A guide to Biol.

102 lecture material:

There will often be questions asked on the slides.
Questions in red font will be discussed immediately
(frequent). Questions in red italic font are those that you
should be able to answer by the end of the topic under
discussion (less frequent). Pre lecture slide sets will
have the questions but not answers. I have inserted
blank slides for takingg notes on answered to these
questions. There may also be changes in slide
order/additions/deletions. The actual slides
shown/lecture will be posted immediately after lecture
on Classes
Slides with a star are for fun and background.

Figure 1.1

All living organisms descended from a common ancestral cell.

Environmental
extremists

There are 3 types of

cells, but Biol. 102 will
focus only on
eukaryotes.

This time
line is
beautifully
explored
in Biol.
iol.
104

Although bacterial cytoplasm is often referred to as noncompartmentalized recent studies have shown that there
compartmentalized,
is a bacterial cytoskeleton and that loci on the bacterial
chromosome can be precisely localized relative to the axis
of the cell.

What type of electron micrograph is this and how was the

specimen prepared?

Organelle compartments
whose membrane and
lumenal proteins are
synthesized by RER are
highlighted

Figure 1-30 Molecular Biology of the Cell, Fifth Edition ( Garland Science 2008)

Lecture Unit 1:
The structure, physical properties
and composition of biological
membranes:
Note that the topic order, lipid bilayer,
then membrane proteins is different than
the presentataion in Karp
Karp.

The fluid mosaic model for biological membranes: A lipid

bilayer within which integral membrane proteins are inserted
into and through the bilayer,
bilayer but unless restrained,
restrained free to move
within the plane of the membrane.

Figure 10.1 Fluid mosaic model of biomembranes.

These are actin filaments;

g about this
what is wrong
depiction?

Structural organization of a lipid bilayer

Experimental evidence for the lipid bilayer:

1. Measurement of lipid content as a function of
membrane surface area:
2. Electron microscopy of OsO4 fixed biological
membranes:
3. X ray diffraction of ordered bilayer arrays.

Gorter and Grendel experiment

p
suggesting the plasma membrane
is made of a lipid bilayer:
1. Calculate surface area of a red
blood cell (why use RBCs?)
2. Extract lipids from a known
number of RBCs using an
organic solvent(How do you
determine number of RBC
used?)
3. Form a lipid monolayer on a
water trough (termed
Langmuir trough)
4. Maximally compress monolayer
with sliding barrier.
5. Measure surface area of
monolayer

Surface area of
monolayer/ surface
area of nRBCs= 2

What type of
micrograph is this?

Note slide for red question

Thin section TEM of OsO4 fixed red blood cell revealing

trilaminar appearance
pp
of the p
plasma membrane

Myelinated membranes surround and insulate axons.

These multilayer sheets of plasma membrane are highly
ordered
d d and
d can b
be purified,
ifi d oriented
i t d by
b sedimentation
di
t ti
and used for X ray diffraction.

X ray diffraction of
myelin sheath
membranes revealed
evenly spaced regions of
high (extracellular
protein) and low (fatty
acid chains of bilayer)
electron density; the
dimension of the low
density region consistent
with a bilayer
arrangement of the fatty
acid tails.

Types of membrane lipids:

phospholipids
sphingomyelin
glycolipids
cholesterol (or other steroid lipids)
pPhosphatidyl choline

Double bonds in tails inhibits close lateral packing

Net neg. charge

Net
charge

Examples of
gy p
glycolipids;
these reside in
the outer leaflet
of the bilayer,
bilayer
often in lipid
rafts.

Cholesterol:

Increases fluidity where hydrophobic tails are

Decreases fluidity where hydrophilic heads are

Types and ratios of lipids present in biological

membranes
b
varies
i dramatically.
d
ti ll

How would you experimentally determine

the relative contributions of a given
g
membrane lipid to the physical properties
of biological membranes? (such as bilayer
fl d solute
fluidity,
l
permeability,
b l llipid
d mobility,
bl
bilayer thickness?)

Factors which affect bilayer fluidity:

lipid head group composition (I.e. head groups
can packk differently)
d ff
l )
Fatty acid chain length (long chains decrease)
Fatty acid chain saturation state (unsat. increases)
temperature (increases with increasing temp)
cholesterol concentration (both increases (inner
portion of leaflets and decreases(outer portion of
l fl ) fluidity
leaflet)
fl idi
What changes in a snapping turtles red blood cell
membranes might occur from September to
January? Assume the turtle lives in a pond in New
Hampshire.

