Physiol Rev 94: 1219 –1248, 2014
doi:10.1152/physrev.00040.2013
DYNAMIC SHAPING OF CELLULAR MEMBRANES BY
PHOSPHOLIPIDS AND MEMBRANE-DEFORMING
PROTEINS
Shiro Suetsugu, Shusaku Kurisu, and Tadaomi Takenawa
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan; Biosignal
Research Center, Kobe University, Kobe, Hyogo, Japan; and Graduate School of Medicine, Kobe University,
Kobe, Hyogo, Japan
Suetsugu S, Kurisu S, Takenawa T. Dynamic Shaping of Cellular Membranes by
Phospholipids and Membrane-Deforming Proteins. Physiol Rev 94: 1219 –1248,
2014; doi:10.1152/physrev.00040.2013.—All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer.
Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia,
podosomes, and other intracellular membrane systems, are molded into their specific submicronscale shapes through various mechanisms. Cells construct their micro-structures on plasma
membrane and execute vital functions for life, such as cell migration, cell division, endocytosis,
exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids,
utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions
with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic
interaction through unstructured polycationic regions, 2) through structured phosphoinositidespecific binding domains, and 3) through structured domains that bind the membrane without
specificity for particular phospholipid. Among the structured domains, there are several that have
membrane-deforming activity, which is essential for the formation of concave or convex membrane
curvature. These domains include the amphipathic helix, which deforms the membrane by hemiinsertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain
superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions
with several BAR proteins as well as other scaffold proteins, resulting in outward and inward
membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and
the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the
precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes.
L
I.
II.
III.
IV.
V.
VI.
VII.
INTRODUCTION
MEMBRANE PHOSPHOLIPIDS
PHOSPHOINOSITIDE-BINDING PROTEINS
GENERAL PRINCIPLES FOR...
THE BAR DOMAIN SUPERFAMILY
BAR PROTEINS AND DISEASE
CONCLUSIONS AND OUTSTANDING...
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I. INTRODUCTION
Life on earth emerged through the appearance of self-sealing, curved, semipermeable membranes, which separate the
inside of a structure from the outside environment. Cells are
separated from the exterior by membrane envelopes, and
thus need to uptake nutrients and excrete wastes through
these barriers to sustain the dynamic activities necessary for
life. Furthermore, cells have to recognize changes in the
surrounding environment and communicate signals across
their membranes, which is especially important for multicellular organisms. To consistently execute these vital functions under variable conditions, cells regulate the shape of
their membranes in a way that reflects the specific needs of
the cell in that environment and/or cellular event (e.g., cell
migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation). Our current knowledge of these cellular structures has been pioneered by using electron microscopy, which enables the visualization of the membrane micro-structures of individual cells. Recently, evidence
concerning the molecular mechanisms underlying the formation of these fine micro-membrane structures has been
revealed.
Eukaryotic cells contain intracellular membrane systems,
whereas most noneukaryotic cell types only have a plasma
membrane and no intracellular membranes. The internal
membranes of eukaryotic cells generally contain numerous
bends, resulting in high levels of membrane curvature. On
0031-9333/14 Copyright © 2014 the American Physiological Society
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SUETSUGU ET AL.
the other hand, the plasma membrane (PM) by itself is a
large, relatively flat structure but contains multiple fine micro-membrane structures, that add high levels of curvature
to the flat PM. These PM structures include outward protrusions, such as filopodia and lamellipodia, as well as invaginations, such as clathrin-coated pits and caveolae, all of
which occur through the deformation of a limited area of
the PM. The process of shaping the membrane is generally
thought to be initialized by the generation of concave (positive) or convex (negative) membrane curvature, resulting in
invaginations and protrusions, respectively. It is increasingly recognized that the formation of these areas of membrane curvature is induced by a variety of protein-driven
mechanisms (3, 140, 174, 184, 255, 258, 274). Most PM
curvature studies have focused on the cytosolic proteins
that utilize electrostatic and hydrophobic interactions to
interface with the negatively charged phospholipids (3, 17).
The phospholipids that form cellular membranes consist of
a hydrophobic tail and a hydrophilic head group. Major
phospholipids possessing the negatively charged head
group include phosphatidic acid (PA), phosphatidylserine
(PS), phosphatidylinositol (PI), and its phosphorylated derivatives, phosphoinositides. PS and PA are thought to be
constitutive components of membranes. PI is also present in
high amounts at the cellular membrane. However, the head
group of PI contains electrically neutral myo-inositol, and
thus PI is not generally considered to contribute to the modulation of cellular functions. In contrast, phosphoinositides
are generated enzymatically by the phosphorylation of PI
through various PI kinases such as PI 3-kinases, and downregulated by PI phosphatases such as PTEN, processes dependent on intracellular signaling in response to extracellular stimuli. The inositol ring of PI can be phosphorylated on
the hydroxyl groups at C-3, C-4, and C-5 positions in different combinations, generating seven different phosphoinositides. The presence of multiple phosphate groups in the
inositol ring results in much stronger negative charges, ⫺2
per one phosphate, than that for PS and PA, both of which
contain net charge of ⫺1 at neutral pH (FIGURE 1A). With
these characteristics and dynamic production dependent on
stimuli, phosphoinositides are considered to be fundamental lipid species for regulation of cellular processes.
These phosphate modifications of inositol allow specific
recognition by phosphoinositide-binding proteins. The
structural folds of these proteins provide pockets for various patterns of inositol ring phosphorylation, which allow
them to recognize specific phosphoinositides (FIGURE 1B).
These domains that recognize specific phosphoinositides include pleckstrin homology (PH), Phox (PX), and epsin
NH2-terminal homology (ENTH) and are found in proteins
that act for signal transduction cascades (10, 23, 50, 292).
In contrast, there are proteins with structural fold, but
without specific recognition of phosphoinositides. The
Bin-Amphiphysin-Rvs (BAR) domain superfamily, which
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include proteins containing BAR, N-BAR, FCH-BAR (FBAR), and inverse-BAR (I-BAR) domains, bind phosphoinositides without strict recognition of the phosphate at the
inositol ring. The structural fold of BAR domains is rather
used to sense and/or to generate tubule-like structures of
phosphoinositide-containing liposomes and cellular membranes (3, 174, 255). However, the structural fold of the
proteins is not always necessary for the binding of proteins to
phosphoinositide or anionic lipids. Some proteins have the
clusters of basic amino acid residues lacking a structural fold
(poly-cationic motif) that are responsible for lipid binding
through nonspecific electrostatic interactions (155, 291).
When we observed the cellular membrane, the PM is supported and stabilized by the cortical actin cytoskeleton, which
is formed and recycled through regulated polymerization and
depolymerization processes. Interestingly, the lipid-binding
proteins provide the connection between membrane curvature
and reorganization of the actin cytoskeleton. For example, the
BAR domain superfamily proteins, which sense and/or induce
the curvature of the membrane, often interact with proteins
that regulate actin reorganization (280, 293). Thus the formation of fine micro-membrane structures is thought to be organized by the binding of membrane-deforming proteins to negatively charged lipids, which leads to the interaction and concentration of actin polymerization regulators.
In this review, we describe the characteristics of membrane
phospholipids, with an emphasis on phosphoinositides, and
then the mechanisms utilized by phosphoinositides to regulate a variety of cellular events through their binding motifs and domains. We also discuss the relationship between
phospholipids and the mechanisms underlying the organization of membrane curvature and micro-membrane structure formation, mainly focusing on the PM.
II. MEMBRANE PHOSPHOLIPIDS
A. Localization of Various Phospholipids in
the PM
The PM is mainly constructed of phospholipids. The phospholipids form a bilayer (⬃5 nm in thickness) by utilizing
their amphipathic characteristics, with the polar head
groups facing the water and the hydrophobic fatty acyl
chains facing each other on the interior of the bilayer. Many
different types of phospholipids can be accommodated into a
single bilayer, and the lipid composition is expected to influence the physiological functions of the membrane (FIGURE 1).
The four primary phospholipids found in eukaryotic membranes are phosphatidylcholine (PC), phosphatidylethanolamine (PE), PS, and sphingomyelin (SM). In addition, two
less abundant species, PI and PA, can also exist in the membranes. We still cannot exclude the existence of trace
amount of other phospholipids, of which function is less
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SHAPING OF CELLULAR MEMBRANES
A
Cylindrical lipids
PC
P
PS
P
Conical lipids
N
NH3
COO
–
Inverted conical (cone-shaped) lipids
PE
P
NH3
PA
P
–
–
P
PI(3,4,5)P3
PI(4,5)P2
P –
P –
–
P
–
P
Acyl chains
Flat bilayer
B
Electrostatic interaction
e.g.,
poly-cationic region
+ + + + +
– –
P
– P
–
Inverted hexagonal phase of tubes
with negative curvature
Lipid-binding domain
e.g.,
PH domain,
PX domain,
ENTH domain,
etc.
–
–
+
– –
P
P
PI
Glycerol
Inositol
Phosphate
Micelles with
positive curvature
Amphipathic helix or
hydrophobic loop insertion
e.g.,
ENTH domain,
ALPS motif,
etc.
+
+
+ –
–
P
P
P –
P –
P –
– –
P
P
BAR domain,
F-BAR domain,
I-BAR domain,
etc.
+
– –
P
P
+
–
+
– –
P
P
+
– –
P
P
–
+
–
FIGURE 1. Phospholipid shape and the associated mechanism of membrane recognition and deformation. A: the
shape of a lipid molecule is classified into three categories: cylindrical, conical, and inverted conical. Alone, each type
of lipid will form a unique membrane structure; however, actual membranes, such as lipid bilayers, are composed
of a mixture of these three types of lipids. Even taking into account the different lipid shapes, biological membranes
are, overall, relatively flat when compared with the submicron scale structures, such as clathrin-coated pits. B: the
protein domains that recognize and bind to lipids are currently classified into four categories: 1) simple, chargedriven interactions without recognition of specific lipid species; 2) lipid species recognition by the protein structure;
3) membrane recognition and deformation with the insertion of an amphipathic helix or a hydrophobic loop; and 4)
protein structure-based deformation of the membrane (e.g., BAR domains).
characterized (246). In addition, PI is used as a source of
various trace amounts of “phosphorylated phosphatidylinositol,” known as phosphoinositide (10).
The various types of lipids show asymmetric distribution in
the inner and outer leaflets of the PM lipid bilayer. PC and SM
are predominantly localized in the outer membrane, whereas
PE is primarily found in the inner membrane (64, 136). Furthermore, PS is exclusively found in the inner leaflet of the PM
along with PI and several phosphoinositides, including PI
4-phosphate [PI(4)P], PI 4,5-bisphosphate [PI(4,5)P2], and PI
3,4,5-trisphosphate [PI(3,4,5)P3] (23, 50). The phospholipid
composition also varies in each cell type and membrane (155,
308). In eukaryotic cells, PC, PE, and SM comprise 45–55,
15–25, and 5–10% of the total quantity of lipids, respectively
(308), while PS and PA only account for 2–10 and 1–2%,
respectively. On the other hand, PI represents ⬃10 –15%,
while phosphoinositides are only present as trace amounts and
are generally less abundant by one order of magnitude.
PI(4,5)P2 is the most abundant phosphoinositide and comprises ⬃0.5–1% of the total quantity of lipids, while the
amounts of other phosphoinositides are even lower.
Although the number of phosphoinositides present in the
membrane is very low, they nonetheless play important
roles in receptor-activated signal transduction as they are
generated (phosphorylated) and degraded (dephosphorylated) in a signal-dependent manner. It is well known that
PI(4,5)P2 is hydrolyzed by phospholipase C to generate two
second messengers, inositol 3,4,5-trisphosphate (IP3) and
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SUETSUGU ET AL.
diacylglycerol (DAG) (13), in response to receptor activation. Furthermore, PI(3,4,5)P3 acts as a key player in PI
3-kinase signaling through PI(3,4,5)P3-binding domains
(309). Other phosphoinositides also regulate a wide variety
of membrane functions and interact with multiple cytosolic
proteins, which are described in detail below. Although
phosphoinositides may play major roles in a wide range of
cellular events, PA has also been shown to bind the polycationic motifs of Rac and DOCK2, a Rac GTP-exchange
factor, leading to Rac activation (23, 192).
