pathways. Functional links of the processes with human diseases will be touched upon.

The course
Outcomes
also will introduce advanced methodologies including various microscopy tools employed in modern
Syllabus cell biology research.

1. Methods used in cell biology: microscopy, cell sorting, fractionation of cellular components,
radioisotopes and antibodies as tools to study cellular functions. All light microscopy platforms
(while light and fluorescence) covering basic principles and applications. Fluorescence activated
cell sorting and radio-isotope/antibody based cellular biochemistry will include isotope based
cellular and molecular fractionation and different immunoblot platforms. [4-5]
2. Cell membrane: organization and composition of the cell membrane, structural property of the
membrane micro-domains. Details of compositions of the membranes of intracellular organelles
and plasma membrane and their properties; and the structural properties of the micro-domains
(lipid rafts etc.) of membranes. Understanding of the functional link of the compositional diversity
of the cell membrane (plasma membrane and intracellular membrane) to cellular processes
pertaining to the organelles and plasma membranes. [2-3]
3. Membrane transport- endocytosis and exocytosis Vesicular transport system and intracellular
trafficking, protein targeting. In depth understanding of the molecular pathways pertaining to
intra-cellular trafficking/transport and their mechanistic insights in model organisms from
unicellular yeast to animal cells, cellular methods/tools/approaches to study these processes.
[4-5]
4. Organelle biogenesis: Understanding the biogenesis of subcellular structures such as
mitochondria, centrosome, kinetochore in cells across eukaryotic kingdom, similarity/diversities
Syllabus in their composition, structural organization and functions. [2-3]
5. Components of the cytoskeleton and their regulations: organization and function of actin,
intermediate filaments, microtubules and motor proteins, integrins, cadherins. Compositions and
cellular/molecular properties of different types of cytoskeletal elements, studies on the
involvement of actin and microtubule cytoskeleton in intra-cellular trafficking, chromosome
organization and cell motility. Functions of actin and microtubule-based motor proteins in
regulating these processes, and the activation/inactivation of signaling molecules associated
with the processes. [4-5]
6. Cell-cell signaling: overview of extracellular signaling, cell surface receptors, cell signaling during
growth and differentiation. overview of different cell surface receptor-based signaling with
emphasis on receptor tyrosine kinase-mediated RAS signaling and its link to cell growth and
division. [4-5]
7. Cell cycle and its control: mechanisms of growth and division of eukaryotic cells, cell cycle
checkpoints. Understanding the molec a ce e /c e ha c ce ge i
to growth/DNA replication/genome segregation phases in eukaryotic cells, mechanisms
underlying activation/inactivation cell cycle check-points and their roles in controlling growth and
division of cells. [6-7]
8. Cell death: Apoptosis and autophagy pathways Canonical and non-canonical apoptosis
pathways, molecular pathways and cellular processes linked to autophagy. [2-3]
Membrane: the live fence
• Reference books:
Cell Biology by Karp
Cellular and Molecular Biology by G. Karp
Molecular Biology of the Cell by B. Alberts
Functions of Cell Membrane
• Compartmentalization
• Site for biochemical reactions
• Provide selectively permeable barrier
• Transport of molecules of various forms
• Respond to external stimuli
• Mediating cell-cell interaction
• Energy metabolism
Basic organization of Composition
 Discovery of Lipid Structure in plasma membrane R
–
117
O P O
O
HCH H

4.1 • Introduction to the Plasma Membrane

HC CH
O O
R
117
C O C O
–
HCH HCH O P O
HCH HCH O
HCH HCH
(b) HCH H

4.1 • Introduction to the Plasma Membrane

HCH HCH
HCH HCH HC CH
HCH HCH
Stationary Movable O O
HCH HCH
barrier barrier CH C O C O
Lipids HCH
CH
HCH
HCH HCH HCH
HCH
HCH HCH HCH
HCH HCH
HCH HCH HCH
(b) HCH
HCH HCH HCH HCH
HCH HCH
H
HCH HCH
H
(a) (c) HCH HCH
Experiment by Red blood cell lipid extraction by Gorter and Grendel
Stationary Movable HCH HCH
FIGURE 4.3 The plasma membrane contains a lipid bilayer. (a) Calculating the surface area of a lipid preparation. When a sample of phospholipids is
barrier barrier CH
Lipids
dissolved in an organic solvent, such as hexane, and spread over an aqueous surface, the phospholipid molecules form a layer over the water that is a single HCH
molecule thick: a monomolecular layer. The molecules in the layer are oriented with their hydrophilic groups bonded to the surface of the water and their CH
HCH
hydrophobic chains directed into the air. To estimate the surface area the lipids would cover if they were part of a membrane, the lipid molecules can be HCH
compressed into the smallest possible area by means of movable barriers. Using this type of apparatus, which is called a Langmuir trough after its inventor, HCH
HCH
Surface area of water covered by extracted lipids: surface area of cell
Gorter and Grendel concluded that red blood cells contained enough lipid to form a layer over their surface that was two molecules thick: a bilayer. (b) As
Gorter and Grendel first proposed, the core of a membrane contains a bimolecular layer of phospholipids oriented with their water‐soluble head groups
HCH HCH
HCH
= 2:1
facing the outer surfaces and their hydrophobic fatty acid tails facing the interior. The structures of the head groups are given in Figure 4.6a. (c) Simulation of
a fully hydrated lipid bilayer composed of the phospholipid phosphatidylcholine. Phospholipid head groups are purple, water molecules are blue, fatty acid
HCH
HCH HCH
chains are green. HCH HCH
SOURCE: (c ) Simulation of a fully hydrated lipid bilayer. Phospholipid head groups are shown in blue and orange, water molecules in red and white, and fatty H H
acid chains
(a)are green. (c)
The first proposal that cellular membranes might contain a lipid lipid bilayer. It was found, for example, that lipid solubility was not
FIGURE 4.3was
bilayer Themade
plasma membrane
in 1925 contains
by two Dutch scientists,a E.
lipid bilayer.
Gorter and (athe
) Calculating the surface
sole determining area
factor as to of a alipid
whether preparation.
substance When a sample of phospholipids is
could pene-
F. Grendel.
dissolved These researchers
in an organic solvent, extracted
such as thehexane,
lipid from human red blood
and spread over trate the plasmasurface,
an aqueous membrane. theSimilarly, the surface
phospholipid tensions of mem-
molecules form a layer over the water that is a single
cells and measured the amount of surface area the lipid would cover branes were calculated to be much lower than those of pure lipid
molecule thick:
when a monomolecular
spread layer.
over the surface of water The molecules
(FIGURE in the layerstructures.
4.3a). Because mature
are oriented with their
This decrease hydrophilic
in surface tension groups
could be bonded
explained to
by the surface of the water and their
hydrophobic
mammalianchains
reddirected
blood cellsinto
lack the
bothair. To and
nuclei estimate the surface
cytoplasmic orga- area the lipids
the presence would
of protein cover
in the if they were part of a membrane, the lipid molecules can be
membrane.
compressed into the smallest possible area by means of movable barriers. Using this type of form
nelles, the plasma membrane is the only lipid‐containing structure in This protein is present in the of individual
apparatus, whichprotein mole-a Langmuir trough after its inventor,
is called
the cell. Consequently, all of the lipids extracted from the cells can be cules and protein complexes that penetrate a fluid lipid bilayer
Gorter and Grendel concluded that red blood cells contained enough lipid to form a layer over their surface that was two molecules thick: a bilayer. (b) As
Lipid dynamics by fluorescent tagging
 Phospholipid
Peripheral
Surface tension of cell membrane found to be much lower than a simple bilayer!
α helix
protein

