Cell

membrane, a fascinating supramolecular aggregate

Zsolt Török
Institute of Biochemistry
Laboratory of Molecular Stress Biology
email: tzsolt@brc.hu
Tel: +36 62 432038

University of Szeged, „Basic Course in Molecular Cell Biology” for PhD students
2nd semester, school year 2015-2016, February 9, 2016.
Cell membrane, a fascinating supramolecular aggregate
Zsolt Török
Institute of Biochemistry
Laboratory of Molecular Stress Biology
email: tzsolt@brc.hu
Tel: +36 62 432038

University of Szeged, „Basic Course in Molecular Cell Biology” for PhD students
2nd semester, school year 2015-2016, February 9, 2016.
 The fascinating membrane

• necessary for life: defines the cell

• protects, insulates every cell, and organelle
• organize the cell – mem-BRAIN
• very thin (4-10 nm)
• highly dynamic
• 50% of all proteins are membrane proteins
• 1-5% of annotated human proteins involved in
lipid biosynthesis
• big repertoire of membrane lipids (thousands of
lipid species)
Cell membrane, a fascinating supramolecular aggregate
Zsolt Török
Institute of Biochemistry
Laboratory of Molecular Stress Biology
email: tzsolt@brc.hu
Tel: +36 62 432038

University of Szeged, „Basic Course in Molecular Cell Biology” for PhD students
2nd semester, school year 2015-2016, February 9, 2016.
Origin of life (Abiogenezis)
Spontaneous generation
Clock of life
 Origin of life (Abiogenesis)
Self-organizing matter

• Primordial soup • Pizza theory

• Lipids store basic • The surface of membranes
information, could serve as an ideal
spontenaous environent
“division”
• primordial cell
Why membranes are needed ?
 What membranes should be able to do?

1. Non permeable wall to define interior

and exterior

2. Elastic (fluid)

3. Selective permeability barrier

Growth,
movements,
4. Ability to adopt to the environment shape changes

5. And many more ….

Membrane types:
•Outer(plasma) membrane
•Iner membranes
The structure of membranes
 History of the membrane model

1925: membrane is a double layer of lipids (Gortel

és Grendel, Hollandia)

1943: lipids are not enough, membrane proteins

should also be there (Danielli, Anglia)

1972: fluid mosaic model (Singer és Nicholson)

2000+: raft model

 Gortel and Grendel experiment, 1924

A lipids
= 2
A RBC

à Lipid bilayer
Langmuir dish
 Definition of fluid mosaic?

• fluid = movement occurs

• mosaic = small pieces fitted together
Fluid mosaic model of biological membranes
Lipids assemble into bilayers in which proteins form the active
components of signal transduction and transport.
First described in 1972 by Singer and Nicolson
Phospholipids are amphiphatic

Membrane lipids differ in:

- head group
- degree of backbone saturation
- length of backbone
- type of backbone
 Major phospholipids in animal cell membranes

Phosphoglycerides (PE, PS and PC) consist of a three-carbon glycerol backbone

linked to:
• Phosphate-head group
• Two long chain fatty acids
 Fatty acyl chains

stearic acid

oleic acid

linoleic acid

linolenic acid

arahidonic acid
Lipid Composition of Some Biological Membranes
 Sphingolipids: Another Major Class of Lipids Found
in Biological Membranes

Black = sphingosine
Black + red = a ceramide
Black + red + blue = a
sphingomyelin

Ceramide
Sphingosine = amino alcohol
with a long hydrocarbon
chain.

Ceramide = sphingosine with

a fatty acid linked by an
amide bond to the amine to
form an N-acyl chain.

Sphingomyelin = ceramides
with a phosphocholine head The myelin sheath that surrounds and
group. electrically insulates many nerve cell
axons is rich in sphingomyelin.
Glycosphingolipids

Cerebrosides = ceramides with a

single sugar residue as head
group.

Gangliosides = ceramides with

attached oligosaccharide as
head group containing at least
one sialic acid residue.
 Gangliosides

Gangliosides constitute a significant fraction (~6%) of brain lipids.

The ABO blood group antigens are also examples of gangliosides.
Cholesterol: The Third Major Class of Lipid in
Biological Membranes
Sterols
Cholesterol Is the Metabolic Precursor of Steroid
Hormones
 How do lipids behave in an aqueous environment?