The distribution of PS and phosphoinositides causes the electrostatic properties of the membrane to vary (17). Membranes
of the early secretory pathway [e.g., endoplasmic reticulum
(ER) and cis-Golgi] have a weakly charged cytosolic leaflet due
to the scarcity of PS and phosphoinositides. In contrast, membranes of the late secretory pathway (e.g., endosome and PM)
have a highly charged cytosolic leaflet because of their abundance of PS and phosphoinositides (17). The electrostatic nature of PS allows it to act as a partial substitute for phosphoinositides in membranes, especially during the interaction with
cationic motifs. Membrane-deforming domains, such as BAR,
F-BAR, and I-BAR, are one such example of cationic motifs
that adapt to curved anionic membrane surfaces using electrostatic interactions (3). However, these domains seem to prefer
PI(4,5)P2 to PS (173, 174). Therefore, phosphoinositides,
rather than PS, are the preferred phospholipids for many proteins, likely owing to their stronger charge. This preferential
interaction has made phosphoinositide a major player in controlling the construction of fine membrane structures utilized
for a variety of cellular functions, including cell migration,
membrane trafficking, and cell proliferation (50, 173).
B. Roles of the Phospholipid Head Group and
Fatty Acid Tail
Phospholipids constituting the majority of lipid bilayers
have a variety of polar head groups, differing in size and
charge. For example, the polar heads of PE are smaller than
those of PS, PC, and SM, whereas phosphoinositides have
very large polar heads because of the presence of the phosphate-bearing inositol ring (FIGURE 1A). The size of the
polar head group causes each of these lipid types to form
different structures (127). PE is defined as a conical-shaped
lipid and by itself forms a structure with negative curvature,
similar to the inverted hexagonal phase of tubes, with the
head groups on the inside and the hydrophobic tails on the
outside (FIGURE 1A). In contrast, PI and phosphoinositides
have an inverted conical shape and form structures with
positive curvatures. PC, PS, and SM are cylindrical-shaped
lipids, which preferentially form flat bilayer structures. In
the eukaryotic PM, PC and PS are the most common constituents of the outer and inner leaflets, respectively. PC and
PS alone form flat or gently curved planar membranes in
vitro, but PE, cholesterol, and phosphoinositides cannot
form bilayers without other lipids. Furthermore, the pres-
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ence of these lipids causes destabilization of the planar
membranes formed by PC and PS, which seems to be required for vesicle budding, fusion, and other shape changes
involving biological membranes (127). Therefore, local accumulation of these lipids in specialized areas of the PM can
modulate the membrane shape with their physical properties, electrostatic nature, and through the recruitment of
membrane-binding proteins.
Lipid geometry also depends on the acyl chains, particularly
the presence of double bonds that produce a kink in the
middle of the chain (17). For example, the oleoyl chain
consists of 18 carbons with one double bond (C18:1) and
occupies a larger volume than a palmitoyl chain (C16:0)
that consists of 16 carbons without any double bond. Thus
the size of the polar heads and species of acyl chain influence
the lipid packing of membranes. Furthermore, cholesterol,
while not a phospholipid, is also abundant in the PM and is
a particularly unique component because it is amphipathic,
with a polar hydroxyl group and a hydrophobic planar
steroid ring. These properties allow it to intercalate between
the phospholipids, with its hydroxyl group near the polar
head groups and its steroid ring parallel to the acyl chains of
the phospholipids, thus improving lipid packing. The PM
contains phospholipids with relatively high-saturated fatty
acids as well as high levels of cholesterol allowing for tight
lipid packing. In contrast, the ER is characterized by loose
lipid packing due to the abundance of unsaturated phospholipids and scarcity of cholesterol.
PI and phosphoinositides contain high levels of polyunsaturated fatty acids, predominantly consisting of 1-stealyl
and 2-arachidonyl acyl chains. An arachidonyl chain
(C20:4) has four double bonds, and therefore, its acyl chain
bends sharply. The large polar head group and abundance
of arachidonic acid in phosphoinositides cause them to disturb lipid membrane packing and, in regions of the membrane where phosphoinositides are concentrated, larger
spots of lipid-packing defects may be formed. Indeed, brain
PI(4,5)P2 [dominantly 1-stearyl-2-arachidonyl PI(4,5)P2]
was found to be a potent modifier of lipid bilayer when
gramicidin A channels’ sensitivity to changes in lipid bilayer properties was measured (233). Furthermore, cytosolic proteins seem to interact electrostatically with
loosely packed lipids more easily than closely packed
lipids (16, 18, 170, 178). In contrast to the loose-packing
nature of individual phosphoinositide molecule, phosphoinositides mixed with other phospholipids in a global
cellular membrane are known to form phosphoinositideenriched microdomains (156, 226). These microdomains
are thought to be further stabilized or reinforced in the
presence of phosphoinositide-clustering proteins (173,
234). Thus cytosolic proteins are probably more apt to
access and interact with phosphoinositides that are concentrated at these “hot spots.”
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SHAPING OF CELLULAR MEMBRANES
sitions 3, 4, and 5) in the inositol ring. These phosphoinositides are synthesized from PI and localize at the restricted membranes (50).
C. Specific Localization of Each
Phosphoinositide
There are seven phosphoinositides found in mammalian
cells (FIGURE 2A): PI 3-phosphate [PI(3)P], PI(4)P, PI
5-phosphate [PI(5)P], PI 3,4-bisphosphate [PI(3,4)P2], PI
3,5-bisphosphate [PI(3,5)P2], PI(4,5)P2, and PI(3,4,5)P3.
The type of phosphoinositide can be identified by determining the combination of phosphate groups at three sites (po-
A
PI 3-phosphatase
e.g.
MTM1
MTMR
PTEN
Sac1
PI3K
Main phosphoinositides found at the PM are PI(4,5)P2, and
less abundant PI(3,4)P2 and PI(3,4,5)P3, but each has its
own specific localization pattern within the PM (FIGURE
2B). PI(4)P is enriched at the Golgi apparatus where it critically regulates Golgi integrity, while PI(3)P and PI(3,5)P2
PI 4-phosphatase
PI(3)P
PI 5-phosphatase
e.g.
SHIP1,2
OCRL1
INPP5
SKIP
PI(3,4)P2
PI4K
PI 3-phosphatase
e.g.
PTEN
PI4K
PI
PI(4)P
PI 4-phosphatase
e.g.
Synaptojanin1, 2
Sac1
PI3K
PI5K
PI 5-phosphatase
e.g.
Synaptojanin1, 2
OCRL1
INPP5
PI3K
PI5K
PI(5)P
PI(3,4,5)P3
PI(3,5)P2
PI 3-phosphatase
e.g.
MTM1
MTMR
PI 3-phosphatase
e.g.
PTEN
TPIP
PI(4,5)P2
PLC
IP3
DAG
B
P
P
P
P
P
P
P
P
P
P
P
P
FIGURE 2. Localization of phosphoinositides. A: the kinases and phosphatases important for the metabolism of a variety of
phosphoinositides are shown. PI(4,5)P2,
PI(3,4)P2, and PI(3,4,5)P3 are exclusively
localized at the PM. The most abundant
phosphoinositide, PI(4,5)P2, is mainly synthesized by PIP5K from PI(4)P, which is
synthesized from PI by PI 4-kinase. The
formed PI(4,5)P2 is further synthesized to
PI(3,4,5)P3 by type I PI 3-kinase or degraded to two second messengers, DAG
and IP3, by phospholipase C in response to
extracellular stimuli. PI(3,4)P2 is formed
from PI(4)P by types I and II PI(4)P 3-kinases or from PI(3,4,5)P3 by PI(3,4,5)P3
5-phosphatases, such as SHIP1, SHIP2,
and SKIP. B: localization of PI(3)P, PI(4)P,
PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3 determined using GFP-tagged domains that
recognize each specific phosphoinositide.
PI(3)P, PI(4)P, PI(4,5)P2, PI(3,4)P2, and
PI(3,4,5)P3 were detected with the Fyve
domain of Hrs and the PH domains of
Fapp1, PLC␦1, Tapp1, and Grp1, respectively. With the use of the Akt PH domain,
PI(3,4)P2 and PI(3,4,5)P3 were detected
simultaneously. In the figure, “2 x” indicates
two domains linked in tandem. Src-transformed cells and mouse fibroblasts were
stained with rhodamine-phalloidin (red) to
visualize the F-actin and with anti-HA or anti-myc antibody (green). PI(3)P is present at
the endosome, while PI(4)P is present at the
Golgi (arrows). PI(4,5)P2 is localized at the
PM, and both PI(3,4)P2 and PI(3,4,5)P3 are
localized at podosome/invadopodia (arrowheads). Bar: 20 ␮m. [Adapted from Oikawa
et al. (193). Copyright 2008 Oikawa et al.
Journal of Cell Biology. 182: 157–169.
doi:10.1083/jcb.200901042.]
P
P
P
P
P
P
P
P
P
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SUETSUGU ET AL.
are enriched at the endosome and the multivesicular body,
respectively. Recently, the role of PI(4)P at the plasma membrane as a pool of anionic phospholipid was discovered
(99). The least characterized PI(5)P is found on the PM, in
endosomes, and in the nucleus (263). It is noteworthy that
each phosphoinositide can be found outside of its wellknown subcellular location. For example, PI(4,5)P2 is
found in the nucleus and is thought to regulate gene expression (177). The different localization of phosphoinositides
in each organelle assists in the targeting of particular proteins to distinct membranes through the specific binding
domains unique to each type of phosphoinositide. This subcellular localization of each phosphoinositide is mostly determined by its lipid kinases and phosphatases that are spatially restricted to specific membranes (119, 243).
PI(4,5)P2 is mainly synthesized from PI(4)P by PI(4)P 5-kinase (PIP5K), which consists of three isoforms, PI(4)P5K␣,
PI(4)P5K␤, and PI(4)P5K␥ (243). Although there is a synthetic pathway to form PI(4,5)P2 from PI(5)P, PI(5)P is very
rare. Therefore, most PI(4,5)P2 is generally considered to be
generated from PI(4)P. Degradation of PI(4,5)P2 is known
to produce two second messengers, IP3 and DAG, when
cells are activated by extracellular stimuli (13) and is an
important step in the inositol lipid signaling system. In this
reaction, receptor-activated phospholipase C is used to hydrolyze PI(4,5)P2 to IP3 and DAG. On the other hand, there
are two types of phosphatases, 5-phosphatase and 4-phosphatase, that dephosphorylate PI(4,5)P2 to mainly control
steady-state levels. PI(4,5)P2 is the most abundant phosphoinositide and presumably plays a pivotal role in cytoskeleton remodeling, endocytosis, and membrane deformation,
which are induced by the interaction between the PM and
certain cytosolic proteins (23, 50, 52, 83, 234).
PI(3,4)P2 is formed from PI(4)P by class I and II PI 3-kinases
or through the dephosphorylation of PI(3,4,5)P3 by
PI(3,4,5)P3 5-phosphatases, such as SHIP1, SHIP2, and
SKIP. PI(3,4)P2 is hydrolyzed to PI(3)P or PI(4)P by 4-phosphatases, such as INPP4A and INPP4B, or the 3-phosphatase PTEN (FIGURE 2A). PI(3,4)P2 serves as a localization
signal for the construction of podosome/invadopodia and
as a regulator for the circular dorsal ruffle formation on the
membrane (105, 193, 195).
PI(3,4,5)P3 is present in negligible levels under resting conditions, but in response to various extracellular stimuli, it is
synthesized at the PM from PI(4,5)P2 by class I PI 3-kinases
(72, 221, 309). Interestingly, PI(3,4,5)P3 is selectively present at the leading edge of the cells. In polarized epithelial
cells, it is abundant in the basolateral membrane, but absent
in the apical membrane (167). Furthermore, PI(3,4,5)P3 recruits cytosolic proteins to the PM through interaction with
specific PI(3,4,5)P3-binding domains, particularly those
containing PH domains.
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The aberrant accumulation of PI(3,4,5)P3, which can be
caused by loss of PI(3,4,5)P3 3-phosphatase activity or an
elevation in PI 3-kinase activity, causes a variety of diseases.
For example, PTEN mutant mice develop several types of
cancer (49), and disruption of the SHIP2 gene causes these
mice to develop modified insulin signaling (268). The precise mechanisms causing these diseases are not yet clear, but
based on these data, it is hypothesized that the development
of each disease occurs through the altered functions of
PI(3,4,5)P3 in different cellular compartments.