GURE 4.4 Plasma mebranes as lipid plus proteins. (a) A repr 118 Oligosaccharide Glycoproteins

lipid bilayer. The external surface of most membrane proteins,

118 Oligosaccharide Glycoproteins
Glycolipid
Glycolipid
CHAPTER 4 • The Structure and Function of the Plasma M
CHAPTER 4 • The Structure and Function of the Plasm

king them glycoproteins and glycolipids. Those portions of the

mposed of hydrophobic amino acids. The two leaflets of the bil Integral
Hydrophobic proteins

Molecular model of the membrane of a synaptic vesicle constru

Integral Phospholipid Cholesterol
Peripheral
Hydrophobic α helix
proteins Phospholipid Cholesterol
Peripheral protein
α helix
protein
(a) (b)

tive numbers obtained from the Proteins

analysis of purified synaptic ve
in synaptic vesicle membrane
(a)

FIGURE 4.4 Plasma

the lipid bilayer. as
mebranes The external
lipid surface of(amost
plus proteins. membrane proteins,
) A representation as wellmembrane
of the plasma
(b)
FIGURE 4.4 Plasma mebranes as lipid plus proteins. (a) A representation of the plasma membrane showing the organization of proteins embedded in
as a small percentage
showing theoforganization
the phospholipids, contain
of proteins short chains
embedded in of sugars,
Membrane Composition
• Phospholipids constituting the bilayer
• proteins interacting with “lipid bilayer”
• Sterols which stabilize the lipid bilayer
• Glycolipids
 HC O C R''
st, all
hasof focused
which are
hilic and hydropho-
on the apparent health benefits of two highly O
C O C R'''

4.2 • The Lipid Composition of Membranes

urated fatty phos-
brane lipids: acids (EPA and DHA) found at high concentration H2
Chemical composition of membrane
h oil. EPA and DHA contain five and six double bonds, respec- (a)
lipids O

and are incorporated primarily into PE and PC molecules of H H

ne lipids contain a O
npidsmembranes,
. Because most
most notably in the brain and retina.H CEPA and
O C (CH ) CH=CH(CH ) CH 2 27 27 3
Sphingosine HO CH2 C C CH CH (CH2)12CH3
O
are described
backbone, as omega‐3 fatty acids because their last
they are HC Odouble
C (CH ) CH=CH(CH ) CH 27 27 3
NH3 OH
+
O
isnlike
situated three carbons from
triglycerides, Dioleoyl the omega (CH3) end O P of
O CHthe fatty –
2 H H
ehain.
not amphipathic,
With fatty acid chains at one
phosphatidic acidend of the molecule
O
and a
–

Ceramide HO CH2 C C CH CH (CH2)12CH3

wo of the hydroxyl
head group at the other
; the third is esteri-
end, all of the phosphoglycerides
Phosphatidic
H
NH OH
acid O C R
t a distinct
any additionalamphipathic
sub- character. O
+
tty acyl chains, the Phosphatidyl- (CH ) N CH CH
33 2 2 HC O C R O H H
2
choline (lecithin) O +
virtually absent in Sphingomyelin (CH3)3N CH2 CH2 O P O CH2 C C CH CH (CH2)12CH3
glycerides have an A less Phosphatidyl-
INGOLIPIDS abundant class H N of membrane lipids,
+ HC O C R'
O –
3 CH CH 2 O NH OH
commonly either, are derivatives of sphingosine, an amino
tsphingolipids serine alcohol
–
COO O P O CH
–
2
O C R
O
ontains
nolaminea(forming
long hydrocarbon chain (Figure 4.6b). Sphingolipids
Phosphatidyl- +