High temp.

Low temp.

Experimental methods for assessing lipid mobility:

Very fast movements: Electron spin resonance measurements of
nitroxide spin labeled lipid analogs: rotation,
rotation flexural rigidity of fatty
acid tails, lipid head group and tail rotation.
~ Slow movements: Fluorescence recovery after photobleaching
(FRAP) of fluorescent lipid probes: determines rates of lateral
diffusion
Very slow movements: rate of lipase cleavage of inner leaflet
lipids; i.e. rate of flip flop

Electron spin resonance: Lipid mobility is measured by using

nitroxide electron spin labels covalently inserted at various
positions along the fatty acid tailsor to the head group.

Classes of lipid mobility in membranes

Why is transbilayer
flip flop of membrane
lipids a rare event?

Fluorescent Probes and Photobleaching

High energy dose of blue
light (fluorescein) green
light (rhodamine) , induces
covalent changes in ring
structure, destroying
capacity to emit additional
photons of green light.
Termed photo bleaching.

Ring electrons
Ri
l
excited by blue
light, emit green
light as electrons
return to lower
energy state
Excited by
green light;
emits red
light.
light
Covalent linkage of
fluorochrome to lipid
head group; protein

FRAP analysis of lipid lateral diffusion

1. Label plasma
membrane with
fluorescently
tagged lipid.
2. Photo bleach a
defined area (at
arrow)
3. Measure rate of
fluorescence
recovery.

fluor. Intensity

Time

Retrograde/rear
ward bulk
membrane flow?
How would you
experimentally
i
t ll
determine if
there is bulk
rearward flow of
lipids and/or
membrane flow
in this moving
f b bl ?
fibroblast?

3 key functions for cholesterol

2
3. Cholesterol also helps stabilize bilayer in part by preventing
phase separation of lipids into lateral arrays of lipids with similar
freezing
g temperatures.
p
How would yyou experimentally
p
y
demonstrate that cholesterol is critical for plasma membrane
integrity?

Asymmetric properties of bilayer lipids:

transbilayer compositional and charge asymmetry
Lipid dependent variations in bilayer thickness
lateral compositional asymmetry

Asymmetric distribution of lipids in inner and outer

leaflet of the bilayer: e.g.
g red blood cell membrane

Negatively charged PS is
exclusively in the
cytoplasmic leaflet

Method for determining transbilayer asymmetry:

e g Vectorial modification of outer leaflet lipids:
e.g.

First determine total lipid composition of a membrane

preparation (e.g. rbc plasma membrane)

Take intact rbcs or right side out sealed membrane

vesicles and digest outside of membranes with various
lipases.(could also use other modes of chemical
modification as long as agents are not membrane
permeant )
permeant.)

Determine levels of digested/modified lipids

compared
p
to total membrane. ((done byy thin layer
y
chromatography)

Outside

Total lipids:
25% Blue lipid
25% Yellow lipid
31% Red lipid
19% orange
Add lipase cocktail to
suspension of intact cells or
sealed
l d membrane
b
vesicles
l to
digest outer leaflet lipids
Outer leaflet contains:
100% of total Blue lipid
50% of total Yellow lipid
40 % of total Red lipid
0% of total Orange lipid
Inner leaflet contains:
0% of total Blue,, 50% of total
Yellow, 60% total Red, and 100%
total Orange

Transbilayer lipid asymmetry

Glycolipids only
in outer leaflet
Some lipids are
f
found
d
predominantly
in one leaflet
Negatively charged PS
only in inner leaflet,
leading to charge
y
y
asymmetry

HC chain saturation can affect bilayer thickness

Bilayer thickness also varies with lipid composition.

Certain transmembrane proteins concentrate/laterally
segregate into membranes of a particular thickness.
What structural feature of a given single spanning
transmembrane protein might drive this segregation?