III. PHOSPHOINOSITIDE-BINDING
PROTEINS
A. Electrostatic Interactions Between
Phosphoinositides and Cytoskeletal
Proteins
Phosphoinositide binding at cationic-charged regions rich
in basic amino acid residues has been observed for a variety
of cytoskeletal proteins (253). Importantly, a lot of actin
regulatory proteins contain basic amino acid residues that
can in fact interact with phosphoinositides, mainly
PI(4,5)P2. Although the net charge of PI(4,5)P2 is less than
that of PI(3,4,5)P3, the contribution of PI(3,4,5)P3 to the
surface charge of the membrane is presumably low because
of the lower amount of PI(3,4,5)P3 found in the membrane
compared with PI(4,5)P2. PI(4,5)P2 has been shown to interact with multiple actin regulatory proteins, including
vinculin (79, 89), talin (106, 296), ␣-actinin (79, 80), cofilin
(327), gelsolin (126), Ezrin-Radixin-Moesin (ERM) (111),
and other actin regulatory proteins. The binding of phosphoinositides to the actin regulatory proteins regulates the
rearrangement of cortical actin filaments, which support
the membrane deformation to construct filopodia and lamellipodia. Furthermore, this binding also activates G-actin
nucleation-promoting factors (NPFs), including neural
Wiskott-Aldrich syndrome protein (N-WASP) and WASPhomologous verprolin homology protein 2 (WAVE2), both
of which are known to be ubiquitously expressed in the cell
(293). These data suggest that inositol lipid signaling influences the remodeling of the actin cytoskeleton and membrane morphology through changes in the levels of
PI(4,5)P2 at the PM. Indeed, increased expression of
PI(4)P5K␣ induces massive actin filament formation (257),
while expression of synaptojanin, a phosphoinositide phosphatase, causes disruption of actin filaments (240). Taken
together, it appears that an increase in the level of PI(4,5)P2
tends to promote actin filament formation, whereas a decrease in the level of PI(4,5)P2 leads to actin depolymerization (125).
Notably, PI(4,5)P2 is unexpectedly concentrated in lipid
rafts (90, 114, 210), where Rho- and ADP-ribosylation factor (Arf)-GTPases facilitate PI(4,5)P2-synthesizing PI 5-ki-
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SHAPING OF CELLULAR MEMBRANES
nases by inducing the recruitment of these kinases to these
sites in the PM. Thus actin assembly can be initiated with
remarkable efficiency at these PI(4,5)P2-rich rafts through
the enhanced recruitment and activation of WASP and
ERM proteins, leading to the promotion of the actin polymerization and actin filament assembly (92). Thus PI(4,5)P2regulated rearrangement of actin filaments in compartmentally restricted areas of the PM seems to concentrate all of
the factors necessary for micro-structure formation.
B. NPFs, N-WASP and WAVEs, Are
Activated by Phosphoinositides in
Combination With Other Factors
When cells start to migrate in response to chemoattractants,
rapid actin polymerization is induced at the leading edge to
form filopodia and lamellipodia. Such rapid actin polymerization is also necessary for forming other micro-membrane
structures, such as endocytic pits. In resting cells, the barbed
ends of actin filaments are capped by large redundant proteins to prevent spontaneous elongation of the filaments.
Therefore, to trigger rapid new actin polymerization during
the rearrangement of the cortical cytoskeleton in response
to extracellular stimuli, cells must form actin nuclei from
which new filaments can be polymerized. Thus actin polymerization takes place through two steps: actin nucleation
and polymerization. First, the actin monomer (G-actin)
forms an actin trimer, which serves as a seed or nucleation
site for the polymerization. This step is time-consuming and
is the rate-limiting step in de novo actin polymerization.
Once a trimer is formed, the actin filament elongates from
the barbed end depending on the concentration of G-actin.
However, to induce quick actin polymerization at the leading edge of migrating cells in response to a stimulus, the
time required for the nucleation step has to be shortened
(200, 212, 283). NPFs that shorten the time required for a
trimer formation have been discovered (231, 293) and include two WASP family proteins, WASP (286) and
N-WASP (179, 180), and three WAVE family proteins,
WAVE1, -2, and -3 (181, 282). Importantly, these proteins
are also phosphoinositide-binding proteins.
N-WASP is anchored and activated by PI(4,5)P2 at the PM
in the presence of Cdc42, inducing quick actin polymerization using the Arp2/3 complex (228). N-WASP also has
several multifunctional domains, including a WASP homology 1 (WH1) domain, a basic region, a GDB/CRIB motif, a
proline-rich region, and a verprolin-homology, cofilin-like,
and acidic domain (VCA) region (293). The WH1 region is
essential for association with WIP/CR16/WICH (or WIRE)
proteins, which are important for stabilization of N-WASP
or WASP (134, 168, 222, 279). The basic region of NWASP is the domain used to bind to PI(4,5)P2, which serves
as an anchor point on the membrane. The VCA region, in
which the V region binds to actin monomers and the CA
region binds to the Arp2/3 complex, is the domain that
plays an important role in shortening nucleation time. The
Arp2/3 complex includes actin-like proteins, Arp2 and
Arp3 (212). Therefore, when two Arps of the Arp2/3 complex and one actin monomer are gathered on the platform
of VCA in N-WASP, the time-consuming nucleation process is skipped. However, it has been shown that full-length
N-WASP does not activate the Arp2/3 complex as
strongly as the VCA domain does alone, suggesting that
the VCA region is masked in inactive N-WASP. Interestingly, the addition of PI(4,5)P2 and Cdc42 appears to
unmask the VCA region, which consequently leads to the
binding of the Arp2/3 complex to N-WASP, resulting in
fast actin nucleation (137, 180, 228). The Arp2/3 complex further assembles actin filaments from the sides of
already existing filaments, thus forming branched, meshlike actin filaments (22, 77).
However, N-WASP forms straight actin filaments, such as
filopodia and actin rods, in cells. Therefore, it is thought
that actin bundling proteins probably reform the branched
filaments into straight filaments after the branched-actin
filaments are generated by the Arp2/3 complex (21, 310). It
is interesting that filopodia-like structures can be reconstituted with N-WASP and Toca-1 or other BAR proteins,
which have membrane-deforming abilities (152). It is likely
that Toca-1 is recruited to the PI(4,5)P2-enriched area and
then activates N-WASP by its membrane-deforming activity
(287). In muscle, N-WASP can form unbranched filaments
by using the multiple actin monomer-binding sites of Nebulin, the SH3-containing protein that binds to N-WASP. In
this case, trimer assembly is thought to be mediated by the
V region and Nebulin in forming the straight filaments
(288).
Unlike N-WASP, WAVE is anchored and activated by
PI(3,4,5)P3 in the presence of Rac, forming lamellipodialike actin filaments (196, 281). There are three isoforms of
WAVE: WAVE1, -2, and -3. All WAVEs also have a VCA
region through which they activate the Arp2/3 complex
(293). However, WAVEs are activated differently than NWASP. Activation occurs by PI(3,4,5)P3 and Rac binding,
inducing lamellipodia and membrane ruffle formation.
WAVE1–3 can form a pentameric protein complex with
HSPC300/BRICK, Abi1, Nap1, and Sra1/PIR121 (34,
120). When cells are activated by chemoattractants or
growth factors, they form lamellipodia at the leading edge
where PI(3,4,5)P3 is concentrated (112), indicating that the
WAVE proteins, particularly the abundantly and ubiquitously expressed WAVE2 isoform, are essential for lamellipodia formation in this context (325).
WASP and WAVE family proteins play crucial roles in
membrane deformation during endocytosis and formation
of outward protrusions by supporting the structures and
generating force by de novo actin polymerization. In addition to these NPFs, other NPFs have been recently discov-
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SUETSUGU ET AL.
ered, but mainly function in intracellular trafficking. These
NPFs include WASP and SCAR homolog (WASH), WASP
homolog associated with actin, membranes, and microtubules (WHAMM), and junction-mediating and regulatory
protein (JMY). Currently, it is unclear whether these proteins are related to the membrane-deforming proteins,
though WASH complex is found to associate with dynamin
and endosomal BAR protein, sorting nexin 1/2 (SNX 1/2)
(47, 93). These new NPFs are discussed in further detail in
other reviews (69, 230).
C. Phosphoinositide-Binding Domains
A variety of membrane-associated proteins have phosphoinositide-binding domains with binding pockets for particular phospholipids. These domains recognize phosphoinositides with structural specificity (9, 291). Among them,
the PH domain was the first phosphoinositide-binding domain reported and is conserved among a large number of
proteins in the human proteome (103). The other phosphoinositide-binding domains are PX (31, 133, 323), Fab1,
YOTB, Vac1p, and EEA1 (FYVE) (84, 202), ENTH (71,
125), AP180 NH2-terminal homology (ANTH) (154, 275),
some of PDZ such as those found in Syntenin, PTP-BL,
CASK, and Tiam-1 (335), Tubby (242), Dock homology
region 1 (DHR-1) (41), GOLPH3 (51, 321), some of C2
such as those found in synaptotagmin and PLAs (37, 38,
45), band 4.1, Ezrin, Radixin, and Moesin (FERM) (111,
191), and glucosyltransferase, Rab-like GTPase activator,
and myotubularin (GRAM) domains (12, 301).
Some of these phosphoinositide-binding domains show specific and high-affinity binding to a particular phosphoinositide. For example, PLC␦1 PH domain specifically binds
PI(4,5)P2 as well as its head group IP3 (103). The PH domain of Grp1 binds exclusively to PI(3,4,5)P3, while that of
Akt/PKB binds both PI(3,4,5)P3 and PI(3,4)P2 (73, 143).
Tapp1 and Fapp1 PH domains specifically bind to PI(3,4)P2
and PI(4)P, respectively (54). The PH domain specificity has
enabled the construction of probes used to detect the localization of these phosphoinositides (FIGURE 2B) (122, 193).
Other than PH domain, GOLPH3, which consists of only
one domain, specifically binds to PI(4)P (51, 321). However, the lipid recognition of the PH domains is generally
less strict compared with that of other domains discussed
here.
The PH domains that recognize PI(4,5)P2, PI(3,4,5)P3, or
PI(3,4)P2 are involved in functions important during a variety of events in the PM. For example, once the PI(3,4,5)P3
hot spots are formed in the PM by the activation of PI
3-kinase, Akt/PKB is recruited to the PM through the PH
domain and is activated. Akt/PKB is a serine/threonine kinase that binds to PI(3,4,5)P3 through its NH2-terminal PH
domain (73, 298) and is activated by phosphorylation at
Thr-308 and Ser-473. Furthermore, activated Akt/PKB
1226
modulates a wide range of cellular functions, such as cell
survival, proliferation, polarity formation, cortical actin
regulation, and glucose metabolism. PI(3,4,5)P3 also modulates the activities of Cdc42 and Rac through the PH domains of their guanine nucleotide exchange factors (GEFs),
such as Tiam1 (241) and Vav (100), leading to membrane
ruffle formation.
Lipid specificity is also observed for the other phosphoinositide-binding domains. The ENTH domain specifically
binds to PI(4,5)P2 (125). PI(3)P is enriched in early endosomes and acts as a recruiter of PI(3)P-binding proteins to
this site, and the FYVE domains of early endosome antigen
1 (EEA1) and hepatocyte growth factor-regulated tyrosine
kinase substrate (Hrs) show exclusive, high-affinity binding
to PI(3)P. However, the FYVE domain of protrudin localizes to endosome and binds to PI(4,5)P2, PI(3,4)P2, and
PI(3,4,5)P3 (87, 262). The PX domains of SNX3, NADPH
oxidase component, and p40phox also bind to PI(3)P rather
than PI(3,4)P2 (31, 56, 133, 319, 323). On the other hand,
the p47phox PX domain binds both lipids, but binds more
strongly to PI(3,4)P2.
