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The organisation of the cell membrane: do proteins rule lipids?
Jeremie Rossy, Yuanqing Ma and Katharina Gaus
Cell membranes are a complex adaptive system: they are
constantly re-organised in response to extra- and intracellular
inputs and their local and global structure ultimately determines
how, where and when these inputs are processed. This
requires a tight coupling of signalling and membranes in
localised and specialised compartments. While lipids are
essential components of cell membranes, they mostly lack a
direct link to the input signals. Here we review how proteins can
deform locally membranes, modify and reorganise lipids to
form membrane domains and regulate properties like
membrane charges and diffusion. From this point-of-view, it
appears that proteins play a central role in regulating
membrane organisation.
Addresses
Centre for Vascular Research and Australian Centre for Nanomedicine,
University of New South Wales, Sydney, Australia
Corresponding authors: Rossy, Jeremie (j.rossy@unsw.edu.au),
Gaus, Katharina (k.gaus@unsw.edu.au)

Current Opinion in Chemical Biology 2014, 20:5459

distinct properties such as composition, charge, curvature

or condensation. These domains then act as signalling
platforms and coordinate cellular events. While lipids
may contribute and determine the plasticity of domain
formation, it is proteins that hold the key to triggering such
membrane re-organisation in an active and regulated
manner. Here we focus on how proteins can locally impact
on membrane organisation and structure, either by acting
on the biophysical properties of cell membranes, for
example via mechanical forces or by introducing delimited changes in the membrane lipid composition.

Pushing, pulling or bending: proteins that

shape membranes
Cellular processes such as endocytosis, exocytosis and
signalling require controlled and localised membrane
deformation [3,4]. Although some lipids have intrinsic
curvature, localised changes in topography are most likely
enforced upon the membranes by specialised proteins. In
the following paragraphs, we describe examples of
proteins that shape cellular membranes.

This review comes from a themed issue on Molecular imaging

Edited by Christian Eggeling and Mike Heilemann
For a complete overview see the Issue and the Editorial
Available online 13th May 2014
http://dx.doi.org/10.1016/j.cbpa.2014.04.009
1367-5931/Crown Copyright # 2014 Published by Elsevier Ltd. All
rights reserved.

Introduction
The plasma membrane is a gateway that coordinates
extracellular signals with intracellular responses and vice
versa, links intracellular processes to cellcell interactions
and tissue organisation. Thus, the plasma membrane
extends to intracellular endosomes and extracellular
vesicles to mediate cell functions such as receptor signalling, presentation of surface proteins and protein
secretion [1]. There is overwhelming evidence that the
plasma membrane is not a homogenous mixture [2], but
how cellular membranes are compartmentalised and
which contribution lipids and proteins make to this compartmentalisation is the topic of on-going debate. The
reason why membrane domains have attracted so much
attention is that membrane organisation conceptualises
how cells can actively coordinate processes to adapt
and respond to their environment. Upon signalling,
cell membranes rearrange in specialised domains, with
Current Opinion in Chemical Biology 2014, 20:5459

The Bin/amphiphysin/Rsv161 (BAR) domain protein

superfamily has emerged as one of the main factors that
control of membrane bending [5]. Various types of BAR
domain proteins can generate a wide range of curvatures.
Although they lack a consensus sequence motif, BAR
domain proteins typically form homo or hetero dimers that
structurally assemble in a curved banana shape. Mechanistically, BAR domain proteins exemplify the two ways
peripheral proteins generally induce curvature: 1) by acting
as a membrane scaffold and bending the membrane due to
their intrinsically curved structure, or 2) by inserting
amphipathic wedges and asymmetrically displacing lipids
in the membrane leaflet with which they interact
(Figure 1A). Consistent with their shape, BAR domain
proteins are also membrane curvature sensors and can act as
protein scaffolds thanks to SH3 domains. These features
give BAR domain proteins not only the ability to enhance
pre-existing membrane curvature, but also to recruit other
proteins to membrane sites with specific curvature. Notably, several members of the BAR proteins superfamily
recruit and activates members of the WASP family to
promote actin polymerisation at site of membrane curvature and to generate tubule elongation [6]. By stabilising
membranes with high curvature, BAR domain proteins can
also inhibit membrane fission [7]. In a mechanism similar to
that of BAR domain proteins, Exo70, a protein of the
exocyst complex, oligomerizes into scaffolds that generate
positive membrane curvature. Exo70, which binds to
phosphatidylinositol 4,5-bisphosphate (PIP2), shapes
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Cell membranes organisation: do proteins rule lipids? Rossy, Ma and Gaus 55

