Transcript BIO 402/502 Advanced Cell & Developmental Biology

BIO 402/502 Advanced
Cell & Developmental
Biology I
Section 1: Dr. Berezney
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…
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)
DPPC bilayer
Below (Tc)
Crystalline Gel
[~ 43 Ǻ wide]
•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
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
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 1030x 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