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Cellular Signalling > Tomlinson - Lipid Rafts > Flashcards

Tomlinson - Lipid Rafts Flashcards

1
Q

Describe the structure of Sphingolipids wrt glycerolipids

A

Sphingolipids possess longer hydrocarbon tails, are fully saturated and therefore more straighter and stiffer than the glycerolipids counterparts

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2
Q

Glycerolipids greater resemble which phase of the bilayer, the ordered or disordered phase?

A

Disordered

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3
Q

What are the three components of a lipid raft?

A

Sphingolipid
Cholesterol
Protein

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4
Q

Most phospholipid bilayers are electrically polarised at..?

A

-60 mV

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5
Q

Phospholipids are amphipathic and form bimolecular sheets, what does “amphipathic” mean?

A

It has a hydrophobic interior (fatty acid tails) and hydrophilic exterior (polar head groups)

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6
Q

What is the phospholipid bilayer generically comprised of?

A

Wholly lipids and proteins, with a few associated carbohydrates

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7
Q

What is the thickness of a phospholipid bilayer?

A

5 nM wide

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8
Q

Describe the structure of glycerolipids wrt sphinoglipids

A

Glycerolipids possess shorter hydrocarbon chains, tend to be unsaturated with C=C Cis double bonds and therefore are bulkier

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9
Q

Sphingolipids greater resemble which phase of the bilayer, the ordered or disordered phase?

A

Ordered

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10
Q

What are the three major classes of membrane residing lipids?

A

Glycerolipids
Sphingolipids
Cholesterol

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11
Q

How do lipid rafts help with efficient signal transduction?

A

For efficient signal transduction to occur, it helps if the associated machinery is co-localised or compartmentalised.

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12
Q

What does cholesterol associate better with, glycerolipids or sphinoglipids?

A

Sphingolipids

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13
Q

In what two ways may a lipid raft micro-domain coalesce to form a larger raft platform?

A

1) via external signal transduction
2) membrane trafficking events which induce:
- lipid-lipid interactions
- lipid-protein interactions
- protein-protein interactions

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14
Q

What is the current definition of a lipid raft?

A
  1. Sphingolipid-Cholesterol-Protein enriched micro domains
  2. Less than 100 nm in length
  3. Preferentially recruit GPI-anchored proteins
  4. Preferentially recruit Palmitoylated proteins (PTM)
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15
Q

From a molecular perspective, why does Cholesterol interact well with Sphingolipid?

A

The Sphingolipids possess long hydrocarbon fully saturated fatty acid chains which are stiff and straight in structure. This accommodates the contrastingly bulkier sterol cholesterol 4-ring structure. There is also opportunity for an extensive hydrogen bonded network to occur, given the dipole-dipole interactions between cholesterol and Sphingolipids

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16
Q

What is the take home message regarding controversies in lipid raft research?

A

“Membrane heterogeneity can be induced or modified when trying to observe it.”

17
Q

State the name of four cholesterol depleting drugs

A

Methyl-beta-cyclodextrin, which has a sugar ring structure wit a hydrophobic interior and interacts well with the sterol group of Cholesterol

Polyene antibiotics such as Filipin and Nystatin

Cholesterol synthesis inhibitors such as HMG-CoA Reductase

18
Q

What is the in vitro evidence to suggest cholesterol interacts well with Sphingolipids?

A

Phosphatidylcholine + Sphingomyelin = freely diffuse structure
Phosphatidylcholine + Sphingomyelin + Cholesterol = Sphingomyelin and Cholesterol assemblies compartmentalised away from PC

19
Q

Are lipid rafts artefacts of detergent use in studies? Discuss

A

Early studies defined raft assemblies as “Detergent Resistance Membranes”, following detergent solubilisation at 4 degrees. Papers often published suggested their proteins resided within the DRM and therefore if they had signalling capability would do so involving raft machinery. However these results were not replicable and detergent solubilisation is artificial. It depends on:

  • The detergent: Triton X-100, CHAPS, SDS
  • Critical Micelle concentration
  • The temperature
  • Solubilisation period

DRMs are therefore not direct descendants of pre-existing lipid rafts

20
Q

State the components of the phospholipid bilayer

A 
Peripheral proteins
Surface proteins
Integral proteins (globular)
Integral proteins (alpha helical)
Cholesterol 
Protein channels 
Glycolipids
Glycoproteins
21
Q

State the names of two Polyene antibiotics which deplete cholesterol levels

A

Filipin and Nystatin

22
Q

Should cholesterol extracting drugs be used to study rafts?