Figure 1

(a)

Shapping membrane
Membrane scaffolds

(b)

Protein crowding

Local control of membrane lipid content

Chemical modifications

(c)

Amphipathic wedges

Flippases

Diffusion barriers

Ca2+ microdomains
Tension

Tension

Current Opinion in Chemical Biology

Local control of membrane shape and composition by proteins. (A) Proteins can shape membranes, either by (from left to right) membrane-bound
scaffolds imprinting their shape onto the membrane, insertion of amphipathic wedges into one leaflet of the bilayer or through steric pressure
generated by the crowding of membrane-bound molecules. (B) Proteins locally regulate the lipid composition of cell membranes. From left to right:
Membrane-bound enzymes modify the chemical structure of lipids, flippases move lipids from one leaflet to the other, plasmalemmal proteins act as
diffusion barriers to create lipid microdomains. (C) In response to membrane tension, transient receptor potential ion channels can trigger ion influxes
that modify the charge of lipids in a restricted area of the cell membrane.

membrane protrusion that can be actin-free and contribute

to the formation of the leading edge in migrating cells [8].
Caveolae are another classical example of membrane
organisers and represent distinct invaginations in the
plasma membrane shaped by 140150 caveolins [9].
Caveolin-1 is the best-characterised caveolin member
and binds to the inner leaflet of the plasma membrane
via palmitoylation. The accepted mechanism is that
caveolin-1 creates membrane curvature through a
putative hairpin domain wedging into the inner leaflet
and its ability to oligomerize. Cavins, a family of cytoplasmic proteins, cooperate with caveolins to drive caveolae formation.
Tetraspanins are structural proteins bearing four transmembrane domains and control the formation of membrane tubules [10]. Similarly to BAR domains proteins,
they can oligomerize and recruit various proteins to
establish functional microdomains. Tetraspanins have
been reported to be involved in almost all cell function
requiring membrane shape changes. Different members
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of the tetraspanins family fulfil different membrane shaping function, either favouring (CD81, CD9, CD151) or
inhibiting (CD82) membrane extension. It is not yet clear
how exactly tetraspanins promote the formation of membrane tubules but evidences point towards their ability to
both generate membrane curvature and control actin
polymerisation [10,11].
The endosomal sorting complex required for transport
(ESCRT) comprises five separate complexes and drives
the formation of multivesicular bodies and cell scission in
cytokinesis. ESCRT 0, I and II recognise phosphatidylinositol (3,4,5)-trisphosphate (PIP3), form stable assemblies and recruit ESCRT-III, which transiently
polymerise to shape the membrane in a mechanism that
we just begin to understand [12]. ESCRT-III starts to
assemble on the inner leaflets in the form of concentric
spirals. Sequential incorporation of ESCRT-III subunits
then triggers a switch in these structures from 2-dimensional spirals to 3-dimensional helixes, pushing the membrane and generating a bud, which eventually severs from
the plasma membrane [12].
Current Opinion in Chemical Biology 2014, 20:5459

