Membrane Phases Flashcards
1
Q
Lipid Raft
Definition
A
- the fluid membrane contains regions, or domains, of a less fluid, more stable phase in which some proteins are located, termed lipid rafts
- sub-domains in lipid bilayers rich in sphingomyelin, cholesterol and saturated lipids
2
Q
Lipid Raft
Function
A
- sites in the membrane where functional proteins tend to be located
- however some membrane proteins seem to be specifically excluded from the rafts
- biological function (and even existence) of lipid rafts in cell membranes is still a very controversial topic
3
Q
Membrane Melting
Definition
A
-melting processes are order transitions between two planar membrane phases or states that display different physical properties
4
Q
Lipid Chains at Low vs High Temperatures
A
- at low temperatures, lipid chains are ordered into an all-trans configuration
- at high temperatures these chains are disordered due to rotations around the C-C bonds within the lipid chains
- the membranes are in an ordered ‘gel’ phase at low temperatures while they are in a disordered ‘fluid phase’ at high temperatures
5
Q
Heat Exchange During Membrane Melting
A
- in the chain melting process, the molecules absorb heat (enthalpy) and the entropy increases due to the increase in the number of possible chain configurations
- the melting process is cooperative, the lipids do not melt independently of each other
- absorption of heat is typically monitored by measuring the heat capacity, cp=(dH/dT)|p
- at melting temperature Tm, is displays a pronounced maximum
6
Q
Lipid Membranes
Gel Phase
A
- high density
- highly ordered, extended chains
- low mobility
- area per molecule ~0.40nm²
- depth of bilayer ~5-5.5 nm
- stiff, solid
- long range translational and positional order
- some rotational modes allowed
7
Q
Lipid Membranes
Fluid Phase
A
- lower density
- well ordered, especially at chain ends
- higher mobility
- area per molecule ~0.5-0.67 nm²
- depth of bilayer ~4-5nm
8
Q
Lipid Phase in Real Cells
A
- the inside of a cell is not clear free flowing ‘bulk’ water, rather a very complex mix of proteins, ions, nucleic acids and sugars which also interact with the water as well as the bilayer
- little free water exists
- internal phase structure of the lamellar bilayer is probably affected
- the existence of stable raft domains in cells is still a controversial area
- however, single particle tracking has recently indicated that zones of high stability exist
9
Q
Head-Group vs Acyl Chain Interactions
A
- head-group interactions, they are hydrated / ionised species: steric interactions, dipolar and electrostatic interactions, H-bonding
- acyl chains interactions, they are homogeneous regions of hydrocarbon chains: van der Waals attractive and steric repulsive forces
10
Q
Single Component vs. Binary Phase Diagram
A
- single component: graph showing the phases of a substance as they relate to temperature and pressure
- binary: diagram showing the phases in a system of two pure components, one variable, T or P, must be fixed in order to render the diagram in 2D
11
Q
Lever Rule
A
-phase separation to:
a/(a+b) quantity of gel phase, composition B
b/(a+b) quantity of fluid phase, composition A
-if one segment is significantly shorter than the other then you must be near the phase boundary
12
Q
Direct Evidence for Phases in Model Sustems
A
- creating giant unilamellar veciles using electroformation from two different phospholipids, DPPC and DOPC
- using fluorescence microscopy, gel-liquid coexistence is observable
- if both co-existing phases are fluid then domains will coalesce eventually merging into only two domains, one of each phase, making up the entire vesicle
13
Q
Is it possible for two liquid phases Lo and Ld to coexist?