Examples of lateral asymmetry of bilayer lipids:

Lipid rafts: Glycolipids, sphingomyelin and cholesterol can be
concentrated in relatively detergent resistant patches of
membrane called lipid rafts. There is also selective lateral
sorting of membrane proteins into these rafts. Rafts are
l
largely
l defined
d fi d based
b d on biochemical
bi h i l studies;
t di iin vivo
i
demonstration of their presence is controversial.
Tight junction mediated segregation of outer leaflet lipids of
the apical and basolateral domains (to be discussed later)

Atomic force microscopic image of liposome outer surface with rafts of

sphingomyelin in a sea of phosphatidyl choline. Peaks are a GPI linked
membrane protein that concentrates in the rafts
rafts. See Fig.
Fig 4 24 in Karp.
Karp

Glycolipids and
GPI anchored
proteins
concentrate in
outer leaflet of
the raft, longer
chain fatty acid
tails
concentrate in
both leaflets.
Cholesterol (not
to scale) also
concentrates in
rafts.
f

Brush border of intestinal

epithelial cell and isolated BB
membrane
b
lipid
li id rafts.
ft

In vivo evidence for the existence of lipid rafts.

~ 50% of the apical
brush border
membrane of the
intestinal epithelial cell
consists of detergent
resistant lipid rafts.
Recent EM studies have
demonstrated that this
membrane
b
consists
i t off
~50% stripes of thick
and 50% thin
membrane. A raft
associated protein
(alkaline phosphatase)
also partitions into
stripes.
stripes

Rafts can serve as clustered platforms for signaling complexes

Structural and functional organization of proteins

in/on biological membranes:

Permeability properties of pure, protein free lipid bilayers

Classes of membrane proteins:

Integral: (1.) single, (2). multiple spanning; (3). Beta barrel pore proteins,
((4).partial
)p
spanning;
p
g ((5, 6)) lipid
p linked
Peripheral: bound to either (8) outer or inner (7) leaflet either through direct
lipid interactions (9) (e.g. to specific lipid patchese.g. PI lipids on inner leaflet)
or to the cytoplasmic or exoplasmic domain of integral proteins

Type 1

9. Peripheral protein bound via specific

i
interaction
i off its
i PH domains
d
i with
ih
phosphatidyl inositol (PI)lipid head groups:

Classes of membrane proteins:

Integral: (1.) single, (2). multiple spanning; (3). Beta barrel pore proteins,
((4).partial
)p
spanning;
p
g ((5, 6)) lipid
p linked
Peripheral: bound to either (8) outer or inner (7) leaflet either through direct
lipid interactions (9) (e.g. to specific lipid patchese.g. PI lipids on inner leaflet)
or to the cytoplasmic or exoplasmic domain of integral proteins
Type 2

9. Peripheral protein bound via specific

i
interaction
i off its
i PH domains
d
i with
ih
phosphatidyl inositol (PI)lipid head groups:

Classes of membrane proteins:

Integral: (1.) single, (2). multiple spanning; (3). Beta barrel pore proteins,
(4).partial spanning; (5, 6) lipid linked
Peripheral: bound to either (8) outer or inner (7) leaflet either through direct
lipid interactions (9) (e.g. to specific lipid patchese.g. PI lipids on inner leaflet)
or to the cytoplasmic or exoplasmic domain of integral proteins

9. Peripheral protein bound via specific

i
interaction
i off its
i PH domains
d
i with
ih
phosphatidyl inositol (PI)lipid head groups:

IInserted
d
during transit
through
RER/Golgi

Includes cytosolic proteins that

can be inserted and removed
posttranslationally

Figure 10.19 Anchoring of plasma-membrane proteins to the bilayer by covalently linked

hydrocarbon groups.

Review from 101:

Hydrophobic alpha
helix is the most
common membrane
spanning
p
g domain.
The spanning helix
is often flanked on
one or both sides of
the bilayer by
charged or polar
amino acids.

Helical transmembrane domains of multi spanners

often have polar residues oriented on one side of the
helix: What is the functional significance?
Hydrophobic
side chains

Polar side
chains

Copyright, , 2002, John Wiley & Son s, Inc.,

Karp/CE LL & M O LE C ULA R BIO LOG Y 3E

Figure 4.20

Hydrophobicity plots of the amino acid sequence of a single and

multiple helix spanning membrane proteins: Sequence analysis can
often predict topographical organization of membrane proteins
proteins:

There are a subset of generally membrane pore

forming proteins that span the membrane as beta
sheets folded into a beta barrel.

Anatomy of an
integral
membrane
protein: a)
extracellular/
lumenal
domain can be
glycosylated
and stabilized
by S S bonds.

The extracellular
surface of many
cells has a thick
sugar coating.
This lymphocyte
has been stained
with a
carbohydrate
stain
t i prior
i to
t
fixation and
embedment.
What are some
potential
functions for this
extracellular
sugar coat?