56 Molecular imaging

Although some members of the BAR domain proteins and

tetraspanins family contribute to flattening the membrane, most of the proteins controlling membrane shape
push or bend the membrane to create curvature. In
contrast, septins, a family of GTP-binding proteins that
assemble in ring-like structures supported by microtubules, tend to prevent membrane remodelling [13]. Septins assemblies, for example, prevent excessive blebbing
in migrating amoeboid cells [14].
In addition to two general mechanisms of membrane
bending described above membrane scaffolds imprinting their shape onto the membrane and insertion of
amphipathic wedges into one leaflet of the bilayer
Stachowiak and colleagues recently proposed an elegant
third alternative [15] (Figure 1A). According to computed
models, amphipathic helixes must cover at least 10% of
the membrane surface to generate curvature, which
means that the membrane covered by proteins with
amphipathic helixes reaches almost 100%. Stachowiak
and colleagues demonstrated that bending can be solely
driven by protein crowding, even without amphipathic
insertion, simply via the steric pressure of membranebound molecules [16]. This model provides interesting
perspectives for the control of curvature by proteins
repulsion or oligomerization. As the authors point out,
the wedge and crowding models are not mutually exclusive and both processes are likely to act in a concerted
manner to drive a curvature-induces-curvature chain of
events: proteins inserting amphipathic helix first create an
initial curvature that triggers the recruitment of curvature-sensing proteins, which in turn bends the membrane
by steric pressure [15].
Last but not least, the actin cytoskeleton constitutes a
formidable membrane shaping system [6,17]. Growing
actin fibbers can directly push the membrane by polymerising at specific sites of the membrane, for example at
the leading edge and other protrusions in migrating cells.
Actin is also required to facilitate processes initiated by
membrane bending proteins, for instance to overcome
pre-existing tension in the membrane or in the case of
endocytosis of cargos too large to be surrounded by a
scaffold [15,18]. As mentioned above, membrane-bending proteins such as BAR domain proteins or tetraspanins
can selectively trigger actin polymerisation at the sites of
membrane deformation. Further, myosin motors
anchored to membrane proteins contribute to membrane
tension and protrusion by sliding on actin fibres, as
exemplified by the blebbing at the leading edge of
amoeboid migrating cells [17].

Proteins that can induce the formation of lipid

domains
Most of the molecular machineries required for membrane bending also require a membrane domain within
which the process takes place. Specific lipid composition
Current Opinion in Chemical Biology 2014, 20:5459

or charge in delimited membrane areas act as signpost for

the recruitment of membrane shaping proteins and complexes. However, lipid domains cannot arise spontaneously, as this would lead to uncoordinated
membrane events. Membrane curvature per se can induce
lipid sorting, but in a process that relies on large-scale
lipidproteins and lipidlipid interactions and not on the
individual properties of lipids [19]. It is therefore crucial
to understand how proteins interact with lipids [20] to
regulate their spatial distribution and chemical modification.
Phosphoinositides are key players in numerous cellular
processes and are particularly important in membrane
remodelling [21]. Phosphoinositides head groups not only
have different charges and structures, directly impacting
on membrane properties, but can also be easily modified
to act as a chemical switch (Figure 1B). Interestingly,
synthesis of phosphoinositides is also linked to phosphatidylcholine (PC) metabolism through phosphatidic acid
(PA). PA, which is synthesised by hydrolysis of PC by
phospholipase D, is an important activator of PIP5K [22].
Typically, local actin polymerisation downstream of PIP2
directly depends on the recruitment and the activation of
PIP5Kg by a pool of PA synthesised by PLD [23]. The
sequential recruitment of proteins supporting clathrinmediated endocytosis represents a good example of how
accurately timed changes in lipid composition are
required for membrane remodelling. During formation
of clathrin-coated pits, the class II phosphatidylinositol-3kinase C2a (PI(3)K C2a), targeted to the plasma membrane by clathrin, spatiotemporally controls the recruitment of a BAR domain protein by generating PI(3,4)P2
puncta. Particularly interesting is that the switch from
PIP2 to PI(3,4)P2, mediated by PI(3)K C2a, regulates
distinct stages of the clathrin-mediated endocytosis [24].
Because their activity can also be spatially organised by
GAP/GEF availability, small GTPases are central to
locally restrict PIP5K activation and hence PIP2 production [21]. In endothelial cells, binding of the
Plexin-B1 receptor by Semaphorin 4D locally activates
RhoA and subsequently PIP5a to produce PIP2 in order
to promote the F-actin polymerisation necessary for
angiogenesis [25]. Similarly, Rac directly interacts with
PIP5Kb to induce a local synthesis of PIP2, leading to
neurite retraction in neuronal cells [26]. Spatiotemporal
activation of signalling proteins is central to T cell activation and phosphoinositides contributes to spatially
organised T cell signalling. Le Floch and colleagues
have identified a two-step process for PIP3 generation
in T cell. First, activation of the T cell receptor (TCR)
triggers the recruitment of PI3-K to TCR microclusters at
the forming immune synapse. Second, PI3-K is activated
by the small GTPase Ras to generate PIP3, which contributes to control actin polymerisation required for T cell
activation [27]. A similar observation was made in macrophages where IL-4 signalling increases the negative
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Cell membranes organisation: do proteins rule lipids? Rossy, Ma and Gaus 57