Lipid bilayer

Lipids aggregate to hide their hydrophobic tail inside and expose

hydrophilic head to the outside (energetically favorable!)
 Lipid bilayers seal spontaneously

Spontaneous “sealing”

A sealed compartment is energetically the most favorable

Do all lipids form bilayers in aqueous environments?

The structure of a lipid determines its packaging in an aqueous

environment
SHAPE CONCEPT MODEL
 Biological membranes are fluid!

• Lipids form a two-dimensional fluid

– lipids are in constant motion

• Lipids:
– can rotate or diffuse laterally
– rarely flip-flop ( can maintain asymmetry)
Experimental Demonstration of Biological
Membrane Fluidity
Lipids are mobile

Lateral diffusion (10-7s)

Flip-flop
(10-3s)

Rotation (10-8s)

Fatty acid tails

lipid headgroups

Water
molecules
 Data for lateral movement of lipids?
Fluorescence Recovery After Photobleaching (FRAP)

•FRAP: Label membrane lipids or

proteins (shown here) with
fluorescent tag

•FRAP experiments can quantify

lateral movements of lipids and
proteins within the plasma membrane
 FRAP data for lateral movement of lipids within a
biomembrane leaflet

• Typical lipid molecule exchanges places with

its neighbors in its leaflet about 107 times /sec

• Lipids can diffuse several micrometers/sec (37°C)

– Membrane lipid could diffuse:
• length typical bacterial cell = 1 sec
• length typical animal cell = 20 sec

– Lipids found in membranes have

similar diffusion rates to
lipids found in olive oil!

http://www.aftouch-cuisine.com/images/produits/Huile-dOlive1.jpg
http://hsb.iitm.ac.in/~jm/ARCHIVES/Jan-
Feb04/articles_files/blood_files/lipid_bilayer.gif
 Fluidity of the membranes is critical for their
function

Fluidity allows for . . .

• fusion of membranes, i.e. fusion of vesicles with

organelles
• diffusion of new lipids and new proteins laterally, so they
are equally distributed
• diffusion of proteins and other molecules laterally across
the membrane in signaling/reactions
• proper separation of membranes during cell division
• Maintain elasticity of the cells
 What regulates membrane fluidity?
Key criterium:
How densely are the lipids packed?

Factors that affect lipid packing ratio:

1. length of the hydrocarbon tail
-lipids with shorter hydrocarbon chain interact less tightly with their
neighbors
(membrane is more fluid)

2. degree of saturation of the hydrocarbon chain

-double bonds in lipid tails add “kinks”, prevent tight packaging
(membrane is more fluid)

3. cholesterol content of membrane

-cholesterol prevents movement lipid tails
membrane less fluid
Cholesterol affects the fluidity of the membrane
•Platelike steroid rings interact with and partially immobilize neighbor hydrocarbon chains;
has a lipid ordering effect (for most lipids) so lipids are less fluid
•Result: cholesterol makes bilayer less deformable in this region, thereby decreasing the
permeability of membrane to small water-soluble molecules

Cholesterol affects the thickness of the membrane

•Membrane thickness depends on: lipid composition, degree of backbone saturation AND
cholesterol content
 Lipids are distributed asymmetrically in a membrane

•PC, SM found outer (exoplasmic) leaflet

•PE, PS found inner (cytosolic) leaflet

• Lateral diffusion of lipids occurs spontaneously and can be measured

• Lipid flip-flop from one leaflet to the opposite leaflet is energetically
unfavorable and does not occur spontaneously
(Flip-flop of lipids requires the help of flippases. Flippases control the distribution of
lipids in the bilayer and are critical for lipid asymmetry.)
Membrane asymmetry
 What is the purpose of lipid asymmetry?

1. Lipid distribution between bilayers

determines the curvature of a
membrane

2. Lipids regulate intracellular signaling events in the cell by selectively

recruiting certain proteins to a subcellular membrane.

3. Phospholipid flip-flop from inner to outer leaflet sends signals to neighbors.

- EXAMPLE: during apoptosis, PS is rapidly translocated to the outer
leaflet. PS in the outer leaflet signals to macrophages to phagocytose this
dying cell.
How is selective membrane permeability achieved?