A new lipid-binding domain, SH3YL1, Ysc84p/Lsb4p,
Lsb3p, and plant FYVE protein (SYLF) domain, was recently identified in SH3YL1 protein (104). This domain
preferentially binds to PI(3,4,5)P3. Furthermore, this domain contains an amphipathic ␣-helix in the NH2 terminus,
which has the capability to deform liposomes into vesicles
(104). The role of amphipathic helices in membrane deformation is described in further detail in a later section. The
SYLF domain has been found to play an important role in
forming circular dorsal ruffles (CDRs), which are characterized by short-lived, dynamic, ring-shaped F-actin-enriched structures thought to be involved in motile cell polarity formation, macropinocytosis, and the internalization
of cell surface receptors. Interestingly, the formation of micro-membrane structures, such as CDRs and podosomes/
invadopodia, has been shown to require PI(3,4)P2 in common (104, 193). In CDR formation, SH3YL1 is recruited to
the dorsal PM region where PI(3,4,5)P3 is newly generated
in response to receptor stimulation. Then, Arf-GTPase activating protein (GAP) with a Rho-GAP domain, an ankyrin
repeat, and a PH domain 1 (ARAP1) is gathered to
PI(3,4,5)P3 -enriched sites through its PH domain, and its
Arf GAP activity is activated. Since SH3YL1 forms a protein complex with the PI(3,4,5)P3 5-phosphatase SHIP2,
PI(3,4,5)P3 at CDRs is hydrolyzed to PI(3,4)P2. These data
indicate that ARAP1 is first activated by the increase in
PI(3,4,5)P3 and then becomes inactivated by its hydrolysis to
PI(3,4)P2. Thus this phosphoinositide conversion leads to
the transition of the nucleotide-binding state of Arfs from a
GDP-bound (CDR expansion) to a GTP-bound form (CDR
closure), which controls the size of the CDRs through the
regulation of ARAP1 substrates, Arf1 and Arf5 (105).
Therefore, conversion of PI(3,4,5)P3 to PI(3,4)P2 is not only
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SHAPING OF CELLULAR MEMBRANES
required for CDR formation but also regulates the dynamic
movement of CDRs. Furthermore, Rac is suggested to be
activated by Tiam1(Rac GEF), which is known to be activated by PI(3,4,5)P3, at the early endosome and is then
delivered to the CDRs (198) where it is involved in the
rearrangement of actin filaments during CDR formation.
It is clear that several of the phosphoinositide-binding domains have the capability to deform liposomes into tubules
and small vesicles. For example, the ENTH domain deforms PI(4,5)P2-containing liposomes to small vesicles by
inserting its NH2-terminal amphipathic helix into the membrane. Furthermore, the BAR, F-BAR, and I-BAR domains
preferentially bind to PI(4,5)P2 and PI(3,4,5)P3, allowing
them to deform PI(4,5)P2/PI(3,4,5)P3-enriched membranes
into vesicles and tubular-like structures, although they can
generally bind negative-charged phospholipids, such as PS.
As discussed later, some BAR and F-BAR domain-containing proteins are accompanied by an extra lipid-binding domain, such as PH and PX domains. The presence of neighboring PH and PX domains may help to target the protein
to sites where a particular phosphoinositide exists. The
membrane deforming activity of BAR, F-BAR, and I-BAR
domains is highly dependent on lipid-binding ability, and
lipid-binding deficient mutants lose membrane-deforming
activity.
IV. GENERAL PRINCIPLES FOR
MEMBRANE CURVATURE FORMATION
The mechanisms of membrane deformation can be classified into three categories: lipid metabolism, protein scaffolding, and binding protein insertion/interaction (174).
The representative proteins that are involved in membrane
deformation are summarized in TABLE 1.
A. Lipid Metabolism
The shape of each lipid molecule, cylindrical, conical, or
inverted conical, is different (FIGURE 1A). Enrichment of
inverted conical or conical lipids is thought to drive membrane deformation. This enrichment can be achieved by
increased lipid metabolism, which is mediated by the synthesis and/or transport of new lipid molecules to the membrane. The metabolism-mediated mechanisms include the
enzymatic conversion of precursor lipids to usable forms as
well as the addition of the head group onto the lipid backbone. The production of phosphoinositides with relatively
large head groups might by itself be enough to induce membrane curvature, but the low concentration of phosphoinositides in the PM indicates that this is not the primary
method used to alter global membrane curvature. However,
phosphoinositides do appear to afford such effects locally in
concentrated areas of the PM, such as clathrin-coated pits,
caveolae, and near membrane receptors. Another metabolic
mechanism employed to alter membrane curvature utilizes
the hydrolysis of the acyl chain, which removes one of the
two acyl chains in the lipid molecule. For example, PA is
normally a conical lipid, but lysophosphatidic acid (LPA) is
an inverted conical lipid. Therefore, conversion of PA to
LPA is assumed to induce curvature formation. It was proposed that PA-hydrolyzing activity of endophilin was one of
such example. However, the activity was the contaminant
from the source of the protein purification, and indeed the
membrane deformation was mediated by the BAR domain
of endophilin described later (82).
The transport of lipid molecules to induce membrane curvature can be mediated by flippases, which induce lipid asymmetry between hemi-layers during cytokinesis and cell migration
(57, 58). During apoptosis, type IV P-type ATPases (P4ATPases) and CDC50 family proteins form a putative phospholipid flippase complex used for the translocation of PS
and PE (135), exposing PS to the outer leaflet of the PM,
which normally does not contain PS. Furthermore, the lipid
scramblase, transmembrane protein 16F (TMEM16F), can
also perform Ca2⫹-dependent scrambling of membrane
phospholipids, resulting in the exposure of PS to the cell
surface leading to apoptosis (285). However, the full mechanism of such enzymatic regulation and its precise effects on
membrane curvature are still unclear.
B. Indirect Protein Scaffolding
Membrane deformation using the scaffold-mediated mechanism was first found for caged vehicles, such as clathrincoated pits, which can be pinched off to form clathrincoated vesicles. Clathrin coats are involved in clathrin-mediated endocytosis from the PM to form endosomes.
Clathrin coats are not membrane-binding proteins, but they
are connected to the membrane through adaptor proteins
(APs) or cargo proteins (3, 50). On the other hand, COPI/II
coats are involved in vesicle transport between the ER and
the Golgi apparatus. Some components of COPI/II coats
directly bind to the membrane, but GTPases such as Arf
play important roles for COPI/II assembly on the membrane (3, 50). In both cases, the formation of rigid lattice
scaffolds, involving both the APs/cargo proteins as well as
the secondary binding proteins, is thought to be the structural determinant of such caged vesicles.
The membrane curvature formation can also be just triggered by the assembly of proteins on the membrane without
specific membrane binding properties. This mechanism is
recently proposed as protein “crowding” for curvature formation (140, 246, 273). In this case, the assembly of proteins on the membrane with some anchoring to the membrane can bend the membrane. However, the existence of
crowding mechanism in vivo is still unclear. The protein
assembly of, for example, AP protein complex at the clathrin-coated pit might be considered to be such example
(273).
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SUETSUGU ET AL.
Table 1. The major membrane deforming proteins
Mode of Membrane Deformation
Classification
ALPS motif
Name of
Protein
Preferred
Lipid
Amphipathic
helix
Loop
insertion
Structural
fold
Multimer
formation
Subcellular
Structures
ArfGAP1
⫹
COPI coat assembly
GMAP-210
⫹
cis-Golgi
Atg14L
⫹
ER
Remarks
Reference Nos.
19, 55
Captures vesicles
205
204
autophagosome
BAR domain
⫹
Nup133
Arfaptin1
N
⫹
⫹?
nuclear envelope
Golgi
Arfaptin2
N
⫹
⫹?
Golgi
Binds to Arl and
Arf
203
86, 294
Binds to Arl and
Arf
189
157, 334
PICK1
N
⫹
⫹?
Spine
APPL1, 2
PI(3,4,5)P3
⫹
⫹?
Endosome
With PH domain
ASAP1
N
⫹
⫹?
Invadopodia
With PH domain
15, 129, 256
SNX4,8,9,18
N
⫹
⫹?
Endosome
With PX domain
Amphiphysin1
PI(4,5)P2
⫹
⫹
⫹?
Clathrin-coated pit
98, 306, 307,
314
131, 208, 225,
271
Amphiphysin2
(Bin1)
PI(4,5)P2
⫹
⫹
⫹?
Clathrin-coated pit
Membrane
insertion
151
T-tubule
helix H0
Endophillin
PI(4,5)P2
⫹
⫹
⫹
Clathrin-coated pit
Membrane
insertion
Bin3
Toca-1
N
PI(4,5)P2
⫹
⫹
⫹?
Lamellipodia
265
⫹
⫹?
Clathrin-coated pit
26, 83, 123,
287
CIP4
PI(4,5)P2
⫹
⫹
Clathrin-coated pit
74, 75, 132,
148, 237,
260, 303
2
binds to Rab5
N-BAR
domain
142, 183, 270
helix H0, H1
F-BAR
domain
Lamellipodia
FBP17
IF-BAR
domain?
I-BAR
domain
Others
PI(4,5)P2
⫹
⫹
Clathrin-coated pit
75, 132, 260,
303
107, 109
FCHo1,2
PI(4,5)P2
⫹
⫹?
Clathrin-coated pit
PSTPIP1,2
PI(4,5)P2
⫹
⫹?
Podosome?
36, 302
PACSIN1-3/
Syndain
1-3
PI(4,5)P2
⫹
⫹?
Clathrin-coated pit
7, 91, 102, 211,
215, 254,
261, 315
⫹
Caveolae
Nostrin
srGAP1-4
N
⫹
FES
N
⫹
FER
IRSp53
N
PI(3,4,5)P3,
PI(4,5)P2
⫹
⫹
Caveolae?
Filopodia
Spines
⫹
Lamellipodia
Outward
protrusions
⫹?
Filopodia, lamellipodia
with FX domain
Outward
Protrusions
MIM
N
⫹
⫹?
Filopodia, lamellipodia
IRSTKS
N
⫹
⫹?
Filopodia, lamellipodia
PinkBAR
N
⫹
⫹?
Epsin
PI(4,5)P2
⫹
Planar membrane
sheets close to
adherence junction
Clathrin-coated pit
␣-Synuclein
⫹
Synaptic vesicle
Arf1
⫹
COPI coat in the Golgi
apparatus
Arf6
SH3YL1
PI(3,4,5)P3
EHD2
PI(4,5)P2
Dynamin
PI(4,5)P2
⫹
⫹
⫹?
Caveolae
⫹
Clathrin-coated pit
149, 244
42, 59, 97, 159
124
1, 172, 234,
284, 332
213
71, 125
128
Small GTPase
Circular dorsal ruffle
⫹
Caveolae
62, 88
104
27, 43
Membrane
scission
molecule
62, 63, 187,
245
⫹
Synaptic vesicle protein
Deformation by
C2 domain
117, 147, 199
Flotillin
⫹
Clathrin-independent
endocytic pathway
Hemi-insertion
loops
52
Caveolin
⫹
Caveolae
Reticulon
⫹
ER
258, 259
Late endosome
118
Synaptotagmin
PI(4,5)P2
ESCRT
complex
Cytokinetic abscission
Autophagosome
N, negatively charged lipids.
1228
Outward
protrusions
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312
SHAPING OF CELLULAR MEMBRANES
Classically, the actin polymerization is thought to be a major source of force generation for membrane curvature formation, especially for protrusive structures such as lamellipodia and filopodia. The actin filaments exist in high density beneath the plasma membrane, and thus can be thought
to be a mechanism for indirect protein scaffolding for membrane curvature formation. The elongating barbed ends of
polymerizing actin filaments are thought to push membrane
by their filling of the space between the filaments and fluctuating membrane, where the filling by actin filaments functions as “ratchet” for membrane protrusion (68, 174, 273).
C. Binding Protein Insertion/Interaction
There are two mechanisms of membrane deformation that
occur through the direct binding of the proteins to the membrane. One utilizes the electrostatic interactions between
the lipid-binding domains on the protein surface and the
negatively charged lipids at the membrane. Two wellknown classes of proteins with structural folds conducive to
this type of electrostatic interaction are the BAR domaincontaining proteins (BAR proteins) and the endosomal sorting complexes required for transport (ESCRT) proteins.
The recently identified BAR proteins are thought to deform
membranes primarily by electrostatic interaction followed
by the recruitment of other membrane-modifying proteins.
The ESCRT proteins utilize their electrostatic interactions
with the membrane to function in membrane budding,
which is essential for releasing virus vesicles to the outside
of the cells as well as the biogenesis of membrane organelles
containing vesicles, otherwise known as the multivesicular
body (8, 108, 188). The details of ESCRT-mediated membrane changes are described in depth in other reviews (118,
219).