phosphatidylserine , ethanolamine H N CH CH
t of sphingosine linked to(cephalin) a fatty acid (R of Figure 4.6b) by its
3 2 2
A cerebroside Gal Ceramide
PI). Each of these
owith
group.
the negatively a ceramide. The
This molecule is Phosphatidyl- H
various sphingosine‐
OH OH

lipids havewater‐
additional groups inositol esterified to OH the terminal alcohol
H H
ms a highly HO
H
H A ganglioside Ceramide
GalNAc Gal Glu
dsphingosine
the head group moiety.
. If the substitution His OH phosphorylcholine, (GM2)
PI have is
olecule an sphingomyelin
overall , which is the only phospholipid Oof the SiA
neutral. In contrast, (b)
brane that is not built with a glycerol backbone. If theH Csubstitu-
ched hydrocarbons
O C R
O
2

a acarbohydrate,
4.6 ). A membrane the molecule is a glycolipid. If the O carbohy-
HC O C R'

FIGURE 4.6 The chemical structures of membrane lipids. (a) The

is a simple
e bonds), sugar, the glycolipid
monoun- is called a cerebroside
Diphosphatidyl-
CH O P O2 ; if it is a
CH 2

olyunsaturated (i.e., that includes

O
–
structures of phosphoglycerides (see also Figure 2.22). (b) The structures of
cluster of sugars glycerolsialic acid, the glycolipid is called
HO C H –
O
hoglycerides often (cardiolipin) CH O P O CH sphingolipids. Sphingomyelin is a phospholipid; cerebrosides and
lioside . Hundreds of different gangliosides have been
acyl chain. Recent O
2
identified 2

gangliosides are glycolipids. A third membrane lipid is cholesterol, which is

HC O C R''
ferences
nefits in highly
of two their carbohydrate chains. Because all sphingolipids O shown in the next figure. (R = fatty acyl chain). [The green portion of each
two long, hydrophobic hydrocarbon chains at one HendO and
high concentration C O C R'''
2 lipid, which represents the hydrophobic tail(s) of the molecule, is actually
(a)
uble bonds,
rophilic region respec- at the other, they are also amphipathic and much longer than the hydrophilic head group (see Figure 4.23).]
nd PC molecules of H H
nd retina. EPA and Sphingosine HO CH C C CH CH (CH ) 2 12CH3

Book: Karp
2

se their last double NH OH

+ 3

CH3) end of the fatty

 Cholesterol

Glycolipid
Fluid-mosaic model

membrane were followed once the two membranes had become 133
Cell Exterior
Transverse diffusion (flip-flop) continuous. To follow the distribution of either the mouse mem-
5
(~10 sec) brane proteins or the human membrane proteins at various times
after fusion, antibodies against one or the other type of protein were

4.7 • The Dynamic Nature of the Plasma Me

prepared and covalently linked to fluorescent dyes. The antibodies
against the mouse proteins were complexed with a dye that fluo-
resces green and the antibodies against human proteins with one
Flex (~10-- 9sec)
that fluoresces red. When the antibodies were added to fused cells,
they bound to the human or mouse proteins and could be located
under a fluorescence light microscope (FIGURE 4.26a). At the time of
fusion, the plasma membrane appeared half human and half mouse;
Lateral shift
-- 6
that is, the two protein types remained segregated in their own hem-
(~10 sec)
isphere (step 3, Figure 4.26a,b). As the time after fusion increased,
Cytosol
the membrane proteins were seen to move laterally within the mem-
FIGURE 4.25 The possible movements of phospholipids in a brane into the opposite hemisphere. By about 40 minutes, each spe-
membrane. The types of movements in which membrane phospholipids cies’ proteins were uniformly distributed around the entire hybrid
cell membrane (step 4, Figure 4.26a). If the same experiment was
Lipid dynamics regulations
Lipid symmetry
 and Function of the Plasma Membrane
Nitroxide-labeled
cysteine residue

pH 3.5 Closed Hydrated Open

Structural Properties of membrane microdomain: permeability and fluidity pH 6.5 K+ ion

Cytoplasm

(a) (b)

FIGURE 4.22 Use of EPR spectroscopy to monitor changes in conformation of a bacterial K+ ion channel as it opens and closes. (a) EPR spectra from
nitroxides that have been attached to cysteine residues near the cytoplasmic end of the four transmembrane helices that line the channel. The cysteine
residue in each helix replaces a glycine residue that is normally at that position. The shapes of the spectra depend on the distances between unpaired
electrons in the nitroxides on different subunits. (Nitroxides are described as “spin‐labels,” and this technique is known as site‐directed spin labeling.)
(b) A highly schematic model of the ion channel in the open and closed states based on the data from part a. Opening of the channel is accompanied by
the movement of the four nitroxide groups apart from one another.
SOURCE: (a) Reprinted by permission from Macmillan Publishers Ltd: From E. Perozo et al., Nature Struct. Biol. 5:468, 1998.

Transition temperature of lipids: Temperature at which lipid can be
converted from gel state to a frozen state
REVIEW
1. Why are detergents necessary to solubilize membrane
proteins? How might one determine the diversity of
integral proteins that reside in a purified membrane
fraction?
2. How can one determine: (1) the location of
transmembrane segments in the amino acid sequence
or (2) the relative locations of transmembrane helices
with access to the external medium?
4.6 Membrane Lipids
and Membrane Fluidity
The physical state of the lipid of a membrane is described by its fluidity
(or viscosity).4 Consider a simple artificial bilayer composed of phos-
phatidylcholine and phosphatidylethanolamine, whose fatty acids are
largely unsaturated. If the temperature of the bilayer is kept relatively
warm (e.g., 37°C), the lipid exists in a relatively fluid state (FIGURE 4.23a).
4
Fluidity and viscosity are inversely related; fluidity is a measure of the ease of flow,
and viscosity is a measure of the resistance to flow.

