Membrane curvature in cell biology: An integration of molecular mechanisms

TLDR: Examples of membrane curvature generation in animals, fungi, and plants are reviewed and lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms the authors expect to see used by plants and protists.

Abstract: Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

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Introduction
Membranes are the frontier between the inside and outside of
cells and separate diverse intracellular compartments while
being hubs of signaling activity. The exchange of molecules
and signals across this barrier requires many processes to bend,
invaginate, protrude, fuse, and break membranes. Further-
more, expanding the surface area of membranes for reactions
is achieved by sculpting folds and tubules into intracellular or-
ganelles. Pure lipid bilayers remain at, and stimulating them to
curve requires energy (Helfrich, 1973; Helfrich and Jakobsson,
1990). The energy needed is provided by modication of the
lipid composition or from the membrane-associated proteins
that make up ∼50% of the membrane surface. Protein mecha-
nisms range from different types of binding interaction to oligo-
merization processes and mechanochemical ATP and GTPases.
Membranes can be positively curved (toward the cytoplasm) or
negatively curved (away from the cytoplasm), and their deform-
ability varies depending on the tension in the membrane (Mc-
Mahon and Gallop, 2005). The local restriction of curvature to
specic areas implies lateral compartmentalization within the
uid mosaic membrane (Singer, 1972; Kusumi et al., 2011).
In the last few years, rapid progress has been made in di-
versifying membrane curvature research beyond membrane traf-
cking to organelle architecture. The molecular mechanisms of
membrane curvature rarely act alone, but instead cooperate in
diverse ways to achieve the highly complex and well-regulated
membrane architectures needed by cells. In this review, we de-
scribe how diverse membranes are shaped in plant, animal, and
fungal cells (Fig.1). First, we give a brief description of the com-
mon molecular mechanisms that are used to bend membranes.
We go on to discuss how the mechanisms are used together to
generate the curvatures present at the plasma membrane (PM)
and intracellular organelles (Table1). Lastly, we look ahead at
some other contexts for membrane curvature that are important
topics for further investigation.
The molecular mechanisms of
membrane bending
It is helpful to consider membrane-intrinsic forces that act by
introducing local asymmetry to the bilayer distinct from mem-
brane-extrinsic forces that are contributed by peripheral inter-
acting proteins acting outside the lipid bilayer itself. One way of
altering the curvature of the membrane is by modifying the local
lipid composition, whether it be the lipid headgroup, tail, or
cholesterol enrichment. The shape of the individual lipids gives
a spontaneous curvature to the membrane (Fig.2A). In model
membranes, lipids of the same kind tend to cluster together, and
protein transmembrane domains prefer to accumulate specic
lipids as a lipid coat. Asymmetry within the bilayer is also in-
troduced by the intrinsic shape of protein membrane-spanning
segments (Fig.2B) or the asymmetric insertion of hydrophobic
protein domains, e.g., hairpins (Fig.2C) or amphipathic helices
(Fig.2, D–F). The contributions made by protein monomers can
be greatly enhanced by oligomerization, which also stabilizes
membrane curvature (Fig.2, C and F).
In addition to membrane-intrinsic mechanisms, various
cytoplasmic membrane-extrinsic protein machineries modify
membrane shape. Inherently curved peripheral binding pro-
teins act as monomeric scaffolds or as homo- or heterodimers.
For example, the Bin-amphiphysin-Rvs (BAR) domain, which
characterizes the protein superfamily with the same name, can
be curved to various degrees, generating either positive or neg-
ative curvature. By individual proteins, curvatures can there-
fore be manipulated at the nanometer scale (Fig.3, A and B).
Both curved and at monomers can also further oligomerize
Curving biological membranes establishes the complex
architecture of the cell and mediates membrane traffic to
control flux through subcellular compartments. Common
molecular mechanisms for bending membranes are evi-
dent in different cell biological contexts across eukaryotic
phyla. These mechanisms can be intrinsic to the mem-
brane bilayer (either the lipid or protein components) or
can be brought about by extrinsic factors, including the
cytoskeleton. Here, we review examples of membrane
curvature generation in animals, fungi, and plants. We
showcase the molecular mechanisms involved and how
they collaborate and go on to highlight contexts of curva-
ture that are exciting areas of future research. Lessons
from how membranes are bent in yeast and mammals
give hints as to the molecular mechanisms we expect to
see used by plants and protists.