Experimental determination of
membrane protein
topography/organization within the
bilayer plane: Freeze Fracture TEM
Freeze fracture of biological
g
membranes: Rapid
p freeze
cells or tissue in lN2 chilled solvent (e.g. Freon;
isopentane): Why not use just lN2
1.

Place specimen on 150oC specimen stage in

vacuum evaporator.evacuate chamber

2.

Cleave frozen specimen with cooled microtome

blade; for etched samples, raise temp. to ~ 115oC to
sublime away ice to expose cytoplasmic structure
and and unfractured membrane surfaces;

3.

Shadow specimen with metal and carbon; thaw and

remove underlying tissue with bleach; view replica
in TEM

Freeze fracture analysis of the lateral topography of integral

membrane p
proteins:

Vacuum evaporator
for coating specimens
with
i h metall (e.g.
(
platinum) and/or
carbon. These
specimens
p
have been
shadowed from a
fixed angle. Better
resolution is obtained
if specimen is rotated
during shadowing.

Freeze
fracture
replica of
RBC
membrane

Thin section of
microvillar
membrane: no
info about
plasma
membrane
structure other
than thickness

Integrall
membrane
proteins are
randomlyy
distributed
along the
length of the
microvillus
i
ill
membrane
but

Membrane
proteins are
excluded
from the tips
of
microvilli
the site of
actin
filament
attachment
to the
membrane.
membrane

Changing the
organization of
the underlying
cytoskeleton
induces a
redistribution of
microvillar
membrane
proteins.
proteins

However, etching depth is

quite limited due to ice
crystal formation and
perturbation of subcellular
structure

Freeze
fractured red
cell and myelin
membranes.
Red cell
membrane
b
contains many
different types
of membrane
proteins.
Myelin
membrane
wrapped
around axons
are essentially
free of integral
membrane
proteins (acts
as cellular
electric
electric tape
tape.

Veryy rapid
p
freezing
prevents ice
crystal
formation,
formation
allowing
removal of
much more
water
revealing
cytoplasmic
structures
underneath.

Experimental analysis assessment of membrane

protein
t i lateral
l t l mobility,
bilit e.g.
redistribution of cell surface antigens after mouse
human cell fusion (Frye and Edidin)
lectin/antibody induced patching and capping of
cell surface proteins
FRAP//FLIP analysis of fluorescently tagged
membrane proteins
p

Why is the lateral movement of membrane proteins a

key functional property of biological membranes?

Cell fusion is
induced by
addition of
Sendai virus.

Frye and
Edidin,
1970

Fused
heterokaryons,
0 time

40 min
min.

Antibody or lectin
induced patching and
capping of cell
surface proteins
demonstrates
induced lateral
movement; Patching
is ATP independent;
capping requires ATP.
What does this
suggest re.
Mechanism?

Lectins are multivalent sugar binding proteins that bind to specific sugars on
glycoproteins;fluorescently tagged lectins can be used for both localization of specific types
of glycoproteins and as tags for FRAP and capping assays

Wheat germ agluttinin

Direct visualization of membrane p

protein mobilityy ((or lack
thereof) by FRAP and FLIP analysis:
methods for fluorescently tagging integral membrane
proteins:
a) monovalent fluorescent lectins (nonspecific) Why
monovalent?
l ?
b) Fluorescently tagged monovalent (FAB fragments)
antibodies to specific protein
c) Transfect cells with construct encoding GFP tagged
membrane protein (can be cytoplasmic or organellar
membrane protein). (we will discuss green fluorescent
protein tagged proteins later)

Fluorescence recovery after photobleaching

Example of restricted lateral

mobility: two different
populations of this
membrane protein, one
mobile, one immobile

One of the early

objections to FRAP
was that the laser
energy might cause
permanent damage
in the bleached
region.
i
TTo address
dd
this, FLIP was
introduced.
Fluorescence loss in photobleaching

Modes of
restricting
lateral
mobility

lateral clustering of
IMP
IMPs

ECM
binding/clustering of
IMPs

cytoskeletal
t k l t l restraint
t i t
of IMPs

Cell adhesion
mediated clustering of
receptors

Lateral segregation of
membrane proteins on surface
of mammalian sperm.
sperm Sperm
membranes are labeled with 3
different antibodies and then
visualized with a fluorescent
secondary antibody (this will
make sense in a week or so!)