(a) (b)

FIGURE 4.23 The structure of the lipid bilayer depends on the temperature. The bilayer shown here is composed of two phospholipids: phosphatidyl-
choline and phosphatidylethanolamine. (a) Above the transition temperature, the lipid molecules and their hydrophobic tails are free to move in certain
directions, even though they retain a considerable degree of order. (b) Below the transition temperature, the movement of the molecules is greatly
restricted, and the entire bilayer can be described as a crystalline gel.
SOURCE: (a, b) R. N. Robertson, The Lively Membranes, Cambridge Univ. Press, 1983, reprinted with permission of Cambridge Univ. Press.

Karp-c04.indd 130 11/23/2015 9:40:10 PM

Permeability is dependent on unsaturation, chain length

It is also modulated by cholesterol, which decreases permeability
Cholesterol
 Fluidity: mostly a property of internal region of the membrane

Fluidity can be altered by:

• By converting saturated bonds to unsaturated ones – Desaturases
• By reshuffling of fatty acid chains by phospholipases and acyl transferases
• By specific phospholipid synthesis
 Lipid-protein microdomain: lipid raft

Usually consists of long fatty acid-containing lipids, and GPI-anchored proteins

132

GPI–anchored
protein
CHAPTER 4 • The Structure and Function of the Plasma Membrane

Signaling
protein
(a) (b)

FIGURE 4.24 Lipid rafts. (a) Image of the upper surface of an artificial lipid bilayer containing phosphatidylcholine, which appears as the black
background, and sphingomyelin molecules, which organize themselves spontaneously into the orange‐colored rafts. The yellow peaks show the
positions of a GPI‐anchored protein, which is almost exclusively raft‐associated. This image is provided by an atomic force microscope, which measures
the height of various parts of the specimen at the molecular level. (b) Schematic model of a lipid raft within a cell. The outer leaflet of the raft consists
primarily of cholesterol (yellow) and sphingolipids (red head groups). Phosphatidylcholine molecules (blue head groups) with long saturated fatty acids
also tend to concentrate in this region. GPI‐anchored proteins are thought to become concentrated in lipid rafts. The lipids in the outer leaflet of
the raft have an organizing effect on the lipids of the inner leaflet. As a result, the inner‐leaflet raft lipids consist primarily of cholesterol and
glycerophospholipids with long saturated fatty acyl tails. The inner leaflet tends to concentrate lipid‐anchored proteins, such as Src kinase, that are involved
in cell signaling. (The controversy over the existence of lipid rafts is discussed in Nature Revs. Mol. Cell Biol. 11:688, 2010 and Science 334:1046, 2011.)
SOURCE: (a) From D. E. Saslowsky, et al., J. Biol. Chem. 277, cover of #30, July 26, 2002; (b) Courtesy of J. Michael Edwardson © 2002 The American Society
for Biochemistry and Molecular Biology.
 The first experiments to demonstrate that membrane proteins using a specific probe, such as a fluorescent antibody. Once labeled,
could move within the plane of the membrane utilized cell fusion, cells are placed under the microscope and irradiated by a sharply
Diffusion of Membrane
and they were reported in 1970 by Larry Frye and Michael Edidin of Proteins
focused laser beam that bleaches the fluorescent molecules in its
Johns Hopkins University. In their experiments, mouse and human path, leaving a circular spot (typically about 1 µm diameter) on the
cells were fused, and the locations of specific proteins of the plasma surface of the cell that is largely devoid of fluorescence. If the labeled

1 2 3 4
Human cell
Addition of 40
sendai minutes
(fusing)
virus

Mouse cell
(a) (b)

FIGURE 4.26 The use of cell fusion to reveal mobility of membrane proteins. (a) Outline of the experiment in which human and mouse cells were fused
(steps 1–2) and the distribution of the proteins on the surface of each cell were followed in the hybrids over time (steps 3–4). Mouse membrane proteins are
indicated by solid circles, human membrane proteins by open circles. Locations of human and mouse proteins in the plasma membranes of the hybrid cells
were monitored by interaction with fluorescent red and fluorescent green antibodies, respectively. (b) Micrograph showing a fused cell in which mouse and
human proteins are still in their respective hemispheres (equivalent to the hybrid in step 3 of part a).
SOURCE: (b) From L. D. Frye and Michael Edidin, J. Cell Science 7:328, 334, 1970, By Permission of The Company of Biologists, Ltd. Courtesy of Michael
Edidin, Johns Hopkins University.http://jcs.biologists.org/content/7/2/319 full.pdf+html?sid=d93ae648-abca-4f5d-90a6- a6d9726d7d30.

Karp-c04.indd 133 11/23/2015 9:40:12 PM

 Both Protein and lipid movements movements are restrcited

Proteins move across the boundaries from one compartment to 135

another through breaks in the fences. Such openings are thought to
appear and disappear along with the dynamic disassembly and reas-
A sembly of parts of the meshwork. Membrane compartments may

4.7 • The Dynamic Nature of the Plasma Membrane

D keep specific combinations of proteins in close enough proximity to
E
B facilitate their interaction.
Integral proteins lacking that portion that would normally pro-
ject into the extracellular space typically move at a much faster rate
than the wild‐type version of the protein. This finding suggests that
the movement of a transmembrane protein through the bilayer is
slowed by extracellular materials that can entangle the external por-
C tion of the protein molecule (Figure 4.28, protein F).

