Biochimica et Biophysica Acta 1858 (2016) 2569–2572

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbamem

Cell membranes: A subjective perspective☆

Kai Simons
Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Cell membranes have developed a tremendous complexity of lipids and proteins geared to perform the functions
Received 26 November 2015 cells require. The lipids have for long remained in the background and are now regaining their role as important
Received in revised form 19 January 2016 building blocks of cells. Their main function is to form the matrix of our cell membranes where they support a
Accepted 21 January 2016
variety of functions essential for life. This 2-dimensional fluid matrix has evolved unexpected material properties
Available online 29 January 2016
that involve both lipid–lipid and lipid–protein interactions. This perspective is a short summary of the challenges
Keywords:
that this field faces and discusses potential ways and means for coming to grips with the properties of this incred-
Lipids ible fluid. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.
Membranes © 2016 Elsevier B.V. All rights reserved.
Lipid rafts
Lipid–protein interactions
Electrostatic properties
Mass spectrometry

An astounding feature of cell membranes is that although the basic
bilayer can be self-assembled from only one single glycerophospholipid
species, the membranes in our cells are made of hundreds of different
lipids. Today we are able to analyze this complexity with a new genera-
tion of mass spectrometers that cope with lipid diversity with unprece-
dented precision and efficiency. The challenge is now to understand
why our cells need to have so many lipids in their membranes. How
do the lipids mingle and interact with the proteins in the membranes?
Why does the lipid matrix differ in the various membranes of the cell?
How do lipids contribute to membrane architecture and cell morpho-
genesis? How do membranes shape themselves into protrusions, invag-
inations, tubules and vesicles or form cubic membranes? Or how do
membranes sub-compartmentalize to form dynamic subdomains?
New methods are being generated such as powerful novel imaging
methods based on a plethora of fluorescent probes but we are far
from understanding how cell membranes can perform all these feats.
This perspective will summarize what we can do to meet this challenge.
1. Lipids in the cell membranes: composition and location
It is now well established that cell membranes are composed of both
proteins and lipids. However, the lipids were long neglected because of
lack of adequate methods to analyze them and probe their role in bio-
chemistry, biophysics and cell biology. When the Gordon Conference
for Molecular Membrane Biology was started over 30 years ago, the pro-
gram was focused on the proteins; the lipids had a backstage role. This
☆ This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen
and Tomasz Róg.
E-mail address: simons@mpi-cbg.de.
trend continued for years. But now with the introduction of new meth-
odologies, lipids are becoming more accessible and their role in mem-
branes is being increasingly explored. Most lipids have polar head
groups and hydrophobic hydrocarbon chains that differ in length, hy-
droxylation and saturation. So far this chemical diversity has received
little attention. Most studies focused on lipid classes (PC, PE, PA, PS, PI,
SM etc.) and not on species and subspecies. We only have a vague un-
derstanding of which enzymes are responsible for synthesizing differ-
ent lipid subspecies and regulating their metabolism. These are now
tasks that can be tackled by our new lipidomics tools to analyze
lipidomes quantitatively and with high coverage [1,2]. Not only tradi-
tional genetics but also CRISPR/Cas9 methodologies can be mobilized
to systematically analyze lipid metabolism at this level in different
cells and organisms [3]. We know roughly where different lipid classes
are made [4]. The endoplasmic reticulum (ER) is the main synthesis site
but many other organelles contribute to the generation of the lipid spec-
trum in cells. The newly synthesized lipids are distributed both by
membrane traffic and by direct delivery across contact sites between or-
ganelles, thereby establishing and maintaining the lipid compositions
that are characteristic to each cell type and their variety of membranes.
We are starting to appreciate that all lipid subspecies are probably as
tightly regulated as sterols are. This implies that lipid metabolism has to
be stringently controlled. How this is achieved remains poorly under-
stood. Clues are starting to emerge about possible sensing mechanisms
that can respond both to the chemical structure [5] and to the physical
properties [6,7] of the lipids and of the bilayer. However, this is only a
humble beginning. These studies will occupy the field for years to
come. We are lacking methods to isolate organellar membranes in
high enough purity so that their lipidomes can be measured. Progress
in this area has been dismally slow. The tools to analyze membrane

http://dx.doi.org/10.1016/j.bbamem.2016.01.023
0005-2736/© 2016 Elsevier B.V. All rights reserved.