A subset of the major membrane

proteins of the RBC are tethered
to the underlying membrane
cytoskeleton (or conversely, the
cytoskeleton is tethered to the
membrane via these interactions.

Cytoplasmic
surface of RBC
membrane

Protein names: not

important: Concept of
membrane cytoskeleton
tethering: important!

Negatively stained
image (????) of RBC
spectrin based
membrane skeleton.
You are looking
down on
cytoplasmic surface
of the membrane

Antibody mediated
patching/capping
kinetics is greatly
enhanced in cells
lacking spectrin, a
major component of
the membrane
skeleton:

Modes of
restricting
lateral
mobility

lateral clustering of
IMP
IMPs

ECM
binding/clustering
of IMPs

cytoskeletal
t k l t l
restraint of IMPs

Cell adhesion
mediated
clustering of
receptors

Freeze fracture image of gap junction. P and E faces are shown of a

hemi junction. Each unit is termed a connexon channel

Experimental characterization of integral membrane proteins:

1. Extract peripheral proteins with a combination of treatments that
differentially solubilize different classes of peripheral proteins:
e.g. High salt, low salt, high pH, low pH, chelation of divalent cations.
2 Extracted membranes
2.
membranes, enriched in integral membrane proteins are then
treated with a mild, nonionic detergent (many to choose from) to
solubilize membrane proteins, hopefully in their native state.
Membrane proteins can then be purified by methods discussed
previously.
3 Purified membrane proteins can be reconstituted into liposomes for
3.
functional studies.
SDS: detergent that is very denaturing
mild, nonionic detergent: polar part not sufficiently active to denature proteins

Outside

To determine what
proteins are p
p
peripherally
p
y
associated with the outer
surface of the plasma
membrane extract intact
membrane,
cells with low salt, or
high salt, or lower or
hi h pH
higher
H conditions.
di i
Only proteins on outside
of cell will be solubilzed.

Outside

To determine what
proteins are p
p
peripherally
p
y
associated with the outer
surface of the plasma
membrane extract intact
membrane,
cells with low salt, or
high salt, or lower or
hi h pH
higher
H conditions.
di i
Only proteins on outside
of cell will be solubilzed.
Collect extracted cells by
sedimentation.

Next to determine what

proteins are p
p
peripherally
p
y
associated with the inner
surface of plasma
membrane mechanically
membrane,
lyse the cells, wash out
cytosolic contents (by
sedimentation);
di
i ) cytosoll
will be in supernate;
membranes in the pellet.

Next to determine what

proteins are p
p
peripherally
p
y
associated with the inner
surface of plasma
membrane mechanically
membrane,
lyse the cells, wash out
cytosolic contents (by
sedimentation);
di
i ) cytosoll
will be in supernate;
membranes in the pellet

Then re extract lysed

membranes as before to
release peripheral
proteins tightly
associated with inner
membrane surface.
Collect extracted
membranes
b
b
by
sedimentation.
Membranes in the pellet,
extracted peripheral
proteins in supernate.

Next to determine what

proteins are p
p
peripherally
p
y
associated with the inner
surface of plasma
membrane mechanically
membrane,
lyse the cells, wash out
cytosolic contents (by
sedimentation);
di
i ) cytosoll
will be in supernate;
membranes in the pellet

Then re extract lysed

membranes as before to
release peripheral
proteins tightly
associated with inner
membrane surface.
Collect extracted
membranes
b
b
by
sedimentation.
Membranes in the pellet,
extracted peripheral
proteins in supernate.

To purify/analyze
p y/
y
integral membrane
proteins, dissolve
bilayer lipids with
non ionic
detergent

Detergent
shell

To purify/analyze
p y/
y
integral membrane
proteins, dissolve
bilayer lipids with
non ionic
detergent. Analyze
proteins
i solubilized
l bili d
(e.g. by gel
electrophoresis)
p
) or
reconstitute into
liposomes for
functional analysis
(e.g. pump or
channel activity)

NO!!

NO!!

Nonionic detergents are

used to isolate
membrane proteins in a
ti conformation.
native
f
ti
Hydrophobic portion of
detergents are shown in
yellow; polar/charged
portions shown in
orange.

reconstitution of an
integral membrane
protein into
liposomes:
p
solubilize with
nonionic detergent
add detergent
solubilized lipids
remove detergent
and hope that the
membrane protein
will properly insert
into the bilayer.