MEMBRANE LIPID MOBILITY Proteins are huge mole-

F cules, so it isn’t surprising that their movement within the lipid
bilayer might be restricted. Phospholipids, by comparison, are small
molecules that make up the very fabric of the lipid bilayer. One
might expect their movements to be completely unfettered, yet a
number of studies have suggested that phospholipid diffusion is also
FIGURE 4.28 Patterns of movement of integral membrane proteins.
restricted. When individual phospholipid molecules of a plasma
Single particle tracking
Depending on the cell type and the conditions, integral membrane proteins
can exhibit several different types of mobility. Protein A is capable of
membrane are tagged and followed under the microscope using
(SPT) of fluorescent tagged
diffusing randomly throughout the membrane, though its rate of movement
ultra‐high‐speed cameras, they are seen to be confined for very brief
periods and then hop from one confined area to another. FIGURE 4.29a
may be limited; protein B is immobilized as the result of its interaction with
protein
the underlying membrane skeleton; protein C is being moved in a particular Total Internal Reflection Fluorescence
shows the path taken by an individual phospholipid within the

(TIRF) microscopy
direction as the result of its interaction with a motor protein at the
cytoplasmic surface of the membrane; movement of protein D is restricted
by other integral proteins of the membrane; movement of protein E is
restricted by fences formed by proteins of the membrane skeleton, but it
can hop into adjacent compartments through transient openings in a fence;
movement of protein F is restrained by extracellular materials. Start
Start

The strongest influences on an integral membrane protein are thought

to be exerted from just beneath the membrane on its cytoplasmic face. Finish
The plasma membranes of many cells possess a fibrillar network, or
“membrane skeleton,” consisting of peripheral proteins situated on the
cytoplasmic surface of the membrane. A certain proportion of a mem-
 JCB

of the Plasma Membrane

Article
MEMBRANE LIPID MOBILITY Proteins are huge mole-
F cules, so it isn’t surprising that their movement within the lipid
Phospholipids undergo hop diffusion in
bilayer might be restricted. Phospholipids, by comparison, are small
compartmentalizedmolecules that make up thecell membrane
very fabric of the lipid bilayer. One
might expect their movements to be completely unfettered, yet a

Downloaded from http://rupress.org/jcb/article-pdf/157/6/1071/1304489/jcb15761071.pdf by IISER Thiruvananthapuram (Indian Institute of Science, Education, and Research) user on 11 April 2022
number
Takahiro Fujiwara, 1 of studies have suggested
Ken Ritchie,1,2 Hideji that phospholipid
Murakoshi, 2 diffusion
Ken Jacobson, 3 is also
and Akihiro Kusumi1,2
FIGURE 4.28 Patterns of movement of integral membrane proteins.
1 restricted. When individual phospholipid molecules of a plasma
Depending on the cell type and the conditions, integral membraneKusumi proteinsMembrane Organizer Project, Exploratory Research for Advanced Technology Organization (ERATO),
Japan Science membrane areCorporation,
and Technology tagged andNagoya
followed underJapan
460-0012, the microscope using
can exhibit several different types of mobility. Protein A is capable 2of
diffusing randomly throughout the membrane, though its rate of movement
3 ultra‐high‐speed cameras, they are seen to be confined for very brief
Department of Biological Science, Nagoya University, Nagoya 464-8602, Japan
Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina,
Chapel
may be limited; protein B is immobilized as the result of its interaction periods
with Hill, NC 27599and then hop from one confined area to another. FIGURE 4.29a
the underlying membrane skeleton; protein C is being moved in a particular shows the path taken by an individual phospholipid within the
direction as the result of its interaction with a motor protein at the