Membrane curvature in cell biology: An integration
of molecular mechanisms
IrisK.Jarsch, FredericDaste, and JenniferL.Gallop
Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, England, UK
© 2016 Jarsch et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the
publication date (see http ://www .rupress .org /terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http ://creativecommons .org /licenses /by -nc -sa /3 .0 /).
Correspondence to Jennifer L.Gallop: j.gallop@gurdon.cam.ac.uk
Abbreviations used: BAR, Bin-amphiphysin-Rvs; CALM, clathrin assembly lym-
phoid myeloid leukemia; CME, clathrin-mediated endocytosis; ESC RT, endo-
somal sorting complex required for transport; GAP, GTPase-activating protein;
GUV, giant unilamellar vesicle; MVB, multivesicular body; Nup, nucleoporin;
PM, plasma membrane; RTN, reticulon.
THE JOURNAL OF CELL BIOLOGY
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Figure 1. Cell shaping through membrane curvature. We highlight prominent examples of membrane curvature in cells where recent progress has re-
vealed insights into the underlying molecular mechanisms. (A) Multiple bending mechanisms in CME; (B) clathrin-independent endocytosis; (C) caveolae for-
mation; (D) induction of negative curvature at the PM for protrusions like filopodia; (E) bulging of the fungal appressorium; (F) constriction of the PM sleeve
and the ER-derived desmotubule at plasmodesmata; (G) dynamic membrane curvature at endosomal tubules; (H) establishment of a protrusion into the MVB
to induce vesicle budding; (I) vesicle budding from the ER and Golgi; (J) shaping of morphologically distinct and highly dynamic ER sheets and tubules; (K)
sculpting and maintenance of mitochondrial cristae membranes; (L) shaping the surface-maximized grana in plant thylakoids; and (M) assembly and per-
sistence of the nuclear pore complex in the highly bent nuclear envelope. Positive curvature is shown in pink lines, and negative curvature is shown in blue.
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How cells bend membranes 377
Table1. An overview of processes and proteins in membrane bending
Process Protein name Curvature Molecular mechanism Figure References
Appressorium Rvs167 Positive BAR domain, scaffolding 3 A Dagdas et al., 2012
Appressorium
Septins Positive
Oligomerization, scaffolding
3 C Bezanilla et al., 2015; Bridges
et al., 2016
Caveolae Caveolin Positive Oligomerization, hairpin insertion 2 C Monier et al., 1995
Caveolae Cavins Positive Oligomerization, clustering 2 C Hansen et al., 2009
Caveolae
EHD2 Positive
Oligomerization, GTPase
3 C Daumke et al., 2007; Morén et
al., 2012
Caveolae PAC SIN2 Positive BAR domain, scaffolding 3 A Senju et al., 2011
Endocytosis, clathrin
mediated
Actin Positive, negative Substrate for motor proteins, membrane
pulling, polymerization pushing against the
membrane, ATPase
3 G & H
Collins et al., 2011; Lewellyn et
al., 2015
Endocytosis, clathrin
mediated
Amphiphysin Positive Amphipathic helix, bilayer asymmetry, BAR
domain scaffolding and oligomerization
2 E Peter et al., 2004; Isas et al.,
2015
Endocytosis, clathrin
mediated
CALM Positive
Amphipathic helix, bilayer asymmetry
2 D
Miller et al., 2015
Endocytosis, clathrin
mediated
CIP4 Positive
F-BAR domain, oligomerization, scaffolding
3 A & C
Frost et al., 2008
Endocytosis, clathrin
mediated
Clathrin Positive
Scaffolding via oligomerization, crowding
3 C & E Fotin et al., 2004; Avinoam et