2570 K. Simons / Biochimica et Biophysica Acta 1858 (2016) 2569–2572
lipidomes are available but the methods for organelle purification have
to be overhauled. Take the secretory pathway as an example. Newly
synthesized proteins and lipids are transported from the ER to the cis-
side of the Golgi complex, moved to the trans-side, and then sorted in
the trans-Golgi network (TGN) into different transport vehicles for de-
livery to the plasma membrane (PM) and other destinations [8]. Each
of these pathways transports different lipid and protein cargoes [9]. A
gradient of cholesterol is established, starting low in the ER where the
sterols are synthesized and increasing over the Golgi complex, reaching
its peak in the PM. Sphingomyelin glycosphingolipids, and gangliosides
are produced in the Golgi complex and transported to the PM where
their concentration is the highest [8]. Also other lipids such as PS are
enriched in the PM. What is lacking are quantitative lipidomes with sub-
species coverage to show how the different lipids are distributed over
the different organellar membranes. We now need to follow how the
whole spectrum of newly synthesized lipid subspecies are distributed
over each cellular membrane and how the cellular lipids add up to
give each cell membrane its unique identity.
Progress towards characterizing temporal and spatial remodeling of
the cellular lipidome has been slow, but emerging observations show
promise to open new frontiers in understanding cellular processes. So
far only two populations of transport vesicles have been isolated in
high enough purity to allow lipid analysis. Brügger et al. [10] isolated
COPI vesicles from the Golgi complex in vitro and demonstrated the
enrichment of SM 18:0. Later it was shown that this SM lipid binds to
the p24 protein of the COPI vesicles and regulates the activity of the
transport machinery [11]. Klemm at al. [12] purified post-Golgi trans-
port vesicles from yeast and demonstrated that the glycosphingolipid
species were specifically enriched by sorting in the TGN [13]. The data
gave direct evidence for the role of the Golgi complex in producing
the gradient of glycosphingolipids, peaking in the PM.
2. Protein–lipid interactions
Understanding the lipid environment where membrane proteins
carry out their function is a starting point for analyzing how lipids con-
tribute to modulating protein function in membranes. Understanding
protein–lipid interactions will be key to understanding membrane orga-
nization and function. Membrane research was long divided into two
camps that rarely met. The protein camp was the dominating one and
caught most of the excitement. It bears repeating that the lipid camp
was isolated and mostly ignored. This dismal situation has changed in
recent years. It has now finally dawned on the field that proteins and
lipids need to be studied together. Most importantly, new methods
have generated tools that have the capacity of analyzing how lipids
interact with proteins. Sophisticated mass spectrometric methods can
now unravel integral membrane proteins in complex with specific
lipid ligands [14]. The binding of peripheral proteins to specific lipids
can be measured by a number of novel screening methods. Chemically
engineered lipids can be employed to identify specific protein binding
partners [15].
Not only x-ray crystallography but also electron cryo-microscopy is
entering the field of protein lipid interactions [16,17]. CryoEM is the
only method that can deliver structures at atomic resolution revealing
how proteins interact with lipids in the membrane itself. The data
emerging show that lipids form a continuous shell around the trans-
membrane domains of the protein. They also mediate contacts between
protein subunits. The lipid hydrocarbon chains localize to grooves on
the protein transmembrane surface to form an electrochemical seal
across the membrane; they occupy the holes that proteins make in the
bilayer. Interestingly, electron crystallography can also provide a mea-
sure for how tightly bound the lipids are. The crystallographic B-factor
represents the statistical disorder of the protein–lipid structure [17]. If
lipids are loosely interacting, representing bulk lipids associating to pro-
tein, the B-factor is higher than for those lipids that are specifically
bound to the protein. Another useful feature of electron crystallography
is that the electrical charge of the protein can be precisely localized.
These exciting developments are bound to stimulate this important
area of membrane research.
3. The structure and dynamic organization of cell membranes
The most challenging feature that membrane research faces, is the
dynamic organization of cell membranes. It was first in the 1970s that
it became clear that the lipids and the proteins can move laterally in
the lipid bilayer i.e. that cell membranes are fluids [18]. At that time, it
was also demonstrated that the lipids are organized asymmetrically in
the two leaflets of the bilayer [19]. The lipids are flipped by specific
flippases from one leaflet to the other to maintain the correct asymme-
try under considerable energy expenditure [20]. Another intriguing
feature was revealed already in the 1960s by Vittorio Luzzati who dem-
onstrated that lipid bilayers can fold into different polymorphisms with
specific symmetries [21]. These are highly curved 3-dimensional struc-
tures that display periodic cubic structures. This remarkable property
showed that lipids have a unique capability to form different architec-
tures. Also important is that the transition from a planar bilayer struc-
ture into cubic membranes requires little energy and this feature
provides ample opportunity for cells to dynamically organize them-
selves. The only polymorphism that has entered biology are the cubic
membranes [22,23] and my prediction is that we will see more of
Luzzati's work in years to come. Cell membrane architecture is bound
to be an important feature in generating form and shape in cellular
architecture.
