CNP 209 Molecular Cell Biology Membrane Structure

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CNP 209 - Molecular Cell Biology - Membrane
Structure
Basic model of membrane structure. Diversity
of lipid classes. Membrane fluidity. Membrane
domains. Membrane fusion.
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Basic Model of Membrane Structure
Singer and Nicolson (1972) Fluid mosaic model
Lipids are fluid, i.e. free to move in two
dimensions. lateral diffusion (107/sec), rotate,
flex, bob, flip-flop 70-80 of membrane
proteins are integral rest peripheral.
Compare with Danielli-Davson (1920s) - lipids
sandwiched between monolayers of protein. Rigid,
static.
DD not thermodynamically favorable. Lipids
and proteins must orient based on thermodynamic
principles Amphipathic. PL spontaneously form
bilayers / proteins fold.
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Rigid to Fluid to Fluid with confined regions
Jacobson, Sheets and Simson (1995) Reevaluates
fluid mosaic model because know that not all
proteins are floating aimlessly in the membrane -
random diffusion (as predicted by SN).
directed movement, i.e. By cytoplasmic domains
interacting w cytoskeletal motors. confinement
by obstacles, i.e. anchored or trapped by
anchored proteins.
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Jacobson, Sheets and Simson (1995) Evidence for
confined proteins FRAP (fluor recovery after
photobleaching) measures lateral mobility. Zap
with laser light, measure return of fluor, calc
fraction of immobile vs. mobile proteins.
Single particle tracking traces movement of
individual proteins, I.e. with fluor-antibody
against one protein. Distinguish between
random/non-random movement. Optical trapping.
Label protein with bead, trap bead with laser
light and drag across cell. Distinguish between
weak/strong barriers by varying strength of
trapping force. Evidence for lipid domains
Lipid-anchored proteins exhibit confined
movement. Egg yolk PtdCho (lecithin)
spontaneously separates into domains (meas by
X-ray diffraction). Energy to create lateral
domain tiny.
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Diversity of Membrane Lipids
1. Classes of membrane lipids - How many
different lipids are in a eukaryotic cell?
Glycerol-based phospholipids. Fatty acids
attached to sn 1 usually saturated
(sterochemical number) sn 2 usually
unsaturated (influences packing) DAG P PA
choline PtdCho Sphingolipids /
glycosphingolipids sphingosine FA ceramide
choline SM sugar glycolipids
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Diversity of Membrane Lipids
2. Lipid composition of subcellular organelles
from rat liver Phospholipid ER Golgi PM Lyso Mi
to PtdCho 59.6 45.3 43.1 41.9 45 PtdIns 10.1
8.7 6.5 5.9 5 PtdSer 3.5 4.2 3.7 -
1 PtdEtn 20.0 17.0 20.5 20.5 36 Cardiolipin
1.2 - - - 12 Sphingomyelin 2.4 12.3 23.1 16.0
2 Chol/PL 0.07 0.15 0.76 0.49 0.10 Percentage of
total PL phosphorus Table 1. Voelker (1996) Vance
and Vance, Chapter 15. Functional difference
between mito and pl membr?
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Diversity of Membrane Lipids
3. Fatty acid composition of lipids -
FA SM PtdCho PtdEtn 160 23.6 31.2 12.9 180 5
.7 11.8 11.5 181 18.9 18.1 182 22.8 7.1 2
00 1.9 203 - 1.9 1.5 220 9.5 1.9 1.5 20
4 1.4 6.7 23.7 230 2.0 240 22.8 224
- 7.5 241 24.0 225 - 4.3
GC analysis of RBC PL, weight percentage of
total Table 1. Culllis, Fenske and Hope (1996)
Vance and Vance Chapter 1.
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Diversity of Membrane Lipids
4. Asymmetric distribution of plasma membrane
lipids - Only know about plasma membrane. Why?
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Diversity of Membrane Lipids
5. Lipid shape - Determined by size of
polar/apolar regions, head gr hydration,
charge Double chain amphiphiles form bilayers
spontaneously cylinders PtdCho, PtdSer,
PtdIns, SM Single chain amphiphiles form
micelles inverted cones Lysophospholipids,
detergents Non-bilayer forming double chain
amphiphiles cones PtdEtn, Cardiolipin Ca2,
PA Ca2, PtdSer at pHlt4
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Membrane Fluidity
At physiological temp, biological membranes are
fluid. Fluidity proportional to
rotation/lateral diffusion inversely
proportional to order or viscosity. At low
temp, artificial lipid bilayer of single PL type
will be rigid or gel-like. Raise temp, changes
to liquid-crystalline at sharp phase transition
temp.
PtdCho Temp 120 120 - 1oC 140 140 23 160
160 41 161 161 - 36 180 180 54 181
181 - 20 Head groups make little difference
Biological membranes have mix of PL, broad
phase transition
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Membrane Fluidity
Cholesterol content affects membrane fluidity -
prevents ordered phase.
ER PtdCho gt PtdEtn gt PtdIns SM and chol
poor. Thin. PM PtdCho gt SM gt PtdEth gt PtdIns
SM and chol rich. Thicker. Outer leaflet of
plasma membrane has a high content of SM, which
has a high phase transition temp. SM causes pl
membr to be less fluid cholesterol fluidizes
SM-rich membrane, causes relative disorder.
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Membrane Domains
Polarized cells epithelia, neurons,
hepatocytes. Epithelial cells (vant Hof and
van Meer, 1994) Protective apical side faces
external world (skin) or digestive tract
(intestine), whereas basolateral side faces
neighboring cells or tissue.
