Lecture 4: Challenging the Fluid Mosaic Model

Question 1

How was membrane fluidity first demonstrated?

Answer 1

two cells, one labelled red, one green, fused by virus and incubated, at first colours were separate but began to merge, suggesting free movement of molecules in the membrane

Question 2

What do you often do to a protein targeted by GFP and what could GFP do?

Answer 2

Over-express it. It is a large protein so could alter the behaviour of the target protein

Question 3

What does FRAP stand for?

Answer 3

Fluorescence recovery after photobleaching

Question 4

How does FRAP work?

Answer 4

Bleach an area irreversibly with a laser. observe the bleached area over time and the recovery of the fluorescence signal. This would be due to the fluorescent, non-bleached molecules moving that area. Half height of recovery is the time for half of the fluorescence to recover.

Question 5

What does FLIP stand for?

Answer 5

Fluorescence loss in photobleaching

Question 6

How does FLIP work?

Answer 6

One region of the cell is continuously bleached, and the fluorescence is monitored over time. Free movement would mean fluorescent molecules move into the bleaching area so a gradual decrease in fluorescence of the cell occurs.

Question 7

What are the two main observations with FRAP? What does this suggest?

Answer 7

1) Membrane proteins move more slowly in PM than in pure lipid bilayers
2) some membrane proteins aren’t free to diffuse to irradiated area eg integral membrane proteins fused to GFP
3) The fluid mosaic model is flawed

Question 8

What are the 3 main limitations of FRAP and FLIP?

Answer 8

1) recovery of fluorescence after FLAP could be due to delivery of new membrane proteins by vesicular transport
2) a protein expressed as a fusion protein is usually over-expressed
3) large GFP molecule could alter behaviour

Question 9

what is single particle tracking?

Answer 9

individual membrane proteins are labelled and followed by video microscopy

Question 10

What factors affect free movement?

Answer 10

attachment to cytoskeleton, interaction with other proteins, extracellular matrix, lipid rafts, interaction with large protein complexes means slower movement than smaller ones

Question 11

What are the 3 models for membrane organisation?

Answer 11

1) fluid mosaic model
2) lipid raft model
3) picket fence model

Question 12

What is the fluid mosaic model?

Answer 12

the random diffusion of proteins and lipids

Question 13

What is the lipid raft model?

Answer 13

Cholesterol and sphingomyelin are enriched in certain domains which are highly ordered and distinct entities in the phospholipids

Question 14

What is the picket fence model?

Where has this been demonstrated?

Answer 14

transmembrane proteins bind underlying actin filaments and lateral diffusion is hindered by membrane associated actin network. Red blood cells

Question 15

Give two examples of membranes that are kept seperated

Answer 15

Apical and basal side has different compositions in epithelial cells. tight junctions keep 2 domains separated

Question 16

give a specific example of where the fluid mosaic model fails

Answer 16

Neurotransmitter receptors limited to the synaptic region

Question 17

Explain the lipid raft concept

Answer 17

Proposed in 1997. Lipids in rafts are in liquid ordered state (Lo) and not in rafts are in liquid disordered state (Ld). Lo domains are rich in cholesterol, GPI anchored proteins, sphingolipids, saturated fatty acids. Unsaturated fatty acids are more fluid. See phase separation at certain temps and when Pc is 60%, SM is 20% and Chol is 20%.

Question 18

What are the potential functions of lipid rafts?

Answer 18

Organisation of proteins, concentration for transport, signalling complexes

Question 19

How are lipid rafts defined with detergent?

Answer 19

Treating cells with cold detergent, non-raft domains will be soluble in this as is less ordered. They can be separated and analysed

Question 20

What is odd about GPI anchored proteins in detergent anlaysis?

Answer 20

They seem to be enriched in the cold detergent resistant fractions but aren’t observed in rafts by microscopy, could be down to limitations in microscopy.

Question 21

What has atomic force microscopy been used for?

Answer 21

Observing thicker sphingomyelin rafts

Question 22

What would proteins with longer Tm domains do?

Answer 22

Associate with rafts due to mismatch between hydrophobic acyl chain length

Question 23

How is cholesterol depletion achieved and what are the effects of this?

Answer 23

With methyl-B-cyclodextrin. has effects on cell signalling

Question 24

What can FRET be used for?

Answer 24

To see if there are different types of raft. Needs to have proteins with the same group interacting to get effects signal.

Question 25

What are caveolae? what to do they form? What do they contain? Where are they found? What might they be involved in?

Answer 25

Deeply invaginated flasks identifiable by EM. form lipid rafts rich in cholesterol, glycosphingolipids and GPI anchored proteins. Contain caveolin structural protein, found in the cytoplasmic leaflet and might be involved in cell stretching.