A
- in a binary system, no
- in a ternary system, i.e. add a third element, with cholesterol a phase transition between high and low temperatures is easy to observe
14
Q
Effect of Cholesterol on Lipid Phase Behaviour
A
- cholesterol induces ordering of lipid acyl tails but retains the liquid-like structure in the plane of the bilayer, a new liquid-ordered phase
- so we now have:
- -solid gel phase
- -liquid ordered phase
- -liquid disordered phase
15
Q
Liquid Ordered Phase
A
- slightly lower density than gel phase, ~0.42-0.5nm²
- less order
- not closely packed
- 5.5nm depth
- lower elastic modulus
- high viscosity liquid
- short range translational order
- no positional order
- highly viscous
16
Q
Liquid Disordered Phase
A
- low density, 0.5-0.75nm²
- no order
- not closely packed
- 4.5nm depth
- much lower elastic modulus
- low viscosity liquid
- liquid, no translational or positional order
- low viscosity
17
Q
Adding Cholesterol to the Solid Phase
A
- cholesterol acts to moderate the structural order in both solid and liquid phases
- it inserts into the closely packed saturated chains of the solid phase
- breaking down perfect order and pushing chains apart
- this weakens van der Waals interactions between adjacent chains enough to break down long range translational order
- it becomes an ordered liquid phase, Lo
18
Q
Adding Cholesterol to the Liquid, Lα/Ld, Phase
A
- cholesterol acts to moderate the structural order in both solid and liquid phases
- the planar cholesterol molecule brings a degree of order to the highly disordered hydrophobic core
- the properties of both phases converge resulting in liquid-liquid coexistence
19
Q
Ternary Phase Diagram of a Synthetic Model Cell Membrane
A
- triangle, each component of the system at a point on the triangle
- percentage is measured from 100% in the corner to 0% on the opposite edge for each component
- fixing temperature and pressure allows the diagram to be represented as a flat 2D triangle
20
Q
AFM on Supported Bilayers
A
- evidence for phases in model systems
- measures differences in height 0.2-1.4nm due to a difference in density between the phases
- in Ld phase, hydrophobic acyl tails are disordered so each lipid molecule occupies more area laterally and is therefore thinner in the vertical plane
- the more ordered Lo or gel domains occupy less area per lipid molecule in the membrane and hence are deeper and protrude from the Ld phase
21
Q
What are the driving forces for phase separation into Lo and Ld phases, they are both liquid, why do they not mix?
A
- the shape factor, if the two components have different shape factors
- head group hydrogen bonding between cholesterol and sphingomyelins
22
Q
Is a binary or ternary lipid system at equilibrium when in the process of separating, or even long after the structure has seemingly stabilised?
A
- often NO
- the derivation behing phase diagrams assumes that phase boundaries do not contribute to the free energy but actually, the interface between two boundaries always carries a free energy contribution
- in a 2D membrane if domains of finite size exist, these contributions can be quite large
- sometimes the domains can become kinetically trapped freezing them in an equilibrium state
- this could be due to high membrane protein content, hindering domains from coalescing or by proximity of the cytoskeleton
23
Q
Critical Points
A
- near the critical point, the physical / chemical difference between the phases is very small
- tension ar boudaries is therefore reduced and phase domains become more fractal in appearance
- at the critical point, the phases are either in the phase co-existence region or a single phase somewhere between Lo and Ld
- the system fluctuates from one to the other depending on small imbalances in temperature, up to microns in scale
- as composition moves away from critical point (or T increases) the size of fluctuations reduces to 100s then 10s of nm, this is the regime that cell membranes could possibly exist within
24
Q
Spinodal Decomposition
A
- if the temperature is reduced and the mixture moves from a single phase melt into a two-phase region and the boundary is crossed in the vicinity of the critical point (where the line tensions are low and boundary penalty is minimal) then the structure that forms will be spinodal in morphology (long, convoluted maze-like boundaries)
- over time this structure will develop as the larger domains absorb the smaller ones
25
Nucleation
- nucleation is a phase transition that is large in degree (compositional change) but small in extent (size) whereas spinal decomposition is small in degree but large in extent
- for nucleation, by contrast to phase separation by spinodal decomposition, the new phase must initiate with a composition that is not near that of the parent phase
- after the nucleus forms, the new phase grows - nucleation and growth
26
Nucleation and Growth of 2 Liquid Phases Far From the Critical Point
- the composition of the two phases are distinct and they have different physical properties, in particular depth (AFM)
- the thinner Ld phase exposes some hydrophobic tails of the thicker Lo (hydrophobic mismatch) which leads to a high line tension around the domain perimeter
- with fluid domains this always results in highly circular domains
27
Nucleation and Growth vs Temperature Cooling Rate in a Binary Lipid Mixture (Solid Domains in a Ld Phase)
- lipid molecule diffusion limits domain growth rate hence controlling the size and distribution of domains
- the faster you move into a region of phase separation, or the deeper into a phase co-existence region you move (the co-existing phases become more different, so the driving force for separation becomes stronger) the less time the molecules have to move into their new equilibrium position
- hence many new domains nucleate and they are small
28
Rules Governing Domain Morphology
- if the temperature quench first passes through a binodal region nucleation and growth will take place (as long as the cooling rate is slow enough that you have not plunged into the spinodal region immediately)
- to reach the spinodal and achieve spinodal decomposition the sample must pass through the critical point, or quench very rapidly through the binodal and spinodal line
29
Critical Fluctuations in the Single Phase Just Above the Critical Point
- could these temporary short-lived fluctuations be the origin of real lipid rafts in cell membranes, perhaps stabilised and coralled by the membrane proteins and cytoskeleton?