Direct protein insertion into the membrane is another
mechanism used to alter membrane curvature. This mechanism of membrane deformation was first characterized for
proteins containing an amphipathic helix, whereby the cylindrical helix is laid down on the membrane, exposing the
hydrophilic surface to the cytosol (3) (FIGURE 1B). Although
the exposure of this surface prevents the penetration of the
helix into the membrane, like that of a transmembrane protein, the hydrophobic surface alone is still inserted into the
hemilayer of the membrane, thereby enlarging the area of
the inner leaflet of the bilayer and causing membrane deformation. If the hydrophilic surface is positively charged, the
helix interacts with anionic phospholipids in the cytosolic
layer of the membrane, while inserting the other side into
the hydrophobic interior, which is the case for the ENTH
domain of epsin and the N-BAR domain of amphiphysin
and endophilin (FIGURE 3). The positively charged, unstructured amino acids in the NH2-terminal region of the ENTH
domain, in combination with the structure region of the
domain, interact specifically with the head group of
PI(4,5)P2. The interaction of ENTH domain and PI(4,5)P2-
containing membrane then turns the last 17 residues into an
amphipathic helix, which is then inserted into the membrane (FIGURE 3) (32, 71, 125, 328).
Furthermore, if the hydrophilic surface of the helix does not
have a strong charge, then the helix has a tendency to function as a sensor for membrane curvature, capturing the
vesicles for intracellular vesicle transport. Such a sensor
function has been found in the amphipathic lipid packing
sensor (ALPS) motif of the ArfGAP1, which is involved in
COPI coat disassembly (19, 55). ALPS motifs have been
found in proteins associated with the nuclear envelope
(Nup133), the ER (Atg14L), and the cis-Golgi (ArfGAP1,
GMAP-210) and thus seem to be involved in the events of
the early secretory pathway (3). The ALPS motif of ArfGAP1 is unstructured in solution, but inserts its bulky hydrophobic residues between loosely packed lipids, and
forms an amphipathic helix on highly curved membranes.
This helix appears to favor insertion at sites of lipid packing
defects that arise in the external leaflet of a liposome when
its curvature increases. Furthermore, the ALPS/ArfGAP1
helix differs from classical amphipathic helices, as there is
an abundance of serine and threonine residues on its polar
face. Thus ALPS motifs favor curvature and lipid-packing
defects rather than curvature and electrostatic interactions.
These ALPS motifs are characterized by their secondary
amphipathic helix structures and are not related to a specific
amino acid sequence (19, 55).
In addition to amphipathic helices, proteins with stretches
of hydrophobic residues are also known to form loop/
wedge-shaped insertions into the membrane, inducing deformation. Caveolins and reticulons are examples of such
membrane-embedded proteins that, after interacting with
the membrane, undergo oligomerization to generate curvature. Furthermore, self-assembly of caveolin and reticulon
can induce membrane remodeling to create plasma membrane invaginations and tubulate the ER (52, 255, 259,
273, 312). A similar protein structure is also found in flotilin, which is a scaffold protein involved in a less-characterized clathrin-independent endocytic pathway (52, 255,
312).
There are two additional important examples for membrane deformation mediated by membrane insertion. One is
the eps15 homology (EH) domain-containing proteins 2
(EHD2) protein. The EHD2 protein is known to oligomerize, and this oligomerization and the insertion appear to be
essential for membrane deformation (43). The C2 domain
from several proteins, such as synaptotagmin, Doc2a/b, and
group IVA cytosolic phospholipase A2 (cPLA2␣), induces
and/or senses membrane curvature likely through membrane insertion, which occurs in Ca2⫹-dependent manner
(117, 166, 317, 331). The roles and mechanisms of C2
domain-mediated membrane deformation are discussed in
other reviews (155, 175).
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SUETSUGU ET AL.
endophilin A1
Epsin (ENTH)
clathrin
Amphiphysin
FBP17
PI(4,5)P2
PH domain
SNX9 (BAR-PX)
FCHo2
PI(4,5)P2 etc
PACSIN2
PI(4,5)P2
P
m
P
P
em
br
an
e
tu
bu
le
APPL1 (BAR-PH)
Dynamin
endosome
monomer
ring
clathrin-coated pit
PI(3,4,5)P3
caveolae
P
P
EHD2
lamellipodia
lysosome
P
P
CIP4
FES
ER
nucleus
IRSp53
SH3YL1
Golgi
filopodia
PinkBar
COPI/II
Arfaptin-Arl
Radixin
(FERM domain)
Arp2/3
actin filament
branched
actin filament
ArfGAP1
WAVE
complex
N-WASP
WASP
PI(4,5)P2
(auto-inhibited
miniWASP)
FIGURE 3. Diagrams of the membrane-deforming proteins during their interaction with the membrane. The
representative structure of the different membrane-deforming proteins and their mode of action are illustrated. The BAR domains, actin regulatory proteins, and dynamin are shown with their structures in contact
with the membrane. Proteins with amphipathic helices are shown with a wheel diagram at the helix. The wheel
diagram was generated by HELIQUEST (85).
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SHAPING OF CELLULAR MEMBRANES
V. THE BAR DOMAIN SUPERFAMILY
A. Identification of BAR Proteins
BAR proteins are classified as having a BAR (N-BAR), FBAR, or I-BAR domain depending on their sequence (TABLE
1). However, all BAR proteins are composed of a helix
bundle, where three helices of one monomer form into a
dimer, producing a six-helix bundle that displays various
degrees of intrinsic curvature (FIGURE 3). Thus the structural classification of these three BAR domain subfamilies is
less important. However, the important distinction between BAR proteins is in the degree of curvature innate to
each BAR domain, which allows a cell to generate a large
range of varying curvature when utilizing a scaffolding mechanism. In all BAR domains, the positively charged residues are
enriched at a particular surface of the dimer, identifying it as
the membrane contact site (280). With the exception of the
I-BAR proteins, the membrane contact surface is found on
the concave side (or the inside of the arc-shaped protein
dimer). Considering only the dimer of the BAR domains,
most BAR domains are thought to fit with negatively
charged lipid membranes through their positive concave
face (FIGURES 1B AND 3). Furthermore, they induce membrane tubulation in vitro. Importantly, the membrane tubules formed by the BAR proteins in these experiments are
topologically the same as those found in the membrane
invaginations of in vivo plasma membranes.
It is still unclear whether BAR domains actually deform
the membrane in cells directly or rather sense the membrane and then recruit other membrane-deforming proteins. However, membrane deformation and membrane
sensing cannot be distinguished in cells. The sensing and
deformation would be inherently linked processes, and
one may be the other under different conditions. It has
been experimentally shown that purified BAR proteins
can deform the membrane in vitro, but it seems that the
concentration of the BAR proteins appears to distinguish
its deforming and sensing activities (271). Furthermore,
in a model experiment where a nanofabricated pattern
was applied onto the plasma membrane, the BAR proteins appeared to be recruited upon membrane deformation, thus supporting the curvature sensing ability of the
BAR proteins (81).
Interestingly, the BAR domains from amphiphysin and
Rvs161/167 have putative amphipathic helices at both
ends of the dimer units of the BAR domain and are thus
called N-BAR domains (208). The amphipathic helix, in
conjugation with the concave structure, is important for
its ability to sense and induce membrane curvature.
Hence, the N-BAR module plays a dual role in regulating
membrane curvature. Insertion of NH2-terminal amphipathic helices into membranes causes changes in lipid
packing and effectively creates local membrane curva-
ture. Thus the NH2-terminal amphipathic stretch plays
pivotal roles in membrane tubulation and shallow helical
fold insertion into the membrane. The scaffolding and
loop/wedge insertion mechanism are independent of each
other; however, both are thought to work simultaneously
to induce membrane curvature. In addition, the amphipathic helix can also function as a sensor for membrane
curvature as it acts as an anchor for the membrane and
can sense high levels of positive curvature as in case of
ALPS motif. Thus N-BAR proteins also could sense
curved membrane domains by probing the surface for
lipid packing defects. Other than that found in amphiphysin, there are only a small number of other BAR
domains that have such insertions. For example, the endophilin BAR domain has a similar amphipathic helix at
the end of its dimer, but also has a helix at the middle of
its dimer (FIGURE 3) (169). The PACSIN1/Syndapin I and
PACSIN2/Syndapin II F-BAR domains also have the
membrane insertion loops at the membrane interacting
surface (FIGURE 4A) (261, 315). The missing-in-metastasis (MIM) I-BAR domain also causes the membrane insertion (234). However, the other BAR domain has not
been reported to have such a membrane insertion mechanism.
Epsin ENTH domain, which has a hydrophobic insertion, is
apt to form small vesicles and extremely narrow tubules. In
contrast, BAR domain scaffolds generally induce membrane tubulation. However, N-BAR domains, which have
both insertions and BAR domain scaffolds, can generate a
mixture of vesicles and tubules. In fact, an increased number of amphipathic helices on a BAR domain correlates with
increased vesiculation and smaller vesicle size, driving
membrane scission (24). Moreover, a high concentration of
BAR domains with amphipathic insertions has been shown
to induce vesiculation of liposomes (24, 91). In terms of
physical chemistry, each lipid molecule is thought to adapt
itself to an energetically lower state. It might be reasonable
to assume that the lower state is achieved by the vesiculation of the tubulated membrane. It is likely that the high
degree of membrane curvature generated by a hydrophobic
insertion can make phase separation of membrane lipids by
clustering specific lipids, which probably leads to the formation of boundaries between lipid clusters on the membrane (232, 267). This boundary might function as cutting
line for vesiculation or scission of the membrane (4, 11,
158, 232). It is proposed that shallow hydrophobic insertions are sufficient for vesicle formation, driving membrane
fission, whereas crescent-like protein scaffolds, such as BAR
domains without amphipathic helix or loop insertion support formation of membrane tubules, hence disfavoring fission.
B. Clathrin-Mediated Endocytosis
The most extensively studied function of BAR domains involves clathrin-mediated endocytosis (CME) (23, 161).
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SUETSUGU ET AL.
A
Clathrin coated pit formation (in mammals)
time
Plasma membrane
actin
Clathrin
FBP17
FCHo2
Amphiphysin
PACSIN2
Endophilin Arp2/3 complex
NPF
(e.g. N-WASP)
Epsin
SNX9
Dynamin
B
C
Transverse section
Filopodia
Lamellipodia
IRSp53
NPF
(N-WASP
or WAVE)
actin
actin and the Arp2/3
Arp2/3 complex
D
Lamellipodia
BB B
P
a
P
P
P
P
P
B
B
B
m embrane
P
P
B
B
B
a me
m
BB
brane
m
pla s m
B
B B
B
Clathrin coated pits
plas
Filopodia
B
B
P
P
FIGURE 4. The interplay between membrane deforming proteins and the cytoskeleton. A: possible sequential
recruitment of membrane deforming proteins and the organization of the actin filaments (40, 107, 295). The
actin filaments induced by F-BAR protein were estimated based on Suetsugu (277). [Part of this schematic is
modified from Suetsugu and Gautreau (280).] B: the I-BAR protein domain and the actin cytoskeleton assembly
at the protruding filopodia and lamellipodia. C: transverse section of the clathrin-coated pit shown in A,
highlighting the direction of actin filament organization. D: actin filaments utilized for protrusion (filopodia and
lamellipodia) formation as well as clathrin-mediated endocytosis are illustrated. The abbreviations B and P
indicate the barbed-end (fast-growing end) and the pointed-end (slow-growing end), respectively. The B ends
always face the membrane, presumably providing the force for membrane shape formation. [Adapted from
Suetsugu (278), by permission of Oxford University Press.]
CME is initialized by clathrin-coated pit formation, which
is the proposed site of sequential recruitment and detachment of the BAR proteins (295). It is hypothesized that first,
an F-BAR protein with shallow curvature, FCHo1/2, is recruited, which initiates the assembly of the clathrin-coated
pits (FIGURE 4A) (107, 305). Then, F-BAR proteins, such as
1232
FBP17 and PACSINs, are recruited, followed by BAR domains with significant curvature, such as endophilin and
amphiphysin (23, 142, 295). The FCHo1/2 proteins are
unique among the BAR proteins because they do not have
the SH3 domain found in other BAR proteins, but instead,
FHCo1/2 has a motif for binding to epsin and other mole-
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SHAPING OF CELLULAR MEMBRANES
cules, providing the scaffold for additional protein binding
at the clathrin-coated pits (107, 304). The other BAR proteins, including those recruited at the clathrin-coated pits
after FCHo1/2, have the SH3 domain that can bind to dynamin and N-WASP. Dynamin is an essential protein that
causes the mechanical scission of the clathrin-coated pits
into clathrin-coated vesicles and has been shown to cooperate with BAR proteins during membrane scission (FIGURE
4A) (176, 190). Furthermore, the sequential recruitment of
BAR proteins at the clathrin-coated pits is also observed in
yeast (131), and while a dynamin-like molecule does exist in
yeast, it is not essential for scission of clathrin-coated pits.