charge of the phagocytic cup by stimulating the production of PIP3 via PI3-K [28].
As lipids diffuse fast within membranes [29], the local
synthesis of a given species is not sufficient to form a
membrane domain. Even in the case of a transient
domain, lipid diffusion must be confined by protein
fences for domain formation (Figure 1B). A great example
of localised synthesis combined with restricted diffusion
has been identified in the budding yeast C. albicans [30].
These buds display a steep gradient of PIP2 from the tip
to the neck. The gradient is achieved by the localised
synthesis of PIP2 by the PIP4 kinase Mss4 at the tip of the
buds and by restriction of PIP2 diffusion at the bud neck.
Fuelling Mss4 with the PIP2 precursor, PI(4)P, by the
PI(4)P kinase Stt4 and actin polymerisation is also
required to maintain this gradient. Containment of the
diffusion of PIP2 in the inner leaflet of the plasma
membrane was also observed in fibroblasts [31]. The
exact mechanism responsible for restriction of PIP2 diffusion was not elucidated in these studies. However, it
has been recently reported that, in addition to membrane
bending and curvature sensing, BAR domain proteins can
directly control phosphoinositide diffusion; BAR domain
proteins stabilise PIP2 microdomains, probably via electrostatic interactions with the PIP2 head group, before
assembling larger proteins complexes and mediate membrane deformation [32]. Interestingly, septins can also
function as a diffusion barrier, for example at the base of
primary cilium [33,34].
While phosphoinositides may be regarded as a special
case, phosphatidylserine (PS) also modulates membrane
charge locally and thus contribute to spatially organise
processes within the cell membrane [35]. Therefore, like
for phosphoinositides, its spatial distribution must be
tightly controlled. PS facilitates signalling within the
various compartments along the secretory pathway and
undergoes a transition from the lumenal leaflet in the ER
to the cytosolic leaflet in the trans Golgi network, probably upon the action of flippases in the Golgi [36]. More
generally, flippases constitute a powerful cellular tool to
locally increase PS concentration (Figure 1B). For
example, flipping of PS generate membrane curvature
and the subsequent recruitment of budding machinery
for endosomal transport in yeast [37]. Spatial control of
the PS distribution is also achieved through containment.
The diffusion of a significant portion of the PS in the
plasma membrane is curtailed; surprisingly, PS confinement relies on the association with protein complexes
immobilised by the cytoskeleton and not on anionic
domains or lipid rafts [38]. PS is also retained in caveolae
[36], most likely through interactions with caveolin-1,
which was shown to define PS domains in liposomes [9].
Concomitantly with its role in mechanically shaping the
cell membranes, actin can act directly on lipid diffusion
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by compartmentalising the membrane as outlined in the

