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

Membrane Organization & Dynamics Lecture 5 Membrane Organization & Dynamics

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

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

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

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).

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]

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

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

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

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

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).

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.

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

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

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.

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