- at 34-37'C, the 2 phase region has shrunk to the bottom right leaving a 'raft' composition in the single phase region
30
Do rafts exist in a real biological membrane?
- what size/shape are they?
- are they equilibrium phase separated domains or something else, kinetically trapped? fluctuations?
- very hard to detect as there are so many confounding signals and they may be extremely small (<100nm) below the optical diffraction limit
31
Fluorescence Correlation Spectroscopy (FCS)
- measures the fluctuation in fluorescent intensity using a temporal autocorrelation function
- measured in a small spot, defined by the optical microscope, the fluorescent molecule concentration fluctuation caused by Brownian motion, diffusion and/or chemical reactions
- if volume is too large technique won't work as the signal is too diffused and spatially averages (not fluctuating)
- used to measure diffusion within domains as long as they are above the size of the pointspread, otherwise will be an average
32
Image Correlation Spectroscopy (ICS)
- correlating multiple points or even whole images
- used when diffusion is too slow (i.e. supported membranes)
- will be able to tell if bilayer is intact, if there is free diffusion, perhaps distinguish difference in diffusion within domain
- if nanoparticles are fluorescently labelled, could measure motion of these
33
NMR
- the chemical shift and splitting of characteristic lines associated with the acyl chains give an indication of order parameter, local chemistry and proximity of neighbouring molecules
- very powerful
- difficult to interpret complex signals
34
Forster Resonance Energy Transfer
- measure of distance between two types of labelled molecule
- use two different fluorophores, one a donor and the other an acceptor
- if acceptor is very close in the near field it will pick up the resonance energy of the donor fluorophore (a dipole-dipole coupling)
- this resonance transfer falls off with an inverse 6th power law so can be used as a molecular ruler, the signal falling away within a few nms
- can be used to determine proximity of different lipids to one another
35
Differential Scanning Calorimetry
- calorimeters measure enthalpy changes in a closed system, hence can study the Tm of various liquids and their mixtures
- then able to construct phase diagrams by performing temperature ramps
- quantitative indication of mixing via analysis of position and breadth of Tm
36
Evidence for Lipid Rafts in Real Cells
1. quantum dots for live cell imaging of raft micro-domains
2. FRET
3. nanoparticles tracking to nm resolution optically by detection of blurred gaussian spot
4. AFM (new)
5. the future - super resolution optical (STED/STORM)
37
Domain Size Data
-all experiments to date indicate that domains in phase separated model systems are large, >1μm, but in the limited data available from real cells, rafts are small <100nm perhaps even as small as 10nm
38
What is the reason for the discrepancy in domain size?
1. although water is in excess in model systems, it might not be quite so abundant in real cells due to concentration of proteins, ions, sugars and some types of lipid, hence lipid phase may be altered into structures more akin to a hydrogel
2. domains in live cells may arise from compositional fluctuations in a single phase in between the stable Lo and Ld phases, perhaps somewhere on the region of the critical point, these may be stabilised by protenis or cell filament anchors
3. small domains are kinetically trapped by high membrane protein content and proximity of the cytoskeleton, no opportunity for coalescence
4. domains are not equilibrium structures but are being constantly remodelled and reshaped by the dynamic action of the cell
5. all of the above!