T
cytoplasmic surface of the membrane; movement of protein D is restricted he diffusion rate of lipids in the cell membrane is The diffusion rate within 230-nm compartments is 5.4
reduced by a factor of 5–100 from that in artificial !m2/s, which is nearly as fast as that in large unilamellar
by other integral proteins of the membrane; movement of protein E is bilayers. This slowing mechanism has puzzled cell vesicles, indicating that the diffusion in the cell membrane
restricted by fences formed by proteins of the membrane skeleton,biologists
but it for the last 25 yr. Here we address this issue by is reduced not because diffusion per se is slow, but because
can hop into adjacent compartments through transient openings instudying a fence;the movement of unsaturated phospholipids in rat the cell membrane is compartmentalized with regard to
kidney fibroblasts at the single molecule level at the temporal lateral diffusion of phospholipids. Such compartmentaliza-
movement of protein F is restrained by extracellular materials. Start
resolution of 25 !s. The cell membrane was Start
found to be tion depends on the actin-based membrane skeleton, but
compartmentalized: phospholipids are confined within not on the extracellular matrix, extracellular domains of
230-nm-diameter (") compartments for 11 ms on average membrane proteins, or cholesterol-enriched rafts. We propose
before hopping to adjacent compartments. These 230-nm that various transmembrane proteins anchored to the actin-
The strongest influences on an integral membrane protein arecompartments
thought exist within greater 750-nm-" compartments based membrane skeleton meshwork act as rows of pickets
where these phospholipids are confined for 0.33 s on average. that temporarily confine phospholipids.
to be exerted from just beneath the membrane on its cytoplasmic face. Finish
The plasma membranes of many cells possess a fibrillar network, or
“membrane skeleton,” consisting of peripheral proteins situated on the
cytoplasmic surface of the membrane. A certain proportion ofIntroduction
a mem-
For the past 25 yr, one of the most serious puzzles in the To address the problem of slowed lipid diffusion and to
brane’s integral protein molecules are either tethered to the membrane
research of membrane dynamics is how the diffusion rate of acquire a better general understanding of the dynamic lipid
skeleton (Figure 4.28, protein B) or otherwise restricted by it.lipids in the cell membrane is reduced by a factor of 5–100 organization in the cell membrane, we observed the move-
from that in artificial bilayers (Swaisgood and Schindler, ment of phospholipids at the single molecule level at a very
Information concerning the presence of membrane barriers has
1989; Lee et al., 1993; Ladha et al., 1996; Sonnleitner et al., high time resolution (up to 25 !s), which turned out to be
been obtained using an innovative technique that allows investiga-
1999). Long-range interactions between lipids and immobi- absolutely necessary to elucidate such a slowing mechanism.
Finish
lized proteins (Sperotto and Mouritsen, 1991; Almeida et As a test molecule, we used 1,2-dioleoyl-sn-glycero-3-phos-
tors to trap integral proteins and drag them through theal.,plasma 1992; Bussell et al., 1995; Dodd et al., 1995) or interactions phoethanolamine (DOPE),* which is a typical nonraft lipid.
(a) (b)
membrane with a known force. This technique, which uses with an appa-
the extracellular matrix (Wier and Edidin, 1986; Lee et Such an unsaturated phospholipid is thought to be most dif-
al., 1993) may be responsible for such a reduction of lipid ficult to have its diffusion rate reduced in the cell membrane.
ratus referred to as optical tweezers, takes advantage of the tiny opti-
diffusion rates. FIGURE 4.29 Experimental
Alternatively, demonstration
cholesterol-enriched lipid thatDOPE
diffusion of phospho-
diffusion as observed at 25-!s resolution revealed that
cal forces that are generated by a focused laser beam. Themicrodomains
proteins to be studied are tagged with antibody‐coated beads,
integral a) on rat cell membrane b) on artificial membrane
calledwithin
lipids “rafts” the(for reviews
plasma
Toomre, 2000; Edidin, 2001) may be involved in such a
whichmechanism labeledbyunsaturated
see Simons
membrane
phospholipidofis raft
and
is confined.
followed
(athe
) Thecell membrane
track of a singleis compartmentalized with regard to
translational diffusion of DOPE. A DOPE molecule is
slowing inducing partitioning lipidsfor 56confined
ms as it within
diffusesa 230-nm
within compartment for 11 ms on average
serve as handles that can be gripped by the laser beam. It is generally
into raft domains the plasma
or by membrane of a rat fibroblast.
becoming diffusion obstaclesPhospholipids
for beforediffuse
hopping freely within
to an adjacent compartment, and by repeating
found that optical tweezers can drag an integral protein for anonraft
limited lipids.
a confined compartment before hopping into a neighboring such temporary confinement and intercompartmental hop
compartment.
movement, it undergoes macroscopic diffusion over many
distance before the protein encounters a barrier that causesThe it online
to be
MonitoringThe rate of diffusion within a compartment is as rapid as that expected by
diffusion
version of this article of fluorescent-tagged1,2-dioleoyl-sn-glycero-3-phos-
includes supplemental material.
released from the laser’s grip. As it is released, the protein Address
typically unhindered
correspondence Brownian
to Akihiro movement.
Kusumi, DepartmentHowever, the overall
of Biological rate of diffusion of
*Abbreviations used in this paper: ", diameter; D , macroscopic
MACRO

springs backward, suggesting that the barriers are elastic structures. the phospholipid
Science, Nagoya University,
phoethanolamine appears Japan.
Nagoya 464-8602,
(DOPE) slowed because
Tel.: the molecule
81-52-789- must
diffusion hop a barrier
coefficient; D , microscopic diffusion coefficient; DOPE,
micro
2969. Fax: 81-52-789-2968. E-mail: akusumi@bio.nagoya-u.ac.jp 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; LUV, large unilamellar
to continue its movement. The movement of the phospholipid withinsquare
eachdisplacement; NRK, normal rat kidney fibro-
One approach to studying factors that affect membraneKey protein
words: cell membrane;
tracking; membrane skeleton
phospholipid; hop diffusion; single particle vesicle; MSD, mean
blastic cell; SPT, single particle tracking; TfR, transferrin receptor.
compartment is represented by a single color. (b) The same experiment
mobility is to genetically modify cells so that they produce altered
shown inPress,
a is carried out for 33 ms in an artificial bilayer, which lacks the
membrane proteins. Integral proteins whose cytoplasmic  portions The Journal
The Rockefeller University of
The Journal of Cell“picket
Cell
Biology, fences”
Biology, Volume
0021-9525/2002/06/1071/11 $5.00157, Number 6, June 10, 2002 1071–1081
Volume 157,present
Numberin6,aJune
cellular membrane.
10, 2002 1071–1081The much more open,

Book: Karp
have been genetically deleted often move much greater distanceshttp://www.jcb.org/cgi/doi/10.1083/jcb.200202050 1071
extended trajectory of the phospholipid can now be explained by simple,
than their intact counterparts, indicating that barriers reside on the unconfined Brownian movement. For the sake of comparison, fake
cytoplasmic side of the membrane. These findings suggest that the
 GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and
microdomains or rafts, transient homodimerization, release from the membrane by cleavage in the GPI

Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized
GPI modification imparts proteins with unique characteristics, such as association with membrane
GPI (Glycosyl Phosphophatidyl Inositol) anchoring of proteins

Functions: as enzymes, adhesion molecules, receptors, protease inhibitors, transporters, etc.

Act as membrane anchors of many cell surface proteins

moiety, and apical sorting in polarized cells.