Require protein-mediated
transport across the lipid
bilayer
Protein, Lipid and Carbohydrate Compositions of
Some Membranes
Proteins associated with lipid bilayer

1. Integral (transmembrane)
2. Lipid anchored
3. Peripheral
Integral membrane proteins are structurally and functionally
diverse

What is a necessary characteristic of a membrane protein?

 Protein domains that are inserted into the
membrane have to be hydrophobic

Typical membrane-embedded α-helix = 20-25 hydrophobic amino acids

 Hydropathy/Hydrophobicity Plots

Glycophorin A

Erythrocyte
glucose
transporter

Bacteriorhodopsin
Various ways in which membrane proteins associate
with the lipid bilayer
QUESTION: How do we
purify and study membrane
proteins?
ANSWER: By solubilizing
membrane proteins with a
mild detergent (detergents
are amphipathic too!)
 Membrane proteins can be glycosylated:
proteoglycans and glycoproteins

Plasma membrane proteins (and lipids) are often decorated

with carbohydrates (sugars) on the exoplasmic surface
 Functions of glycosylation?

• Protect cells from chemical and mechanical

damage
• Keep other cells at a distance
• Prevent undesirable cell-cell interactions
• Cell recognition processes
– Transient cell-cell adhesion processes such as:
• Sperm-egg interactions
• Blood clotting
• Inflammatory responses
 Lipid Rafts

Lipid rafts: small, specialized areas (microdomains) in membranes

where some lipids (SM), cholesterol and proteins are concentrated
•lipid rafts are resistant to non-ionic detergents (“mild” detergents such as
Triton X-100 )
•when such a detergent is added to cells, the fluid membrane will dissolve
while the lipid rafts may remain intact and can be extracted
Szfingomielin raftok (narancs) és a velük asszociálódó fehérjék (citrom) a
plazmamembrán hátterében (fekete)
 Certain proteins associated with cellular signaling
processes have been shown to associate with lipid rafts

•Transient accumulations of SM, cholesterol

•Raft regions are thicker than surrounding membrane regions, can better accommodate
certain “larger” membrane proteins
•RESULT: organization/accumulation of these “larger” proteins (cell-surface receptor
proteins, signaling proteins) facilitates detection of signals from the environment and
subsequent activation of cytosolic events
 Important terms/concepts

• Biological membranes consist of a lipid bilayer in which membrane

proteins are embedded (fluid mosaic model)

• The lipid bilayer is fluid, most lipids moving freely within a leaflet (data
from FRAP experiment). Certain lipids (SM) can be organized into
lipid rafts which appear to be involved in signaling.

• A biological membrane is asymmetric in respect to lipid and protein

composition. Bilayer asymmetry is important for cell function.

• Selective membrane permeability is mediated by integral

transmembrane proteins. Transmembrane proteins must contain
stretches of hydrophobic amino acids in order to traverse the
hydrophobic lipid tail region.

• Detergents, which are also amphipathic, can be used to study

membrane lipids and proteins.
Vesicular transport: many compartments,
interconnected by trafficking routes
Two Key Steps:
1. sorting during vesicle formation
2. targeting during vesicle fusion
Morphological Experiments Suggest Role of Vesicle
Coats

COP: coat protein complex

Each type acts at distinct locations
 Bulk transport across the plasma membrane occurs by
exocytosis and endocytosis

• Small molecules and water enter or leave the cell

through the lipid bilayer or by transport proteins
• Large molecules, such as polysaccharides and proteins,
cross the membrane via vesicles
 Exocytosis

• In exocytosis, transport vesicles migrate to the

membrane, fuse with it, and release their contents
• Many secretory cells use exocytosis to export their
products
• Three types of endocytosis:
– Phagocytosis (“cellular eating”): Cell engulfs particle
in a vacuole
– Pinocytosis (“cellular drinking”): Cell creates vesicle
around fluid
– Receptor-mediated endocytosis: Binding of ligands
to receptors triggers vesicle formation
 Phagocytosis EXTRACELLULAR
FLUID
Pseudopodium Solutes
of amoeba
Pseudopodium

Bacterium

1 mm
Food vacuole “Food”
or other
An amoeba engulfing a bacterium particle
via phagocytosis (TEM)

