analyse lipid rafts?

Answer 16

Treated them with detergent at 4C
Placed them on a sucrose gradient and span for 16 hours
Took off fractions and ran them on a gel
Western blotted for caveolin to identify the caveolae fractions
Analysed the signalling proteins that are enriched in caveolae

Question 17

What proteins did Lisanti find to be enriched in caveolae?

Answer 17

c-Src
c-Yes
Lyn-
Fyn
Lck
G beta
c-Fyr
JAK -2
G alpha s
G alpha i
G alpha q

Question 18

What proteins did Lisanti find to be excluded from caveolae?

Answer 18

PLC gamma
Ras
Rho A
Rho B

Question 19

What is the caveolae/raft signalling hypothesis?

Answer 19

These specialized regions of the membrane act to accumulate and compartmentalize different interacting signalling molecules into signalling microdomains. Proteins may also localize to lipid rafts/caveolae due tot he specialized lipid microenvironment and/or interaction with caveolae/raft associated proteins

Question 20

How does caveolin act as a signalling scaffold?

Answer 20

Signalling molecules binds to the caveolin scaffolding domain in their inactive state. Caveolin keeps them quiescent until activation leads to conformational change and release of the signalling proteins

Question 21

Who found caveolins function as a signalling scaffold in 1996?

Answer 21

Li

Question 22

GIve a function for lipid rafts

Answer 22

In cardiac myocytes Beta adrenergic receptors efficiently activate adenylyl cyclase 6 while prostaglandin E2 receptors do not, despite both being Gas receptors. This is because Beta adrenergic receptos are localised to caveolae and EP2 receptors are free in the bilayer.

OR

In cardiac myocytes L-type calcium channels are present in caveolae and so are beta adrenoceptors, leading to beta adrenoceptors efficient influx of calcium

Question 23

What are the three non-exclusive models for caveolae function?

Answer 23

Scaffolds for signalling events
Intracellular transport vesicles
Buffers