(about GPI 150 proteins)

Fig. 1. GPI-anchored protein structure, b

thesis, and trafficking. A: Schematic depicts c
cal structure of full-length GPI-ethanolamine

by neurological abnormalities.
to terminal mannose of a trimannose oligos
ride via phosphodiester linkage, in turn coup
glucosamine PI. Reproduced with permission
(5). B: Schematic represents biosynthesis an
ficking of remodeled GPI-anchored proteins
association to membrane rafts, and diseases cau

immune system.
defective GPI synthesis. Adapted from T. Kin
unpublished observations. PNH, paroxysmal n
nal hemoglobinuria. C: Schematic shows the s
mechanism of GPI-anchored proteins at the
Golgi network in polarized epithelial cells. Th
mary level of sorting involves the segregati
remodeled GPI-anchored proteins into chole
and sphingolipid-enriched domains and oth
proteins. The next level of sorting occurs m
due to oligomerization effected by the inter
of different receptors with GPI-anchored pro
which, in turn, might help in vesicle formatio
budding. LAM, lipoarabinomannan; LM, lipoma
PIG, phosphatidylinositol glycan; PIM, phos
dylinositol mannoside; PM, plasma memb
Adapted from (132).
 thematic review series

Thematic Review Series: Glycosylphosphatidylinositol (GPI) Anchors:

Biochemistry and Cell Biology

GPI-anchored protein organization and dynamics at

the cell surface
1, 1, ,† 2, ,§
Suvrajit Saha, * Anupama Ambika Anilkumar, * and Satyajit Mayor *
National Centre for Biological Sciences (Tata Institute of Fundamental Research),* Bangalore 560065,
India; Shanmugha Arts, Science, Technology and Research Academy,† Thanjavur 613401, India; and
Institute for Stem Cell Biology and Regenerative Medicine (inStem),§ Bangalore 560065, India

Abstract The surface of eukaryotic cells is a multi-component
fluid bilayer in which glycosylphosphatidylinositol (GPI)-
anchored proteins are an abundant constituent. In this re-
view, we discuss the complex nature of the organization and
dynamics of GPI-anchored proteins at multiple spatial and
temporal scales. Different biophysical techniques have been
utilized for understanding this organization, including fluo-
rescence correlation spectroscopy, fluorescence recovery
after photobleaching, single particle tracking, and a num-
ber of super resolution methods. Major insights into the
organization and dynamics have also come from exploring
the short-range interactions of GPI-anchored proteins by
fluorescence (or Förster) resonance energy transfer micros-
copy. Based on the nanometer to micron scale organization,
at the microsecond to the second time scale dynamics, a pic-
ture of the membrane bilayer emerges where the lipid bi-
layer appears inextricably intertwined with the underlying
dynamic cytoskeleton. These observations have prompted
a revision of the current models of plasma membrane orga-
nization, and suggest an active actin-membrane composite.—
Saha, S., A. A. Anilkumar, and S. Mayor. GPI-anchored
protein organization and dynamics at the cell surface.
J. Lipid Res. 2016. 57: 159–175.
Supplementary key words lipid rafts • glycosylphosphatidylinositol-
Paper for extra reading and assignment
anchored protein • nanoscale • diffusion • homo-fluorescence reso-
nance energy transfer • super-resolution • signal transduction •
microdomains • actomyosin • activity
of the protein called glycosylphosphatidylinositol (GPI)-
anchored proteins. The basic structure of a GPI-anchored
protein consists of phosphatidylinositol (PI) linked to an
unusual non-N-acetyl glucosamine, which, in turn, is linked
to three mannose residues followed by an ethanolamine
covalently linked to the protein via an amide linkage
(EtNP-6Man!1-2Man!1-6Man!1-4GlcN!1-6myoinositol-
phospholipid, Fig. 1A). Depending on the species and
functional context, there may exist variations in the side
chain associated with the glycan core. These have been
summarized in (1). The lipid moiety is necessary for the
incorporation of GPI-anchored proteins into so-called
lipid rafts/microdomains (2, 3), which can serve as a sort-
ing station for a number of cell signaling molecules,
thereby functioning as a reaction center. GPI-anchored
proteins can exist in different forms depending on the
context and the tissue in which they are expressed. Alter-
nate splicing can cause the same protein to exhibit TM,
soluble, or GPI-anchored forms; for example, neural cell
adhesion molecule (NCAM) can exist in its GPI-anchored
and soluble form when expressed in muscles; whereas, it
takes up a TM form instead of the soluble form in brain.
GPI anchoring of proteins occurs at the luminal face of
Charge properties of phospholipids
PI3 kinase-mediated activation of intracellular signaling happens only when the
cytosolic layer has phosphatidylinositol
 In apoptotic cells, Phosphatidylserines translocate to the outer layer

R B Birge et al., Cell Death & Differentiation, 2016

LIPID compositional diversity in different cellular membranes
Lipids in PM
Plasma Membranes of higher eukaryotes
~1,000 chemically different types of lipid molecules
(glycerophospholipids with diverse head groups and acyl chains), tissue-
specific sphingolipids (SLs) with more than 500 carbohydrate structures
as head groups, and sterols.

The main glycerophospholipids:

phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), cardiolipin (CL), phosphatidyc acid (PA), and
phosphatidylinositol (PI)
Lipids in ER
Rough ER performs synthesis and translocation across membranes of
transmembrane and secretory proteins.
Smooth ER participates in lipid and carbohydrate metabolism and
detoxification.

In contrast to other cellular membranes, ER membranes have a nearly

symmetric lipid distribution between both leaflets and a relatively loose
packing of lipids that are mainly composed of PC (50-70%), PE (15-
30%), PI (~10%), PS (~7%) with a minor level of other lipids and
cholesterol.