so-called pickets-fences model [39,40]. Transmembrane
proteins anchored to underlying actin fibres act as pickets
to limit movements of lipids they interact with. In
addition, the cortical actin network acts as diffusion
fences that stand in the way of diffusing lipids. Interestingly, it has been suggested that short (<250 nm) cortical
actin snippets can dynamically organised into asters by
motor proteins that are capable of organising lipids in the
adjacent plasma membrane into nanoclusters [41]. Two
studies using an interesting approach based on imaging
mass spectrometry to map lipids in the plasma membrane
further illustrate how actin can organise membrane lipids.
Frisz et al. have identified 200 nm sphingolipids microdomains whose integrity was independent of the membrane cholesterol but relied on the actin cytoskeleton
[42]. Using the same methodology, an Arachidonic acidPC gradient supported by actin dynamic was detected
along the axon of cultured neurons [43]. It should be
noted that specialised proteins that interact both with
lipids in the inner leaflet of the plasma membrane and
cortical actin contribute to compartmentalise the plasma
membrane. Ezrin/Radixin/Moesin [44] proteins and
membranes of the annexin family are especially interesting as a molecular switches governing the linkage of PIP2
in the membrane and the actin cytoskeleton.

Localised membrane charges a new kind of

membrane domain?
An emerging view in membrane biology is that membrane charges, but not necessarily specific lipids, facilitate
distinct processes such as protein recruitment to the
plasma membrane [35] and conformational change of
plasmalemmal proteins [45,46]. Corralling negatively
charges lipids as described above is one way to create a
charged membrane domain. The transient and localised influx of ions by channels located within the membrane could represent a new mechanism to create
domains that differ in membrane charge via the local
calcium concentration from the surrounding membrane
(Figure 1C). Transient receptor potential ion channels
can respond to mechanical stress induced by tension
resulting from membrane deformation or differences in
lipid packing [47], triggering the influx of Ca2+ that can
non-covalently interact with negatively charged lipids in
the membrane [46]. The divalent cation channel
TRMP7, for example, drives the formation of Ca2+
microdomains at the plasma membrane by facilitating
the influx of extracellular calcium during invadosome
formation in neuroblastoma cells [48]. Similar TRMP7induced Ca2+ microdomains have been previously
observed in the leading edge of migrating cells [49].
Interestingly, it has recently been shown that Ca2+
oscillation are coupled to PIP2 and actin oscillation in
mast cells [50] opening the possibility that Ca2+ microdomains are linked to other mechanisms of protein-driven
lipid domains and membrane compartmentalisation.
Current Opinion in Chemical Biology 2014, 20:5459

58 Molecular imaging

Conclusion
Proteins that shape the membrane, generate lipid species
and restrict diffusion with membranes are often recruited
to lipid domains [3,4,21]. Typically, BAR domain
proteins, tetraspanins, ESCRT, septins or actin remodel
cellular membranes at sites of high local PIP2 or PIP3
concentrations but are also responsible for establishing
phosphoinositide domains. Hence, the common view is
that proteins and lipids in membrane processes are
mutually dependent on each other. Indeed, recruitment
and activation of membrane proteins, local lipid synthesis,
flipping and restricted diffusion of lipids all involve the
spatial organisation of lipids and proteins. While the lipid
domains as organisers of proteins has attracted widespread attention [2], the question whether proteins control lipids or lipids control proteins in cell membranes may
not to be the chicken-and-egg problem as it is so often
portrayed. Functionally speaking, it is the proteins that
execute most of the cellular function and allow the plasma
membrane to act as a gatekeeping in the coordination of
extracellular and intracellular responses. The fact that
proteins exploit the biophysical properties of lipids including lipid charge and phase separation for membrane
function is one of the reasons why membrane biology is
such a fascinating area of research.

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