A

In order to determine whether lipid rafts are important for a particular proces, studies have utilised cholesterol extracting drugs I.e. Filipin, nystatin, methylbetacyclodextrin to remove cholesterol from rafts and observe the effects. However it also removes cholesterol from the rest of the membrane too (40% cholesterol component) which can have effects such as unwanted membrane depolarisation and calcium signalling

23
Q

Describe how lipid rafts are used in HIV assembly

A
  1. Gag binds to PIP2, and flips its own myristoylation into the inner leaflet of the plasma membrane, replacing the unsaturated fatty acids of PIP2.
  2. This drives lipid raft formation at the plasma membrane of the host cell.
  3. Gag multimerisation drives the assembly of the virus particle.
24
Q

State the name of three detergents

A

Triton-X100, SDS, CHAPS

25
Q

Describe the differences between STED, SIM, and dSTORM

A

STED: confocal based technique
SIM & dSTORM: widefield/camera based

All 3 can visualise 3D and 2D specimens in multicolour
STED & SIM: live technique
dSTORM: fixed

26
Q

Describe the principle of using Super Resolution Microscopy to study lipid rafts

A

Super resolution microscopy allows objects to be distinguished as separate entities which otherwise could not be resolved by conventional microscopy techniques: essentially breaking the “diffraction limit”.

27
Q

State three super resolution microscopy techniques

A

STED
SIM
dSRORM

28
Q

What are the resolutions achieved with STED?

A

x axis: 50nm
y axis: 50 nm
z axis: 100 nm

29
Q

Using STED Microscopy, what could be elucidated regarding lipid rafts?

A

It was found that GPI anchored proteins and sphingomyelin were transiently trapped in a cholesterol dependent manner

30
Q

Describe how STED microscopy works

A
  1. Focused excitation light spot with a size of 250 nm on specimen
  2. Excited molecules in the outer circle are switched off using intense depletion light
  3. This leaves a focused region in the centre with excited light about 50nm in diameter
31
Q

Although DRMs are not wholly considered the same as lipid rafts, why is it useful to know what proteins are localised within this DRM fraction following solubilisation?

A

Proteins residing in DRMs reflect the high affinity preference it has for ordered membrane states, which are found in lipid rafts.

32
Q

Apart from just the plasma membrane, where else have lipid rafts been documented to be found?

A

In the lysosomes and Golgi apparatus

33
Q

Describe how Cholera B toxin subunit has been used to give evidence for lipid raft existence.

A

Fluorophores are bound to the Cholera B toxin subunit. This subunit binds extensively to the raft constituent ganglioside GM1.

By addition of Cholera to giant unilamellar vesicles (GUVs) and to plasma membrane spheres (PMS), phase partition can be observed. This separation is based on non-raft and raft areas.

34
Q

Simply state three functions of lipid rafts

A
  • T cell Receptor Signalling
  • HIV assembly
  • Intracellular Trafficking
35
Q

What are three flaws in using synthetic membranes to model lipid rafts?

A

Artificial membranes have lower concentrations of protein than biological membranes.

It is difficult to model membrane-cytoskeletal interactions in synthetic membranes.

Artificial membranes lack natural asymmetry.

36
Q

How much more cholesterol in enriched in the lipid rafts wrt the plasma membrane?

A

3-5 fold more

37
Q

How do we know lipid rafts are involved in T cell receptor signalling? State four pieces of evidence

A
  1. Cross linking of GPI anchored proteins leads to T-Cell Activation
  2. TCR Signalling proteins are localised to the DRM band
  3. If palmitoylation sites were mutated, this caused solubilisation and failure to signal
  4. If cholesterol was depleted, this also led to signal failure
38
Q

Using lipid rafts, how do T-cell receptors signal?

A
  1. T-cell receptors in the resting state form nanoscale assemblies with lipid rafts
  2. These nanoscale rafts may coalesce into larger rafts following activation by an antigen presenting cell
  3. This promotes signalling by raft associated proteins i.e. Lat and Lck

Cellular Signalling (5 decks)

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