Instead, WASP-homolog-mediated actin polymerization
plays an essential role during this process (272). However,
detailed understanding of how this sequential recruitment is
regulated is currently limited.
It is known that PI(4,5)P2 plays a crucial role in CME.
Clathrin-coated pit formation is initiated in PI(4,5)P2-enriched membranes, and PI(4,5)P2 is essential for the early
steps in CME, namely, nucleation, cargo selection, and coat
assembly, whereas the latter steps, such as scission and uncoating, depend on PI(4,5)P2 elimination. A lot of proteins
associated with clathrin-coated pits bind directly to
PI(4,5)P2, but clathrin does not. The clathrin-binding proteins, such as epsin and AP180, have ENTH and ANTH
domains, respectively, that interact with PI(4,5)P2. Classical BAR domain-containing proteins, such as endophilin
and amphiphysin, also bind to PI(4,5)P2 and can deform
PI(4,5)P2-containing membranes (329). However, it should
be noted that BAR domains generally do not recognize
PI(4,5)P2 specifically, rather bind to membrane with electrostatic interactions. Furthermore, dynamin also binds to
PI(4,5)P2 through its PH domain. After the early steps of
CME are complete, endophilin is thought to recruit the
PI(4,5)P2 phosphatase synaptojanin, which depletes
PI(4,5)P2 from the clathrin-coated pits, thereby inducing
clathrin uncoating and scission (207, 249). Thus the CME
process is closely regulated by PI(4,5)P2.
Furthermore, the BAR proteins appear to finely regulate
actin polymerization during CME, depending on the membrane size and structure. The first example, demonstrating
the dependency of actin polymerization on the size of the
membrane, indicated that actin polymerization by Toca-1
and FBP17 in combination with N-WASP was affected by
the diameter of the liposome (287). The curvature-dependent formation of actin filaments was also observed when
actin polymerization was induced by the BAR-PX domain
of SNX 9 on phosphoinositide-coated silica beads of a defined curvature (83). In this case, the actin filaments were
found to be elongated toward the neck of constricting clathrin-coated pits (FIGURE 4C) (40, 277). Therefore, actin polymerization was found to push the membrane in analogous
ways during both filopodia, lamellipodia, and clathrincoated pit formation, presumably under the regulation of a
diverse set of BAR domain proteins
(280).
(FIGURE 4, B AND D)
Along with the BAR proteins, N-WASP is thought to
induce actin polymerization at the clathrin-coated pits,
as indicated in the several examples described above
(FIGURE 4, A, C, AND D) (40). However, N-WASP-mediated
actin polymerization by itself is not required for endocytosis
in cultured cells under normal conditions without membrane tension. Notably, this actin polymerization is required for endocytosis under membrane tension, presumably to support the membrane during scission (25, 67, 322),
indicating a context specific need for N-WASP-mediated
polymerization. In addition to N-WASP, other actin regulatory proteins, such as WAVE, formin, VASP, and Cobl,
can also function downstream of the BAR proteins (74, 78,
94, 150, 250, 326).
C. Other Invagination/Endocytotic
Structures
The involvement of BAR domain proteins in the formation
of other membrane structures is less clear. Caveolae are
flask-shaped invaginations that are enriched in cholesterol,
sphingolipids, and PI(4,5)P2 (76, 258). The major structural
component of caveolae is the integral membrane protein
caveolin, a membrane-embedded protein that undergoes
oligomerization to generate curvature (229). Caveolins
have transmembrane domains that contain an amphipathic
helix inserted into the bilayer (201). This helix also binds
cholesterol and could cause the raftlike lipid composition of
the caveolae (61). Recent work has identified a protein family associated with caveolae, which includes cavin1– 4.
Cavin proteins interact with caveolin in mature caveolae,
and the deletion of cavin results in the loss of caveolae (101,
110). Thus this cavin complex may function as a scaffold
for caveolin and other components of caveolae.
With regard to F-BAR-domain proteins, PACSIN2 was
shown to be present in caveolae and appears to be important for morphogenesis of the caveolae structure (102, 146,
254). Indeed, depletion of PACSIN2 results in the loss of
morphologically defined caveolae. Similar to the BAR proteins in the clathrin-coated pits, PACSIN2 binds to dynamin
and N-WASP through its SH3 domain (102, 254). There is
an Asn-Pro-Phe (NPF) motif in PACSIN2 that can bind to
EHD2 (43, 186), which is thought to stabilize the caveolae
through the association with actin filaments (276), likely
induced by PI(4,5)P2 (FIGURE 3) (76, 266). Thus it is clear
that PACSIN2 plays an important role in the formation of
caveolae. Another F-BAR protein, NOSTRIN, is also reported to be localized at caveolae, although it is not clear
specifically how NOSTRIN functions in this membrane
structure (244).
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SUETSUGU ET AL.
Among the variety of endocytotic pathways that internalize
numerous cargoes, several appear to proceed independently
of the canonical protein clathrin. The clathrin-independent
carriers (CLIC) and GPI-enriched endocytic compartments
(GEEC) pathways play major roles in such uptake (141,
235). GTPase regulator associated with focal adhesion kinase-1 (GRAF1) protein, which contains an NH2-terminal
BAR domain, a PH domain, a Rho-GAP domain, a prolinerich domain, and a COOH-terminal SH3 domain, is shown
to be important in coordinating small G protein signaling
and membrane remodeling to facilitate internalization of
CLIC/GEEC pathway cargoes (162). In this pathway,
GRAF1 localizes to PI(4,5)P2-enriched sites in the membrane via its NH2-terminal BAR and PH domains, and deforms the membrane to facilitate endocytic intermediate
formation.
D. Intracellular Organelles
The membranes of intracellular organelles have also
emerged as a site of BAR domain function. For example, the
PX-BAR domain of SNX18 is shown to function in autophagosome formation (144). Furthermore, the endosome
contains SNX1, -4, -8, -9, and -18, all of which have BAR
domains, and appear to be important during the formation
of tubular networks and in endosome trafficking (98, 299,
306, 307). Endosomes also contain adaptor protein containing a PH domain, phosphotyrosine-binding domain,
and leucine zipper motif 1 (APPL1), which contains an
NH2-terminal BAR domain, a central PH domain, and a
COOH-terminal phosphotyrosine-binding (PTB) domain
(FIGURE 3). The crystal structure of the BAR domain dimer
of APPL1 revealed that it contains two four-helical bundles
(FIGURE 3), whereas other BAR domain dimers have only
three helices in each bundle (157, 334). The PH domain is
located at the ends of the BAR domain dimer, and all of the
BAR, PH, and PTB domains have shown preferential binding to PI(3,4,5)P3. The BAR-PH domain has also been
shown to fold together, forming the binding site for the
small GTP-binding protein Rab5.
The Golgi network also contains BAR proteins, such as
Arfaptin, which is the BAR domain only protein and modulates small GTPases, such as Arl and Arf, for Golgi trafficking (86, 294). For example, Arfaptin 2 binds to Arl1
through its BAR domain (FIGURE 3) and is then recruited to
the Golgi membrane, where it induces membrane tubules.
Interestingly, the Arl1 in complex with the BAR domain of
Arfaptin 2 resides at a similar position as the PX domain in
the PX-BAR unit of SNX9 (FIGURE 3) (189), suggesting a
similar mechanism underlying BAR domain targeting to
specific organelle membranes.
E. Membrane Protrusions
The protrusive structures found on membrane surfaces had
previously been believed to be organized solely by the
1234
power of actin polymerization (160). Therefore, it was surprising that membrane deformation was found to be, at
least in part, mediated by BAR proteins. Currently, there
are two classes of BAR proteins with protrusion forming
ability, proteins containing an I-BAR, and some of the FBAR domain-containing proteins, which comprise only a
small population of the entire BAR protein superfamily.
The I-BAR domain, which has a kink in the helix, was first
found in IRSp53 and then later discovered in MIM (172,
284). The kink produces the surface of the inverted membrane, to the opposite direction than do classical BAR domains. The I-BARs can deform membranes in vitro into
protrusion-like shapes, consequently clarifying the role of
I-BAR domain in negative curvature formation (FIGURE 3).
However, it is still uncertain whether such membrane deformations are actually taking place in vivo, although several researchers have concluded that I-BAR proteins are
localized at and involved in the formation of filopodia and
lamellipodia (1, 20, 251) (FIGURE 4B). In other words, it is
still unknown whether protrusions forming independently
from actin filaments actually exist in the natural cellular
environment. As Ahmed et al. (1) pointed out, the artificial
filopodia induced by the I-BAR domain in cell culture and
the “genuine” filopodia observed in vivo show distinctively
different dynamics: the former is relatively stable and less
motile with a lifetime of more than 10 min, compared with
the latter with a lifetime of 79 –142 s. Importantly, the
full-length IRSp53 has the scaffolding ability to generate
actin-mediated forces, which may explain the differences in
the filopodia dynamics. The relative contribution of curvature-sensing, -generating, and scaffolding is currently unknown, and further studies are necessary to provide additional insight into the mechanism of IRSp53-mediated
filopodium formation.
Recently, another I-BAR function has been proposed in
Drosophila, where I-BAR proteins indirectly help protrusion formation by preventing invagination for endocytosis
from occurring at these membrane sites (218). In addition,
Pinkbar, a relative of IRSp53, has more flat surface for
membrane interaction and functions in generating planar
membrane sheets in intestinal cells (FIGURE 3) (213).
Another class of BAR domains with protrusion-forming
activity was found in srGAP proteins that were classically
called as Slit-Robo GTPase activating proteins. The srGAP
family of proteins consists of four family members, srGAP1,
2, 3, and 4, which have from the NH2 terminus an F-BAR
domain, a Rho-GAP, and an SH3 domain. Although each
family member contains a GAP domain, there are differences in GTPase activity between the proteins. The RhoGAP domain of srGAP1 has been shown to promote GTP
hydrolysis of Cdc42 and RhoA (320), whereas the GAP
domains of srGAP2 and srGAP3 are both specific for Rac1
(97, 269), and srGAP4 can act on both Cdc42 and Rac1
(311). All four family members display spatially and tem-
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SHAPING OF CELLULAR MEMBRANES
porally distinct patterns of expression in the central nervous
system (5, 70) and have been shown to regulate cell migration and neuronal morphology in mammalian cells. Surprisingly, the overexpression of the F-BAR of srGAP2 did not
cause invagination, but instead led to outward protrusions
along the cell surface (29, 42, 59, 97). As well, srGAP1 and
srGAP3 induce filopodia formation, although they are less
potent than srGAP2. And incubating this domain with liposomes caused deformations of the membrane similar to
those created by the IRSp53 I-BAR domain in vitro. These
data were unexpected because F-BAR domains, in general,
had been believed to induce positive membrane curvature.
However, it is plausible that the F-BAR domain of srGAP
functions like I-BAR, although there is no reported structure for the F-BAR domain of those proteins to corroborate
this hypothesis. Thus this family of Rho-GAP is now defined as inverse F-BAR (IF-BAR), which is functionally distinct from other F-BAR domains.
Recent studies show that the F-BAR-containing srGAP family of proteins can regulate dendritic spine morphogenesis.
Dendritic spines, small bulbous protrusions found on neuronal dendrites, are postsynaptic structures that receive inputs from axons. Spine formation, remodeling, and shape
change are associated with the brain’s ability to store and
process information in response to an experience. With the
use of primary cultures of neurons, both srGAP2 and srGAP3 were shown to promote dendritic spine maturation,
presumably by facilitating dendritic filopodia formation at
the site of developing spines (29, 30). The overexpression of
the GAS7 F-BAR domain has also been reported to induce
cellular protrusions, which are suggested to be related to
neurite extension (330). However, the mechanisms of these
relationships remain unclear because the structures have
not been solved yet.
The Fes or Fer proteins had first been characterized as members of nonreceptor protein tyrosine kinases, which play
important roles for intracellular signal transduction for cell
adhesion and migration (95). The Fes or Fer proteins have
the F-BAR domain, SH2 domain, and tyrosine kinase domain. The Fes or Fer has a recently characterized neighboring FX domain. Whereas the F-BAR domain of Fer alone is
not sufficient for membrane binding, the FX domain is
found to be required for efficient binding. Furthermore, the
FX domain specifically binds to PA, but not PI(4,5)P2, and
functions in concert with the F-BAR domain (124). Indeed,
the addition of PA seems to enhance the tyrosine kinase
activity of Fer in vitro, and phospholipase D (PLD)-generated PA appears to serve as an activator in response to
extracellular stimuli, since tyrosine kinase activation was
suppressed by the addition of PLD inhibitors. Furthermore,
the FX domain may be important for targeting the F-BAR
domain of Fer to the lamellipodia formation sites in the
membrane as the F-BAR-FX domain was found to be necessary for lamellipodia formation by Fer.