al., 2015
Endocytosis, clathrin
mediated
Dynamin Positive
Oligomerization, scaffolding, GTPase
3 C Chappie et al., 2011; Morlot et
al., 2012
Endocytosis, clathrin
mediated
Epsin Positive
Amphipathic helix, bilayer asymmetry,
crowding
2 D, 3 E Ford et al., 2002; Stachowiak
et al., 2012, 2013; Busch et
al., 2015
Endocytosis, clathrin
mediated
FCHo1/2 Positive
F-BAR domain, scaffolding
3 A
Henne et al., 2007, 2010
Endocytosis, Shiga
toxin uptake
Gb3 Negative
Lipid receptor, clustering, crowding
2 C
Römer et al., 2007
Endocytosis, Shiga
toxin uptake
Shiga toxin Negative
Clustering, crowding
2 C
Römer et al., 2007
Endosomal tubules
Dynein Positive Microtubule motor, pulling of membrane
compartments, ATPase
3 H
Day et al., 2015
Endosomal tubules
Kinesin Positive Microtubule motor, pulling of membrane
compartments, ATPase
3 H Roux et al., 2002; Delevoye et
al., 2014
Endosomal tubules SNX1 Positive BAR domain, scaffolding, oligomerization 3 A Carlton et al., 2004
ER shaping
Actin Positive
Substrate for motor proteins, membrane
pulling, ATPase
3 H Quader et al., 1989; Liebe and
Menzel, 1995; Estrada et al.,
2003; Sparkes et al., 2009
ER shaping
Atlastins Positive Hydrophobic domain insertion, clustering,
GTPase
2 C
Hu et al., 2009
ER shaping
DP1/YOP1 Positive Insertion of wedge-shaped membrane-binding
domain
2 C
Voeltz et al., 2006
ER shaping
Microtubules Positive
Substrate for motor proteins, membrane pulling
3 H Waterman-Storer and Salmon,
1998
ER shaping Myosin Positive Actin motor protein, ER tubule pulling, ATPase 3 H Estrada et al., 2003
ER shaping RTNs Positive Insertion of hairpin membrane-binding domain 2 C Voeltz et al., 2006
ER shaping Sey1 Positive Hydrophobic domain insertion, clustering 2 C Hu et al., 2009
ER, anterograde
trafficking
COP II Positive
Scaffolding via oligomerization
3 C Lee et al., 2005; Manneville et
al., 2008
ER, anterograde
trafficking
Sar1 Positive
Amphipathic helix, dimerization, scaffolding
2 D & F; 3 C Lee et al., 2005; Beck et al.,
2008; Krauss et al., 2008
ER, anterograde
trafficking
Sec12/31 Positive
Oligomerization, scaffolding, crowding
3 C & E
Copic et al., 2012
ER, anterograde
trafficking
Sec23/24 Positive Curved membrane-binding domain,
scaffolding
3 A
Bi et al., 2002
Filopodia
Actin Negative Polymerization within membrane compartment,
membrane pushing
3 F
Liu et al., 2008
Filopodia
Dopamine
transporter
Negative
Shaped transmembrane domain
2 B
Caltagarone et al., 2015
Filopodia IRSp53 Negative I-BAR domain, scaffolding 3 B Mattila et al., 2007
Filopodia
MIM Negative
I-BAR domain, scaffolding
3 B Mattila et al., 2007;
Saarikangas et al., 2009,
2015
Filopodia srGAP2 Negative F-BAR domain, scaffolding 3 B Guerrier et al., 2009
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to larger curved scaffolds that manipulate shape at up to a mi-
crometer scale (Fig.3, C and D; for more details, see McMa-
hon and Boucrot, 2015). In contrast to true scaffolding, protein
crowding involves large, unstructured protein regions introduc-
ing asymmetry in the membrane by sheer volume increase and
steric constraint (Fig.3E). Cytoskeletal elements can push or
pull membranes by polymerization or with the help of motor
proteins (Fig.3, F–H).
In the following sections of this review, we examine a
range of cellular contexts to illustrate how the core molecular
mechanisms of membrane curvature are combined by cells to
sculpt membrane architecture.