These properties of the lipid bilayer facilitate insertion of newly

synthesized lipid and protein molecules into ER membranes. Lipids and
proteins are then distributed from ER to PM, endosomes, lysosome,
and mitochondria.
Lipids in Golgi
Made of stacks of vesicular cisternae with tubular connections that participate in
vesicular transport in the secretory, lysosomal, and endocytic pathways.
Glycosylation enzymes from Golgi complex attach sugar moieties to proteins
moving from ER to the plasma membrane via Golgi apparatus in a direction from
the cis-Golgi network (CGN) to the trans-Golgi network (TGN).

From cisternae of TGN, proteins are packaging into vesicles destined to lysosomes,
secretory vesicles, or plasma membrane.

TGN is a main sorting station in the secretory pathway which confers 5 to 10-fold
enrichment of sphingolipids and sterols in PM as compared to ER.

TGN have highly asymmetric lipid distribution with PS and PE accumulated at the
cytoplasmic side, and PC and SM in the inner side.

Based on yeast data, Golgi primarily composed of PC (20-30%), PI (25-30%),

glycosylinositol-phosphoceramide (11%), and ergosterol (10-22%).
Specific lipids can induce specific membrane curvature
 REVIEWS
Understanding the diversity
of membrane lipid composition
If interested in extra reading
Takeshi Harayama and Howard Riezman
Abstract | Cellular membranes are formed from a chemically diverse set of lipids present in
various amounts and proportions. A high lipid diversity is universal in eukaryotes and is seen from
the scale of a membrane leaflet to that of a whole organism, highlighting its importance and
suggesting that membrane lipids fulfil many functions. Indeed, alterations of membrane lipid
homeostasis are linked to various diseases. While many of their functions remain unknown,
interdisciplinary approaches have begun to reveal novel functions of lipids and their interactions.
We are beginning to understand why even small changes in lipid structures and in composition
Phosphophatidylcholine
Endosomes

MVB: multivesicular bodies

Lipid composition of early endosomes resembles those of PM.

Multivesicular bodies (MVBs), which are spherical vesicles with a diameter of

~400-500 nm, originate from early endosomes by the inward invagination of
the limiting membrane into the endosomal lumen followed by bud scission.

MVBs fuse with either PM, where their content is released into extracellular
space (as exosomes), or with late endosomes that transform into lysosomes,
where their content undergoes degradation.

MVBs targeted to degradation are presumed to undergo the ubiquitination of

proteins and release into the lysosome lumen.
 Mitochondria

Mitochondria outer membrane contains phospholipids in both leaflets, are composed of

PC (46-55%), PE (28-33 %), PI (9-13 %), and small amount of PS , and CL (Cardiolipin) (1-
6%).

Mitochondrial inner membranes (MIM) form highly folded structure with cristae, the internal
mitochondrial compartments.

MIM contains PC (~40%), PE (25-40 %), PI (~16%), and CL (10-23%) as major phospholipids.

Unlike the variable lipid composition of membranes in diverse eukaryotic cells, the phospholipid
levels of the MIM is rather constant in different tissues, and its alterations are not tolerated but
cause diseases.
 Cardiolipin in Mitochondria

CL is critically required for biogenesis and stabilization of respiratory chain

supercomplexes, as well as for mobilization of cytochrome C on inner membrane, thus
preventing the appoptosis.

Depletion of CL due to absence of tafazzin, an enzyme essential for the formation of CL,
causes the severe mitochondrial dysfunction with defective ATP formation, which is
manifested as Barth syndrome.
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that cytochrome
Role of cardiolipin in cytochrome c release from step process, in
mitochondria
proton gradient generated by the stepwise transport of protein from the
electrons from oxidizable substrates to molecular oxygen permeabilization
M Ott1, B Zhivotovsky1 and S Orrenius*,1
mediated by the mitochondrial respiratory chain. One compo- c into the extramit
nentCellofDeath
the and Differentiation (2007) 14, 1243–1247; doi:10.1038/sj.cdd.4402135; published online 13 April 2007
respiratory chain is cytochrome c, which transfers indicate that car
Mitochondria play a pivotal role in the regulation of apoptotic including energy supply.1,2 The latter is achieved by oxidative
Figure 2 tBid-induced cytochrome c release from mitochondria. tBid, formed by proteolytic cleavage of Bid, for example by caspase-8, might exert multiple effects at the level of
mitochondria. Hence, tBid can induce insertion and oligomerization of Bax in the OMM. This leads to the formation of pores that allow the release of cytochrome c (Cyt c) into the
cytosol. tBid can also directly induce oligomerization of membrane-associated Bak leading to pore formation. In addition, tBid might interact with cardiolipin to influence its
distribution between the mitochondrial membranes or induce changes in the ultrastructure of mitochondria. Cytochrome c is normally bound to the IMM by cardiolipin (CL) and
transports electrons from Complex III to Complex IV. During apoptosis, cytochrome c detaches from cardiolipin and appears as a soluble protein in the intermembrane space.
Detachment of cytochrome c from cardiolipin can be mediated either by direct formation of oxidized cardiolipin (CL-OOH) induced by the cytochrome c-cardiolipin peroxidase, or
through oxidative modification of cardiolipin by ROS originating from the respiratory chain or by phospholipase-A2 (PLA2)-mediated formation of lyso-cardiolipin (lyso-CL)

21
Membrane Proteins in Lipid Bilayer
Integral proteins consist of tranmembrane
domains
In Most Transmembrane Proteins, the Polypeptide Chain Crosses the Lipid Bilayer in
an α-Helical Conformation

About 20-22 predominantly non-polar amino acids

Membrane Proteins Can Be Solubilized and Purified in Detergents