F. How Is the BAR Domain Superfamily
Regulated?
The BAR domain superfamily is unique in its structural
characteristics and its structure-membrane curvature interactions. Therefore, because of their numerous biological
functions, it is important to understand how the BAR domain superfamily is regulated. A large number of cytosolic
proteins are known to be activated and transduce signals
upon phosphorylation or the addition of a charge to the
protein. Recently, the BAR domains of ACAP4 have been
reported to be activated by phosphorylation during membrane association in a manner dependent on epidermal
growth factor (EGF) stimulation (333). However, when we
consider the increase of negative charge brought by phosphorylation, the consequence might be the opposite, because an addition of negative charge may enhance repulsion
from anionic lipids of the membrane. Drosophila syndapin/
PACSIN is also reported to be phosphorylated, but in this
case, the phosphorylation inactivates the membrane tubulation ability (289). Mammalian PACSIN1 and PACSIN2
are also reported to be phosphorylated, which results in
defective neuronal morphologies and/or functions (2, 217).
Because phosphorylation introduces a strong negative
charge, it is assumed that the electrostatic membrane interactions are destroyed, thereby leading to weaker membrane
affinity.
Autoinhibition is a widely used mechanism for the regulation of multidomain proteins. For example, N-WASP is
regulated by the intramolecular interaction between the
VCA and the CRIB motif (228). This interaction is released
by the binding of PI(4,5)P2 and Cdc42, thereby freeing the
VCA to associate with the Arp2/3 complex. WAVE is also
regulated by protein complex formation, where the VCA
region is exposed through phosphorylation and binding of
the small GTPase Rac (197). However, among the BAR
proteins, such autoinhibition has not been reported. Only
one curious example is PACSIN1, in which the SH3 domain
is proposed to interact with the F-BAR domain intramolecularly; however, the lack of a 1:1 stoichiometry for the
F-BAR and the SH3 domains in the crystal structure indicates that this is a weak interaction (224).
It would not be surprising if the regulation of BAR proteins
depended on molecule-molecule interactions (i.e., interactions with other proteins or lipids). This type of mechanism
is known to be utilized in the regulation of SH3 binding
proteins. In this process, if IRSp53 is phosphorylated by
Par1b kinase or an unidentified kinase downstream of
GSK3␤, then the binding of the SH3 domain is hindered by
the binding of 14 –3-3 protein (39, 130, 227). However, to
date, no regulatory mechanism controlling the membrane
deforming ability of the BAR proteins has been discovered.
So far, it is not clear how BAR domain assembly, which is
required for forming a stable membrane micro-structures, is
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SUETSUGU ET AL.
regulated. Membrane bending due to thermal fluctuation
may be sufficient for triggering BAR domain assembly. This
initial BAR assembly likely results in the assembly of several
additional molecules, which may lock the membrane into
the conformational structure formed by the protein domains. In this case, BAR proteins are pure sensors of membrane curvatures, of which no particular regulation might
be required. However, this theory has not yet been tested
directly. If BAR proteins are actively engaged in the formation of membrane curvature, then it is still unclear how the
membrane deforming ability of the BAR proteins is regulated, even 9 years after the structural determination of the
amphiphysin BAR domain.
higher ordered structure is proposed to function for formation of membrane curvature (213, 224). Especially, Trp141
of Pinkbar is shown to be involved in lateral contact for
formation of planar sheet of the I-BAR domain. It is important to note that such insights into the assembly of BAR
proteins into higher order structures are typically based on
the protein packing observed in the crystalized form. In a
crystal, the proteins must interact with each other to form a
stabilized crystal structure, and the contact sites between
molecules can be determined from the spatial organization
of the proteins in these structures. However, it is still unclear whether such higher order structures actually exist for
the membrane-deforming proteins.
G. Higher Order Structure Assembly
Furthermore, the localization of the BAR protein is also
expected to affect its ability to form higher order structures,
and, importantly, proteins with similar structures do not
necessarily localize in the same areas of the cellular membrane. For example, the F-BAR protein FBP17 is reported
to localize at clathrin-coated pits (260), while its homolog,
CIP4, can localize to lamellipodia (163, 236, 297). In addition, although the three PACSIN isoforms have a similar
structure, PACSIN2 and -3 were localized at caveolae,
while PACSIN1 was not (254). PACSIN2 is also involved in
EGF receptor internalization, presumably through clathrincoated pits (46, 295). These data suggest that the BAR
domain is not the only determinant of BAR protein localization. Rather, the formation of a protein complex might
be more important than the BAR domain-membrane interaction for the localization of the protein.
When we consider how proteins with positively charged
surfaces and/or amphipathic helices deform membranes, it
is not likely that a single molecule or single dimer could act
alone to deform the membrane in such a specific and effective manner. Instead, the assembly of many molecules at the
same place of the membrane is certainly required for membrane deformation. For such assembly, it is quite reasonable
to expect that the enzymatic production of phosphoinositides, such as PI(4,5)P2, would recruit membrane-deforming
proteins. The assembly of several of these molecules may
trigger the “polymerization” of membrane deforming proteins necessary for definite curvature generation (123, 260).
In such a scenario, the concentration of the membranedeforming protein will need to be fairly high, presumably in
the micromolar range.
When the structures of BAR, F-BAR, and I-BAR proteins
were discovered, it was obvious that a dimer structure of the
BAR domains was responsible for binding the protein to the
curved membranes. However, BAR domains not only bind
to membranes of the appropriate curvature to stabilize
them, but can also act to deform membranes into such a
shape (75, 183). Therefore, it is reasonable to suggest that
major curvature deformations (e.g., protrusions, invaginations, etc.) might be formed by the assembly of the BAR
domains into chains, sheets, and/or spirals. Thus the shape
of membrane contact surface is determined by not only the
dimer unit structure, but also the assembly of higher order
structures on the surface of the membrane.
The first example of higher order assembly formation found
in nature was the putative filament formed by the F-BAR
domains of CIP4 and FBP17, where tightly packed F-BAR
spirals were observed on the surface of the tubules (FIGURE
3) (260). Subsequent research has identified spiral formation by endophilin and has further revealed some unique
spacing patterns among the spirals of this endophilin assembly on the membrane tubules (FIGURE 3) (183). However,
this filament formation was not clearly observed for other
BAR domains. With regard to PACSIN1 and Pinkbar, the
1236
The exploration of BAR protein localization and assembly
formation is limited by the current methods used to visualize live cells (e.g., microscope resolution and membrane/
protein visualization methods). Furthermore, the diffraction limit of a live cell also causes difficulties, as most of the
membrane structures formed by BAR proteins are below
this limit, thus preventing the use of conventional microscopy to examine BAR protein-mediated deformation. Recently, superresolution microscopy, such as stochastic optical reconstruction microscopy (STORM), photo-activated
localization microscopy (PALM), and structured illumination microscopy (SIM), has been used to try to overcome
such limitations, and preliminary studies have been initiated to reveal how membrane structure is built up by cytosolic proteins (14, 116). However, superresolution microscopy is not without its own limitations, and although the
membrane micro-structure probes can be rapidly developed, the signal density still needs to be increased to effectively observe the shape of the membrane. However, this
methodology represents a large step toward visualizing protein expression and localization, as accomplished with conventional microscopy, in an electron microscopy-grade image of the cellular structures. This type of detailed visualization of proteins and membranes will be indispensable to
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increase understanding of the function and regulation of
BAR proteins during membrane morphogenesis.
VI. BAR PROTEINS AND DISEASE
The members of the BAR protein superfamily play crucial
roles in the formation of fine membrane micro-structures,
such as membrane protrusions, filopodia, lamellipodia, and
endocytotic invaginations. Therefore, defects in the function of these proteins are likely related to a variety of diseases. Cell culture and animal model knockout studies are
powerful tools used to determine the physiological role of a
protein in vivo. Here, we describe several BAR protein mutations connected to disease pathology as well as the phenotypic consequences of lacking one or more BAR protein
in a mouse model.
A. Cancer
Cancer cells have different morphology compared with the
parental normal cells. Although the relationships between
shape formation by BAR proteins and cancer formation is
not clear, several reports showed the involvement of BAR
proteins in cancer. The amphiphysin-like protein Bin1 has
been reported to contribute to the Myc and Raf pathways
which play a crucial role in apoptosis and senescence (214),
and one isoform appears to act as a tumor suppressor by
inhibiting c-Myc. Loss of Bin1 in mosaic mice was also
associated with overall organ tissue inflammation and an
increased occurrence of lung and liver carcinomas (33). Deletion of Bin3 has also been associated with increased lymphomas in aging animals as well as juvenile cataract formation, with a near total loss of F-actin in the lens fiber cells
(220).
Numerous BAR proteins are known to be involved in podosome/invadopodia formation in cancer cells. Formation
of these F-actin-rich fingerlike membrane protrusions
drives cell migration and invasion into the extracellular matrix (ECM) (185). Podosomes/invadopodia were initially
discovered in cells transformed with the Rous sarcoma virus
as well as in monocyte-derived cells, such as osteoclasts and
macrophages (44, 164, 165). The BAR protein CIP4 has
been shown to be localized at the leading edge and podosomes and may be involved in ECM invasion (115, 209). In
addition to CIP4, FBP17 is also localized and appears to
function in the podosomes (300). Furthermore, both proteins have been shown to be involved in invadopodia formation in invasive breast and bladder cancer cells, respectively (209, 324), and CIP4 are expressed at high levels in
the invasive breast cancer cell line MDA-MB 231, while
lower levels are found in the less invasive cell line MCF-7. It
has been suggested that CIP4 expression promotes breast
cancer cell invasion and invadopodia formation through
the activation of N-WASP (209).
CIP4 is also expressed at abnormally high levels in chronic
lymphocytic leukemia (CLL) cells compared with normal B
cells or other subtypes of B-cell malignancies. Experimental
CIP4 knockdown in CLL cell lines causes decreased activation of WASP, but increased activation of PAK1 and p38
mitogen-activated protein kinase (MAPK), resulting in impaired lamellipodium assembly and loss of directional migration (163). In addition, overexpression of CIP4 in CLL
patients seems to be associated with aggressiveness of the
disease and a shorter survival. However, the inconsistent
evidence is also reported. According to that report, CIP4
may function as a suppressor of Src-induced invadopodia
and invasion in breast tumor cells by promoting endocytosis of MT1-MMP (115). Therefore, it is likely that CIP4
plays different roles dependent on the types of cancer cells.
As well, CIP4 gene knockout mice only cause delayed and
decreased endocytosis (65). Although such partial suppression of endocytosis probably implicates the redundancy of
these proteins and limitation of knockout studies, CIP4mediated endocytosis might be involved in some forms of
cancer progression.
The BAR and GAP protein, ASAP1, as well as the I-BAR
protein IRSp53 are also involved in cancer cell podosome/
invadopodia formation (15, 194). ASAP1 is an Arf-GAP
known to regulate membrane trafficking and actin cytoskeleton organization (121, 223). It contains an NH2-terminal
BAR domain, followed by a PH domain, an Arf GAP domain, an ankyrin repeat, a proline-rich region, and an SH3
domain. On the membrane surface, ASAP1 catalyzes the
hydrolysis of GTP bound to Arf family GTP-binding proteins (Arfs), preferably to Arf6. Arf6 is localized at the PM
where it functions in a wide range of processes, such as
endocytosis, cytokinesis, and the reorganization of the actin
cytoskeleton, possibly through the action of increasing
PI(4,5)P2 synthesis and Rho family G protein regulation
(88). The BAR domain of ASAP1 is critical for in vivo
function of ASAP1, and the PH domain selectively binds to
PI(4,5)P2 (129). The PH domain is functionally integrated
with the GAP domain, raising the possibility that the BAR
domain affects GAP activity through the PH-BAR folding
mechanism. It has also been reported that ASAP1 is a substrate of the tyrosine kinase Src and binds to a variety of
actin regulatory proteins, including cortactin, which is a
required component for podosome formation (15). Indeed,
ASAP1, particularly the BAR domain was found to be required for podosome formation in breast cancer cells and
NIH 3T3 expressing active c-Src. Furthermore, such podosome formation was inhibited by the overexpression of the
BAR-PH domain (15). These results suggest that the recognition of PI(4,5)P2 by the BAR-PH domains trigger subsequent signaling events in podosome formation including the
regulation of Rho family G proteins, the reorganization of
actin filaments, and possibly the membrane curvature generation.