Membrane invaginations at the PM
Endocytosis is the main process by which eukaryotic cells inter-
nalize extracellular material. As clathrin-mediated endocytosis
(CME) has been an important model for elucidating how mem-
branes deform to make vesicles, we consider this pathway in
detail (Table1 and Fig.1A). The FCH-BAR (F-BAR) domain
proteins FCHo1/2, which have a shallowly curved shape, are
early arrivals to incipient sites of CME, yet depart from the bud-
ding intermediate (Fig.3A; Henne et al., 2010). In vitro, these
proteins form variable 20–130-nm-wide tubules from PI(4,5)
P
2
liposomes by orienting the F-BAR domain differently on the
membrane, which is twisted relative to similar domains in other
proteins (Henne et al., 2007). Some F-BAR domain proteins that
are recruited later to sites of CME, including Cdc42-interacting
protein 4 (CIP4), take advantage of both curvature scaffolding
and higher oligomerization mechanisms. Their membrane bind-
ing leads to activation of actin polymerization, probably at re-
gions where membrane tension is high (Fig.3, A and C; Frost et
al., 2008; Boulant et al., 2011; Collins et al., 2011).
As the major PM phosphoinositide, PI(4,5)P
2
is used
during CME for recruiting proteins and is also metabolized in
response to curvature, which tunes this function (Chang-Ileto
et al., 2011; Schmid and Mettlen, 2013). Three key PI(4,5)P
2
binding proteins implicated in membrane curvature are epsin,
CALM (clathrin assembly lymphoid myeloid leukemia), and
the N-BAR domain containing protein amphiphysin. They have
amphipathic helices at their N termini, which fold and protrude
hydrophobic residues upon membrane binding (Fig.2D; Ford
et al., 2002; Peter et al., 2004; Miller et al., 2015). All three
proteins likely help to convert an early, shallowly curved in-
termediate into a deeply invaginated pit (Fig. 2 E). CALM,
epsin, and amphiphysin also bind to clathrin and adapters via
long, natively unstructured regions that contain short motifs for
protein–protein interactions and are expected to cause protein
crowding (Fig.3 E). In vitro studies show that the crowding
of epsin makes a greater contribution to curvature than its am-
phipathic helix (Stachowiak et al., 2012; Busch et al., 2015).
However, endocytic cargo is also expected to be glycosylated
on the outer surface (which should promote crowding in the
opposite direction), so the contribution of crowding in vivo is
not yet clear (Stachowiak et al., 2013).
The clathrin lattice itself initially assembles at fairly at
membrane areas, recruited there by adapter proteins. Dynamic
rearrangements of the lattice occur as the membrane bends to
stabilize the curvature of the nascent vesicle (Fig. 3C; Fotin
et al., 2004; Avinoam et al., 2015). As the vesicle curves more
deeply, the force generated by actin polymerization is impli-
cated in further constriction in mammalian cells (Fig.3G; Col-
lins et al., 2011). In yeast, actin–myosin interactions and myosin
motor activity are essential for endocytic membrane deforma-
tion (Fig.3H; Lewellyn et al., 2015). In the last phase of CME,
the deeply invaginated pit has a highly curved neck region, ex-
hibiting high positive curvature in one direction and negative
curvature in the other (a saddle-shaped intermediate; McMa-
hon and Gallop, 2005). Dynamin oligomers assemble in helices
around the neck of endocytic vesicles (Fig. 3 C), constricting
and increasing pitch to break the membrane using energy from
GTP hydrolysis (Chappie et al., 2011; Morlot et al., 2012).
