1. BIO 402/502 Advanced Cell & Developmental Biology I
Section 1: Dr. Berezney

2. Membrane Organization & Dynamics
Lecture 5
Membrane Organization & Dynamics

3. MEMBRANE ORGANIZATION
Fluid Mosaic Model – is the basic paradigm for the organization and dynamics of biological membranes. Core structure of biological membranes is the phospholipid bilayer (“membrane bilayer”).
Trifold Concept of: Membrane Amphipathy, Membrane Fluidity and Membrane Asymmetry

4. Membrane organization contd…
Bilayer organization is a consequence of the amphiphatic nature of phospholipids (PLPs)
Glycerol based PLPs are a modification of triglycerides in which one of the three fatty acid chains attached to the glycerol backbone is replaced by a polar headgroup consisting of phosphate and another polar moiety.
Fluid state of the lipid bilayer is critical for membrane function
Fluid lipid bilayer
Rigid lipid bilayer

5. Liposomes
Preparation involves sonication treatment of lipids in aqeous solution
Liposomes are in vitro lipid bilayer vesicles
Membrane proteins can be incorporated into the liposomal vesicles. This enables direct testing of the properties of specific membrane proteins in an artificially created lipid bilayer membrane.
Inserting proteins into liposomes

6. Fluidity of the Lipid Membrane
At physiological temperatures the lipids in the membrane bilayer are very dynamic exhibiting vibrational , rotational (10-9 sec), lateral movement (10-6 sec) & “flip-flop” (105 sec or every 28 hr).

7. Lipid Membrane Fluidity contd… Crystalline Gel [~ 43 Ǻ wide]
Transition temperature
(Tc) is the temperature at
which the transition from a
crystalline gel-like phase
to a liquid crystalline phase
occurs (similar to a
transition from solid to a
liquid phase)
The (Tc) is characteristic of
each lipid mixture.
Tc is measured with a
differential scanning
calorimeter that measures
the differential change in
enthalpy as a function of
temperature.
DPPC bilayer
Below (Tc)
Crystalline Gel [~ 43 Ǻ wide]
DPPC bilayer
Above (Tc)
Liquid
Crystalline Gel
[~ 38 Ǻ wide]

8. Lipid Membrane Fluidity contd…
The (Tc) increases proportionally at the chain length of fatty acids increase
Introduction of double bonds in fatty acids (unsaturation) greatly depresses (Tc).
DSC
Profiles

9. Effect of cholesterol on Tc for DPPC bilayer
Cholesterol as the phase transition “Abolisher”
Cholesterol is a major lipid of
the cell surface.
Composed of a 4 member
hydrocarbon ring structure and
a tail that inserts into the lipid
layer and a hydroxyl group on
the peripheral ring which
interacts with the polar head
groups of the PLP’s.
Cholesterol with its rigid
steroid structure is a disrupter
of cooperative effects of the
fatty acid chains in the bilayer
and therefore below the phase
transition it makes the
PLP layer more fluid and
above it, it makes the PLP layer
less fluid.
Structure of cholesterol
Effect of cholesterol on Tc for DPPC bilayer

10. Membrane Proteins Protein Amphipathy Protein Asymmetry
Protein Mobility
Membrane proteins are asymmetrically arranged with respect to the two membrane sides. This is absolute and enables distinctive functions to occur on the two sides as well as across the membrane
Peripheral proteins are found along the surfaces of the membranes either by direct interaction with the polar head of lipids or with other membrane proteins

11. Membrane proteins contd…
Integral transmembrane proteins contain membrane spanning domains of ~ 22 hydrophobic rich a.a sequences that typically form alpha helices. 2 or more alpha helices form coiled-coil structures.
They can also form beta strand barrels where the polar a.a. point inward into a central channel and non polar a.a. point towards the nonpolar lipid bilayer.
Thus they form polar lined channels that penetrate through the lipid bilayer membrane (e.g., porin complexes of E.coli).
Porin monomer

12. Membrane Proteins contd…
Lipid anchored proteins (a) Glycolipid covalent attachment by glycophosphatidylinositol (GPI anchored proteins) (b) Covalent attachment of the protein to fatty acid like myristic acid or palmitic acid or the prenyl group (15- C franesyl hydrocarbons with repeating vinyl groups).

13. Peripheral Proteins
Form extensive networks that line the surface of a membrane and may involve multiple interactions with other proteins that are integral, peripheral, lipid anchored or transmembrane associated.
Spectrin network on RBC surface
Nuclear lamina along the inner nuclear membrane
Phosynthetic center of Rhodopseudomonas viridis consists of a complex of three different transmembrane proteins and a peripheral cytochrome protein.

14. Membrane Protein Mobility
The Fyre-Edidin experiment showed mixing of mouse and human membrane proteins after cell fusion and the temperature (lipid fluidity) dependency of this translational movement and thus provided evidence for the fluid-mosaic model
Broad phase transition
membrane- cholesterol

15. Fluorescence Recovery After Photobleaching (FRAP):
measuring the diffusion rates of membrane proteins and lipids

16. Results of FRAP & Other Translational Movement Measurements
PROTEINS: 30-90% of the cell surface proteins are freely diffusing but the rate of this movement is 10-30x less than in after inserting the protein in a liposome
LIPIDS: Freely distribute over a distance of about 0.5 microns but severely limited after that suggesting the presence of lipid-rich and protein-rich domains in the membrane in which the protein-rich domains inhibit lipid diffusion. Also the protein-rich domains exhibit more limited diffusion. The existence of specifiic lipid domains is supported by the findings of “lipid rafts” which are enriched in spingomyelin and cholesterol and often concentrated in certain proteins.

17. FRAP and other measurements contd..
Patterns of movement of integral membrane proteins
Mobility of the membrane proteins is dependent on the bilayer fluidity (A), the degree of anchoring of proteins to an underlying: cytoskeleton (B), motor proteins (C), association with other membrane proteins (D) and/or microdomains in the membrane that restrict long range mobility (E) [e.g., “lipid rafts”], (F) extracellular matrix (ECM) components