The focus of membrane research has so far been on local modula-
tions of membrane organization. For example, how do membranes
form vesicles and tubules and how do these fuse with the correct target
membrane? As in so many other areas of biological research, the main
achievement so far has been to identify the proteins – the parts list –
regulating these processes and to characterize these players functional-
ly. The role of the lipids was so far more or less neglected. Obviously,
electron crystallography is starting to provide exact information on
the interplay between the proteins and the lipids involved. However,
this research is still in its infancy.
An interesting emerging area is to define how physical parameters
such as membrane charge density, membrane curvature and lipid pack-
ing and thickness are being employed in regulating membrane function
[24]. Using different surface probes and protein markers as well as by
analyzing lipid composition, two territories have been defined in the
secretory pathway. One is the early secretory pathway (the ER,
nuclear envelope and the cis-Golgi). This territory is enriched in lipids,
having monounsaturated acyl chains. It is also low in cholesterol and
sphingolipids. This lipid composition is disordered and leads to voids
and defects in the bilayer, thus constituting a membrane environment
that allows the accommodation of different polypeptide chains in the
bilayer. These are important properties for translocation and folding in
the ER and for transport to the next station in the pathway, the Golgi
complex [24–26]. The late secretory pathway (TGN, PM and the
endosomes) forms the second territory. This is characterized by high
electrostatics and by lipids that form more tightly packed and ordered
bilayers. Generally there is gradient of lipids that have more saturated
hydrocarbon chains (acyls and sphingosines) in the secretory pathway,
peaking towards the PM. Negatively charged PS, phosphoinositides,
cholesterol and sphingolipids are enriched in this territory [8]. Interest-
ingly, PS in the luminal leaflet in the ER and the Golgi complex and is
flipped to the cytosolic leaflet in the TGN [27]. PS is maintained exposed
cytosolically in the PM and in the endosomes. The negatively charged
cytosolic membrane surface facilitates binding of many peripheral
proteins, required for different signal transduction processes and for
regulating membrane shape and traffic [24].
Another interesting feature is the role of polyunsaturated acyl chains
such as C20:4 and C22:6 [28]. They are known to be enriched in lipids of
the photoreceptor membranes and synaptic vesicles. These fatty acids
 K. Simons / Biochimica et Biophysica Acta 1858 (2016) 2569–2572
have been shown to flexibly adopt different conformations. Thereby,
they are able to adapt to rapid structural changes in the bilayer by buck-
ling up along the protein transmembrane surface to keep the membrane
sealed. High concentration of lipids with polyunsaturated fatty acyls
such as in photoreceptor membranes also speeds up lateral diffusion
of transmembrane [29] and peripheral proteins bound to the
membrane.
These differences in lipid composition play an important role in
defining the properties of the cell membranes in different organelles.
They also contribute to endowing these territories with unique sub-
compartmentalization capabilities. The propensity of forming lipid
rafts is a characteristic of the post-Golgi membranes [30]. Lipid rafts
are dynamic assemblies of sterols, sphingolipids, and saturated PC, and
they can be formed in the secretory pathway, when the concentration
of these lipids increases towards the trans-side of the Golgi complex
[31]. In the TGN they form dynamic platforms in the membrane that
bend into transport vehicles for transport to the cell surface [32]. This
sub-compartmentalization principle is generally operating to provide
post-Golgi membranes with multitasking capabilities to simultaneously
create local subdomains, operating in specific signal transduction pro-
cesses or in other membrane functions as well as in endocytosis and
recycling.
The basis for membrane sub-compartmentalization lies in the
capability of post-Golgi membranes to undergo liquid–liquid phase
separation in the membrane plane. Plasma membrane blebs and
balloons—giant PM vesicles(GPMVs) have been shown to separate
into two fluid domains at the micrometer length scale, mimicking the
phase separation seen in simple lipid membrane model systems, sepa-
rating into liquid-ordered and -disordered phases [33]. In the GPMVs
both raft lipids and proteins are enriching in the more ordered phase,
de-mixing from the other less ordered membrane domain. Thus, plasma
membranes have lipid and protein composition, poised close to a phase
transition boundary. In living cells, this tendency to phase separate
enables the formation of specific functionalized subdomains with little
added energy, operating in membrane traffic, signaling and other
post-Golgi membrane functions. The activation is usually brought
about protein ligands that oligomerize raft lipids or proteins. The coales-
cence of raft assemblies into large micrometer phase separated domains
is possible in GPMVs only because the cortical actin network has disso-
ciated from the membrane. In intact cells, cortical actin filaments are hy-
pothesized to trap the raft platforms and to prevent further coalescence
[34,35]. Veatch et al. have demonstrated that the compositions of
GPMVs are not only close to a phase boundary but seem to be even
more precisely regulated, looming close to a critical point, perhaps
explaining the fluctuations of the raft nano-assemblies taking place in
the resting state and their stabilization by clustered platforms [36].