GSL 33 mole PL 33 (PtdCho 8/SM 3) Chol
33 All GPI-anchored proteins Lipids in outer
leaflet cannot diffuse past tight
junctions. GSL 8 PL 66 (PtdCho 34/SM 7) Chol 26
Apical
Basolateral
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Membrane Domains
Neurons (Kobayashi, 1992) Cultured hippocampal
cells. GSL targeted to axons (like apical).
Infect cells with fowl plague virus. Viral
protein expressed on axons. Make liposomes of
fluorescent lipids influenza hemagglutinin.
Liposomes bind to viral protein on axon, fuse,
lights up axonal membrane. Lipids do not
diffuse to cell body.
Functional barrier to diffusion on outer and
inner leaflet between cell body and axon. What
is the physical barrier?
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Membrane Domains
Viral budding (Luan, Yang and Glaser, 1995) VSV
buds from host plasma membrane. Always thought
was random nip of PM. One of first methods to
analyze lipid composition of non-RBC PM. But VSV
membrane is enriched in PtdSer, PA, SM, chol
depleted in PC. Do viral proteins (G and M)
influence lipid domains as virus buds?
Patching prepare LUV of PtdCho dansylPA
diffuse add G patch add M patch prepare
LUV of PtdCho dansylPtdSer diffuse add G
diffuse add M patch patching of NBD-SM
requires PA G M Fluorescence resonance
energy transfer prepare LUV of PtdCho, PA, SM,
dansyl-SM and G (tryptophan) no energy transfer
from Trp to dansyl-SM add M see energy
transfer
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Membrane Domains
Excluding prepare LUV of dansylPtdCho
NBD-PA or NBD-PtdSer
or NBD-SM add G M dansyl and NBD patch
separately (PtdCho excluded)
Patching prepare LUV of PtdCho, PA, SM,
NBD-chol NBD-chol diffuse add G or M
diffuse add G M patches leave out SM or PA
diffuse
Does NBD-chol behave like chol does? What
about dansylPtdCho NBD-PtdCho
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Membrane Domains
Detergent resistant membranes / Rafts /
Caveolae Separate domains exist in biological
membranes (Brown and London, 1998) Solubilize
cells with cold non-ionic detergent (TX-100).
Large chunks of membrane not dissolved. Could
comprise gt 50 of PM area. Consist of GSL, SM,
chol, GPI-linked and acylated proteins.
Isolation - detergent vs. density DRMs vs.
caveolae vs. rafts Many signal transduction
molecules in cav
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Membrane Budding and Fusion
How does fusion occur? energetically
unfavorable does not happen spontaneously a
departure from normal bilayer structure must be
induced How does directed fusion occur? ER
vesicles fuse with Golgi, not plasma membrane or
mitochondria
Example Exocytic vesicle fusing with pl membr.
PtdSer/PtdEtn rich membranes hydrate poorly, may
allow close apposition of membr. postulated to
involve fusogens, which induce HII phase, disrupt
bilayer. Annexins - assoc w membr surface,
self-assoc w apposing annexin. Forms stable
aggregate, still needs fusogen, i.e. arachidonic
acid (204).
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Membrane Budding and Fusion
Burger - Greasing Membrane Fusion and Fission
Machineries Traffic 2000 1605-613 Stalk
model for intermediate stages during membrane
fusion adhesion / close apposition repulsive
force from water bound to PL head groups vs.
attractive force between hydrocarbon membrane
interiors. introduction of defects in membrane
packing, due to curvature, change in lipid
composition, insertion of stretch of
hydrophobic amino acids. semifusion, cis
monolayers continuous, trans monolayers
separate. stalk develops into trans monolayer
contact. pore flickering, then irreversible
expansion.
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Membrane Budding and Fusion
Role of lipids in membrane fusion fusion
stimulated by cis-unsaturated fatty acids
(cone) fusion inhibited by lysoPL (inverted
cone) fusion inhibited by inhibitors of
phospholipase-A2
stimulate
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Membrane Budding and Fusion
Role of lipids in membrane fission Fission
requires strong membrane bending and highly
constricted neck. Usually involves coat proteins
on cytoplasmic (trans) leaflet. Membrane
deformation driven by membrane microdomains
(A-1). lipid translocation (A-2). Lipids flex,
dont stretch. phospholipase action (A-3). DAG
and ceramide flip-flop is rapid. lipid transfer
proteins (A-4). lipid exchange (A-5). Global
difference in lateral pressure could drive local
bending with membrane microdomains acting as
nucleation sites.
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Other sources for lecture material Biochemistry
of Lipids, Lipoproteins and Membranes. 1996.
Vance and Vance, eds. Singer and Nicolson (1972)
The fluid mosaic model of the structure of cell
membranes. Science 175 720-731.
Jacobson, Sheets and Simson (1995) Revisiting
the fluid mosaic model of membranes. Science
268 1441-1442. vant Hof and van Meer
(1994) Lipid polarity and sorting in epithelial
cells. Current Topics in Membranes 40
539-563. Kobayashi, Storrie, Simons and
Dotti (1992) A functional barrier to movement of
lipids in polarized neurons. Nature 359
647-650. Luan, Yang, and Glaser (1995)
Formation of membrane domains created during the
budding of vesicular stomatitis virus. A model
for selective lipid and protein sorting in
biological membranes. Biochemistry 34
9874-9883. Brown and London (1998)
Structure of detergent-resistant membrane
domains Does phase separation occur in
biological membranes? Biochem. Biophys. Res.
Comm. 240 1-7. Burger (2000) Greasing
Membrane Fusion and Fission Machineries. Traffic
1605-613