Whereas CME generally leads to the formation of uniform
vesicles, clathrin-independent endocytic mechanisms invaginate
membranes with varying morphologies, from small tubular or
vesicular structures to large macropinosomes (Fig.1B). Clath-
rin-independent pathways are diverse and are typically named
either after the ligand that is taken up or after a dominant protein
used to invaginate the endocytic intermediate. In the clathrin-in-
Process Protein name Curvature Molecular mechanism Figure References
Golgi, retrograde
trafficking
Arf1 Positive Amphipathic helix, dimerization, scaffolding
via oligomerization
2 D & F; 3 C Lee et al., 2005; Beck et al.,
2008; Krauss et al., 2008
Golgi, retrograde
trafficking
ArfGAP1 Positive
Amphipathic helix, bilayer asymmetry
2 D Bigay et al., 2003; Antonny et
al., 2005
Golgi, retrograde
trafficking
COPI Positive
Scaffolding via oligomerization
3 C Lee et al., 2005; Manneville et
al., 2008
Mitochondrial cristae
F1Fo-ATPase Positive Dimerization, shaping via transmembrane
domain
2 B & C
Jiko et al., 2015
Mitochondrial cristae
Mic10 Positive Insertion of hairpin transmembrane domain,
clustering
2 C Harner et al., 2011; Barbot et
al., 2015
MVBs
ESC RT-III Negative
Scaffolding, helical oligomerization
3 D Hanson et al., 2008; Henne et
al., 2012; Cashikar et al.,
2014; McCullough et al.,
2015
MVBs
Lysobisphosphatidic
acid
Negative
Lipid shape
2 A
Matsuo et al., 2004
Nuclear pore Nup53 Positive Amphipathic helix, bilayer asymmetry 2 F Vollmer et al., 2012
Nuclear pore Nup133 Positive Amphipathic helix, bilayer asymmetry 2 F Doucet and Hetzer, 2010
Nuclear pore
Nup153 Positive
Amphipathic helix, bilayer asymmetry
2 F Mészáros et al., 2015; Vollmer
et al., 2015
Thylakoid shaping CURT1 Positive Amphipathic helix, bilayer asymmetry 2 D Armbruster et al., 2013
Table1. An overview of processes and proteins in membrane bending (Continued)
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How cells bend membranes 379
dependent carriers/glycosylphosphatidylinositol-enriched early
endosomal compartment pathway, which is activated through
small G proteins, the BAR domain containing protein GRAF1
localizes to PI(4,5)P
2
-enriched tubular membranes, stabilizing
their curvature (Table1 and Fig.3A; Lundmark et al., 2008).
Likewise, BAR domains are used by fast endophilin-mediated
endocytosis (Boucrot et al., 2015) for endocytic membrane
bending and scission in neurons and for Shiga and cholera toxin
uptake (Llobet et al., 2011; Renard et al., 2015). In this case, the
key protein, endophilin, combines a curved BAR domain with
insertion of two amphipathic helices to bend the membrane
(Fig.2E). Endophilin uses its src-homology 3 (SH3) domain
to recruit dynamin for scission (Gallop et al., 2006; Masuda et
al., 2006; Jao et al., 2010). Lipid reorganization mechanisms
also participate in shiga toxin uptake. The internalization of
the toxin requires its binding to the glycosphingolipid receptor
globotriaosyl ceramide (Gb3). The clustering of Shiga toxin
and Gb3 promotes invagination by inducing asymmetric stress
in the external leaet of the membranes, both in cells and in
articial membranes (Fig.2C; Römer et al., 2007). Clustering
is not sufcient to induce membrane bending during the uptake
of cholera toxin B subunit. Here, tubulation of the PM is driven
by dynein and dynactin (Fig.3G; Day et al., 2015). In otil-
lin-dependent endocytosis, otillins associate with membranes
via hydrophobic hairpin insertions into the inner leaet (Mor-
row and Parton, 2005) and form homo-/heterooligomers that
colocalize with PM-invaginated microdomains (Fig.2C; Frick
et al., 2007). These microdomains are cholesterol enriched
(Fig.2A) and have a similar morphology to caveolae.
Caveolae are ∼70-nm-wide pit-like membrane invagina-
tions, likely used as a buffering mechanism in response to vari-
ations in PM tension (Fig.1C; Cheng et al., 2015). Caveolin
Figure 2. Mechanisms of direct membrane bending within the lipid bilayer. (A) The shape of the lipid molecules gives rise to spontaneous curvature of the
membrane, and individual lipids can be curvature promoting. (B) Transmembrane proteins can introduce curvature into the bilayer by the shape of their
transmembrane domain. (C) Individual transmembrane protein without curved shapes can bend the membrane via oligomerization and clustering. Interac-
tions with the membrane can be (a) through the formation of single leaflet hairpins; (b) via transmembrane regions; or (c) via protein interactions with lipid
receptors. (D) Many curvature-inducing proteins have peptide sequences that are disordered in solution and that fold into α-helices upon interacting with
the cell membrane, creating one hydrophobic and one polar face, termed amphipathic helices. The hydrophobic side of the helix penetrates like a wedge
into the outer leaflet of the membrane, inducing curvature. (E) Amphipathic helices can cooperate with other proteins, such as the curved scaffold proteins
that contain a BAR domain. (F) Amphipathic helices can also be present in the component proteins of large membrane-associated complexes (also called
protein coats) and thus anchor them tightly onto the bilayer.
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