There are many open issues to be pursued to understand how this
sub-compartmentalization principle works. What makes transmem-
brane protein raftophilic? We now have methodology to study this
question by the use of GPMVs. Most interestingly, electron crystallogra-
phy is bound to give exact information on how raft lipids interact with
raft proteins. Attractive starting points would be to study the envelopes
of raft viruses such as influenza and HIV at atomic resolution. The big-
gest challenge in this area of research is to move to functionality. We
need detailed analysis of how rafts support specific membrane func-
tions, employing the full repertoire of biochemical and biophysical
methods now available.
4. Conclusions
This brief survey highlights the astounding capabilities of the
2-dimensional liquids that form the cell membrane in our cells. They
contain hundreds of different lipids and proteins that intermingle
to give the membrane remarkable properties. It is now becoming
clear that the functions of membranes not only need their protein
2571
complement, but also the different lipids that these liquids require for
functionality.
One of the most interesting properties of membranes is that the
lipids and proteins behave as collectives [33]. For instance, the collective
properties of the lipids and proteins that form raft assemblies will be
key to understanding their structure and function. What are the detailed
material properties of membranes of different lipid compositions? Espe-
cially intriguing is to understand the properties of the cytosolic surfaces
in different subdomains of the membrane.
Another phase separation phenomenon is emerging as a novel prin-
ciple in cytoplasmic organization. A number of organelles that are not
membrane-bounded, including P-granules and the nucleolus have
been shown to behave like condensed liquid phases [37,38]. They are
composed of RNA and proteins and form liquid droplets in the cyto-
plasm. Also pure protein complexes have been shown to form such liq-
uid drops [39,40]. This astounding discovery changes our concepts of
cell organization. We now have to introduce new methods to under-
stand how proteins, RNA and other molecules interact with each
other. What are the structural principles driving and regulating these
phase separations? These cellular structures could not be studied by
the conventional methods of biochemistry and therefore little progress
was possible. Why? Because the RNA and/or the proteins forming the
condensed liquids associate like raft lipids and proteins by weak
interacting forces and dissociated when the cell was homogenized.
Now when we comprehend that not only membranes can phase
segregate into different liquid domains, but that also many processes
in cells employ phase separation to form functionalized states, new
vistas open up. One could for instance imagine that epithelial polariza-
tion would involve the formation of an apical pole [41], driven by a
phase separation in the polarizing plasma membrane, linked to a
concomitant 3-dimensional phase separation of cortical proteins into a
cortical liquid. The mature apical membrane of epithelial MDCK cells
has been shown to behave like a percolating raft phase [42]. Apical trans-
membrane proteins could bind to peripheral cortical proteins, inducing a
coordinated phase separation to define the apical pole. The induction of
the phase separation in the polarizing plasma membrane could be trig-
gered by ligands such as galectin-9 that has been demonstrated to be in-
volved in polarization, binding to the Forsmann glycolipid [43]. This
glycolipid starts to be synthesized during the onset of epithelial polariza-
tion [44]. Polarization processes could also be initiated from the cortical
side by nucleating the cortical condensation process. The most exciting
quality is that we now can conceive completely new scenarios for gener-
ating working hypothesis for cellular processes that eluded us until now.
If we examine the methods available to study many of the questions
discussed, it is obvious that the most difficult aspect to get a handle on is
the dynamics of membranes. One method that has access to the time
and spatial scale needed to analyze membrane functions is molecular
(MD) simulation. There is rapid progress in the field, which this issue
of BBA Biomembranes on “Biosimulations of lipid membranes coupled
to experiments” attests to. The MD method can provide information
on atomic scale mechanisms that we need to analyze if we want to un-
derstand how different membrane functions work dynamically. We
clearly need high resolution studies to come to grips with how lipids in-
teract with proteins. With more structural data delivered by electron
crystallography, the dynamics of how transmembrane proteins change
conformations e.g. to become raftophilic or activated to perform func-
tions, will be interesting and demanding challenges. Also the issue of
lipid asymmetry will demand atomic scale simulations. What are the
chemical principles underlying this property of cell membranes? It
must be fundamental since so much energy has to be invested in main-
taining the correct asymmetry [20]. Also coarse-grained molecular
models will be important to develop. We need methodology to simulate
phase separation and collective behavior. When we understand
more about how lipid subspecies are generated by different enzymes
and how they are regulated, it will be important to simulate how
lipid subspecies collectively behave in membranes. Obviously, new
2572 K. Simons / Biochimica et Biophysica Acta 1858 (2016) 2569–2572
biosimulation methods will emerge in the future to expand our reper-
toire of methods to meet these challenges.
Membrane research is in an exciting stage. The most encouraging
trend is that the fragmentation of the membrane research is coming
to an end. We are now starting to study membranes as a whole not
only statically but also dynamically. Here we are becoming models for
other fields of similar complexity. Hand in hand, with experimentalists
and MD simulators working together, the membrane field will blossom.
Transparency document
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found, in the online version.
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