Food
vacuole

CYTOPLASM
 Pinocytosis

Plasma
membrane

0.25 mm
Coat
protein
Pinocytotic vesicles forming
(TEMs) Coated
pit

Coated
vesicle
 Receptor-Mediated
Endocytosis

Plasma Coat
membrane protein Receptor

0.25 mm

Top: A coated pit Bottom: A coated

vesicle forming during receptor-
mediated endocytosis (TEMs)
Experimental techniques
Preparation of Vesicles and Bilayers
The complexity of lipidome
Lipid analysis – GC-MS
• Thin Layer Chromatography (TLC) to separate lipid classes
• Analysis time– days
• Sample requirement (milligrams of protein equivalent)
(x10,000,000)

1.50 • Fatty acid composition of lipid classes

• Lipid classes composition

1.25

1.00

0.75

0.50

0.25

0.00
15:0 IS 16:0 18:0 18:1 n-9 20:4 n-6 22:6 n-3
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0
Lipidomics

Orbitrap Elite (Since 2012 September)

Shotgun lipidomics – Survey scan (Orbitrap)
• Great mass resolution →
lipid species with fatty
B16_conc_sor_1x_dneg_ acid RT:
#133-311 composition
1.91-4.34 AV: 179 NL: 2.34E6
T: FTMS - c NSI Full ms [400.00-1100.00]
742.5387 MS1, negative
PE(34:1) PE(36:2)
2200000

2000000

1800000 PE(P-34:1)
IS 700.5284
1600000 662.4764
1400000
Intensity

1200000
PI(36:1)
1000000
861.5490
800000 IS PG(36:2) 885.5488
PI(34:1)
619.4158 773.5330 PI(38:4)
PS(36:2)
600000
656.4249 788.5440
400000 794.5466
IS 835.5332
200000 591.4029
898.6980
0
600 650 700 750 800 850 900
m/z
Cellular stress response – and the lipidome

Principal component analysis (PCA)

PC3, mild vs severe heat

PC1, stress vs control PC2, BA vs heat

Stress results in significant, stressor specific alterations

in the lipidome

Balogh et al. BBA (2010)

Reconstitution of the Ca2+ Pump
Freeze Fracture
DSC
Monolayer: constant surface area

balance

Surface pressure
"Interaction"

Δπ

Time
 Infrared spectroscopy

2853.0

2852.5
CH2 stretch (cm )
-1

2852.0

2851.5
DMPS
2851.0 HSP17
AC
2850.5
ν

2850.0

2849.5
20 25 30 35 40 45 50
Temperature (°C) lipid / sHSP = 55
PNAS (2002) 99, 13504
 Heat induced alterations in the membrane
structure

Fiziológiás állapot „Hiperfluid" állapot Nem kettősréteg

szerkezet
Ultra-sensitive fluorescence microscopy
Mapping plasma membrane rafts

B16 cells were labeled with Bodipy-SM

Fast ultrasensitive detection of fluorescently labeled
membrane rafts by TIRF microscopy

10 µm

B16 cells labeled with fPEG-Cholesterol

 … zooming in
with high spatial resolution

10 µm

B16 cells labeled with fPEG-Cholesterol

 …. the details
by ultrasensitive fast fluorescence microscopy
10 µm

10 µm
 Raft dynamics

Real time(10 fps)

B16 cells, Bodipy-SM label

 TOCCSL – Thinning Out Clusters
while Conserving the Stoichiometry of Labelling

TIRF

Moertelmaier, Brameshuber et al., APL (2005)

Physiological temperature defines optimal membrane domain
structure

Dimer fractions of GPI-mGFP in CHO cells

0.4

0.3
dimer fraction (%)

0.2

0.1

0
25 30 35 40
temperature (°C)

37ºC
Single dye tracing with ultrasensitive fluorescence
microscopy

Model membrane GPI-GFP in CHO cells

 (Bottom view of the same GUV).

Coinducer promotes, or prevents disruption of domain

formation . POPC:SM:Ch (1:1:1)
Heat sensing membranes: rafts on the stormy sea

Gericault: Le Radeau de la Méduse (Raft of the Medusa)

Membrane-lipid therapy
Membrane-lipid therapy

LipidArt Ltd, 2014

Zsolt Török,
email: tzsolt@brc.hu