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Another phosphoinositide, PI(3,4)P2, is required for podosome/invadopodia formation in a unique way. Tks5/FISH,
a scaffold protein, has been shown to play an essential role
in invadopodia formation and is upregulated in invasive
forms of cancer (252). This protein consists of one PX domain and five SH3 domains and binds through PX domain
to PI(3,4)P2 (193, 195). Tks5 is essential for podosome/
invadopodia formation in many different cell types (38,
193, 252, 321). However, it is not clear yet how Tks5 plays
a role in the podosome/invadopodia formation. Recently, a
new sequential model of invadopodia assembly is presented
(242) based on high-resolution spatiotemporal live cell imaging. According to that model, cortactin, N-WASP, cofilin,
and actin arrive together to form the invadopodia precursor, followed by Tks5 recruitment. Tks5 then forms a complex with N-WASP, cortactin, cofilin, and actin. Next,
PI(3,4,5)P3 gets enriched in a ring around the precursor
core. SHIP2, a PI(3,4,5)P3 5-phosphatase, also localizes at
the invadopodia core and hydrolyzes PI(3,4,5)P3 to
PI(3,4)P2 which anchors the Tks5 complex to the precursor
core and stabilizes the invadopodia (FIGURE 2).
The I-BAR domain-containing protein IRSp53, which can
produce negative curvature, may also act as a scaffold protein, rather than a membrane-deforming protein, during
podosome formation, although its full function is still unclear (194). IRSp53 consists of an I-BAR domain, a CRIB
motif, and an SH3 domain. The CRIB motif binds to Cdc42
and the SH3 domain binds to Eps8, N-WASP, WAVE2,
MENA, and VASP (251), solidifying its potential as a scaffold protein, on which a variety of actin regulatory proteins
can be assembled for inducing actin polymerization.
Some BAR family proteins act as tumor suppressors. FBAR-only protein, PSTPIP2, antagonizes actin polymerization in the podosome by competing with FBP17 at the membrane (302). Furthermore, the I-BAR containing protein
MIM is present in normal tissues, but is missing in several
metastatic cell lines, and this downregulation is associated
with the progression of bladder transitional carcinomas
(316). Thus MIM has been proposed as a suppressor of
metastasis genes in bladder cancer (153).
These accumulating evidences clearly indicate the involvement of BAR proteins in cancer. However, how the membrane morphology generated by the BAR protein relates to
cancer formation remains unclear.
B. Immune System Disorders
Multiple F-BAR proteins affect immune cell function
through their effects on membrane cytoskeleton remodeling. For example, an alternatively spliced form of CIP4
localizes in the phagocytic cup of RAW murine macrophage
cells, indicating a potential function in phagocytosis (53).
Furthermore, CIP4 is necessary for integrin-dependent T
1238
cell trafficking, and CIP4-deficient T lymphocytes cause impaired T-cell-dependent antibody response, impaired contact hypersensitivity, and defective adhesion to immobilized
VCAM1 and ICAM1 in endothelial cells, leading to impaired transendothelial migration (148).
Two additional F-BAR proteins, PSTPIP1 and PSTPIP2,
containing an F-BAR and SH3 domain and a sole F-BAR
domain, respectively, are also involved in immune function.
PSTPIP1 recruits PTP-PEST to immune synapses where it
negatively regulates the antigen-dependent activation of T
cells. Point mutations of PSTPIP1 are found in conjunction
with pyogenic sterile arthritis, pyoderma gangrenosum, and
acne (PAPA) syndrome (318), where the binding to PTPPEST was markedly reduced. In contrast, the interaction of
the mutated PSTPIP1with pyrin protein, which mediates
the inflammatory response and is responsible for the familial Mediterranean fever protein, was markedly increased
(264). PSTPIP2 also appears to be a negative regulator of
the immune system as it antagonizes FBP17 by competing
for membrane binding sites, resulting in the suppression of
immune system hyperactivation (302). PSTPIP2 point mutations and protein deficiency in mice in fact induce autoinflammatory disease and exhibit a similar phenotype to
chronic recurrent multifocal osteomyelitis (35, 66, 96).
C. Muscle Disorders
The membrane-deforming ability of the BAR proteins was
pioneered by the study of amphiphysin with mutation defective in muscle formation. Amphiphysin is necessary for
the organization of the excitation-contraction coupling machinery (t tubule), which is long invagination in muscles,
but it is not necessary for synaptic vesicle endocytosis in
Drosophila (225). In mammals, mutation of amphiphysin 2
(Bin1) causes autosomal recessive centronuclear myopathy
by interfering with the remodeling of t tubules and/or endocytic membranes (152, 225). In this case, two missense
mutations affecting the BAR domain disrupt the membrane
tubulation properties in transfected cells (206).
Bin3, consisting only of an N-BAR domain, is known to be
required for proper formation of multinucleated muscles
both in vitro and in vivo. During early myogenesis, Bin3
promotes migration of differentiated muscle cells, where it
colocalizes with F-actin in lamellipodia. Importantly, Bin3
forms a complex with Rho GTPases, such as Rac1 and
Cdc42, which are essential for myotube formation (265).
Bin3 knockout mice showed the delayed muscle regeneration (265). Interestingly, lack of Bin3 in differentiated muscle cells prior to myotube formation led to defects in lamellipodia formation and cell migration in addition to significantly decreased levels of active Rac1 and Cdc42. These
data suggest a major role for a Bin3-dependent signaling
pathway in regulating Rac1- and Cdc42-dependent processes during myotube formation (265).
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SHAPING OF CELLULAR MEMBRANES
D. Neuronal Diseases
The endocytosis and exocytosis of neurotransmitters and
their receptors are crucial for a large number of neuronal
functions. Dynamin, endophilin, and PACSIN1/Syndapin I
are essentially involved in such processes, presumably
through their functions in scission, subsequent uncoating of
cargo, and/or recycling. PACSIN1 is crucially involved in
the morphogenesis of neuronal cells in cooperation with
N-WASP and dynamin (48). Furthermore, PACSIN1
knockout mice suffer from seizures, a phenotype consistent
with excessive hippocampal network activity (145) as well
as defects in presynaptic membrane trafficking processes.
At the molecular level, it appears that PACSIN1 plays an
important role in the recruitment of all dynamin isoforms
and is a central player in vesicle-membrane fission reactions
(145, 216). Thus PACSIN1 acts as a pivotal membraneanchoring factor for the dynamins during regeneration of
synaptic vesicles.
Unlike PACSIN1, triple knockout of all three mouse endophilin proteins did not cause defects in endocytotic vesicle
scission, but led to defects in the uncoating of clathrincoated vesicles, thereby reducing the dynamics of synapse
transmission (182). This defect is likely caused by the loss of
the ability of endophilin to bind to and recruit synaptojanin, the PI(4,5)P2 phosphatase that functions to uncoat
these vesicles (207, 249). However, further studies are
needed to elucidate the precise mechanisms involved in this
process (6, 145, 171, 182, 290).
Interestingly, CIP4 appears to function in the formation of
neurite, although it is unclear how CIP4 functions in endocytosis or other processes in neurons. CIP4-null neurons are
specifically precocious in forming neurites (stage 1–2 transition), but not in polarization (stage 2–3 transition), indicating that CIP4 expression inhibits neuritogenesis. If CIP4
is a negative regulator of neuritogenesis, then the CIP4-null
neurons would be expected to form longer neurites than
those of the wild-type controls, and this is indeed the case.
The CIP4-null cortical neurons extend axons 1.5 times longer than the controls at 1 day in vitro (236). One additional
link between CIP4 and neuronal health is through the huntingtin gene, which CIP4 can bind to, thus potentially affecting the symptoms and progression of Huntington’s disease (113).
As described above, srGAPs which have Rho-GAP activities
are important multifunctional adaptor proteins involved in
various aspects of neuronal development, including axon
guidance, neuronal migration, spine maturation, and synaptic plasticity. Therefore, the defects of srGAP genes are
thought to link to some neurodevelopmental disorders,
such as mental retardation, schizophrenia, and seizure. The
srGAP3 protein, alternative name of mental-disorder associated GAP protein (MEGAP), is reported to be disrupted
and functionally inactivated by a translocation breakpoint
in a patient who shares some characteristic clinical features,
such as hypotonia and severe mental retardation, with the
3p⫺ syndrome (caused by deletions affecting many genes at
the terminal end of chromosome 3p) (60). Loss of srGAP3
was found to result in reduced density of spines and be
linked to impaired learning and memory (29). Its knockout
mice lead to mismigration of postnatal neural progenitors
and blockage of the cerebral aqueduct, inducing lethal hydrocephalus (139) or schizophrenia-related behaviors
(313). On the other hand, srGAP2 is implicated in a severe
neurodevelopmental syndrome causing early infantile epileptic encephalopathy (239). Interestingly, there are human
specific splicing isoforms of srGAP2, which might be involved in human specific brain development (30).
Intriguingly, the role of PACSIN1/Syndapin I also extends
to cilia formation in the sensory hair cells of the inner ear in
zebrafish, where it is required for formation of both microtubule-dependent kinocilia and F-actin-rich stereocilia
(247). Furthermore, two less characterized F-BAR domaincontaining proteins, FCHSD1 and FCHSD2, are also reported to be involved in hair cell stereocilia function
through regulation of actin polymerization (28).
VII. CONCLUSIONS AND OUTSTANDING
QUESTIONS
All cellular organelles have a membrane separating it from
the rest of the cell, with the cell itself being surrounded by
the PM. Although the development of the electron microscope has helped to reveal many of the micro-structures
present on these membranes, these structures are often complex, difficult to visualize, and short-lived. Thus we only
have a fragmented understanding of how these membrane
structures are generated. Many of these structures are
thought to be generated through protein-lipid interactions,
and numerous factors in the genome appear to encode proteins for such functions. Recently, noncoding RNAs have
been shown to function in various aspects of membrane
biochemistry/morphology, mainly through manipulation of
transcription. However, RNA and other nucleic acids do
have a strong negative charge, making it possible, albeit at
high concentrations, for them to affect the interactions between the anionic membrane lipids and the cationic residues
of membrane deforming proteins. However, it is not clear
whether RNA and other nucleic acids interact with membrane-deforming proteins.
Although we have a basic understanding of the membranebinding proteins at present, there are no genome-wide studies identifying the whole list of proteins with lipid-binding
abilities. Several attempts have been made to begin the
membrane-binding domain screening process (138); however, few membrane-binding gene families have been found
to date. When we consider the total number of human proteins, then the total number of known membrane curvature-
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SUETSUGU ET AL.
related proteins might be small in percentage. The occurrence of the proteins containing an ALPS motif in the whole
genome is totally 385 among the 14,049 human proteins
analyzed, suggesting 2.7% of the total number of human
proteins according to the prediction program (55). Furthermore, the total number of BAR domain-containing proteins
in the human genome, including BAR, F-BAR, and I-BAR,
is ⬃70 (238), while the number of ENTH domain-containing proteins is only 21, according to the SMART database
(248). With such a low percentage of proteins being able to
interact with the membrane, the functional loss of just one
can lead to any number of cellular defects or diseases. However, we know that the cluster of the basic-charged amino
acid residues is sufficient for membrane binding, and there
are thought to be a lot of proteins with such amino acid
sequence. Therefore, we can assume that we only know a
part of the whole list of the membrane binding protein.
Thus it is increasingly important to determine the possible
membrane-binding ability of all of the proteins to understand how proteins are regulating membrane morphology
in a particular organism, such as humans.
Next Generation World-Leading Researchers (NEXT Program)” initiated by the Council for Science and Technology
Policy (to S. Suetsugu), JSPS Grants-in-Aid for Scientific
Research Grant 25860215 (to S. Kurisu), and JSPS Grantsin-Aid for Scientific Research Grant 23227005 (to T. Takenawa).
Our ability to study membrane-protein interactions is entirely dependent on the methodology used to visualize this
relationship. The structural determination of protein domains, combined with superresolution microscopy (which
enables resolution as high as 10 nm, corresponding to the
size of a single protein), will likely revolutionize the field,
pushing our understanding of membrane structure formation forward. With these techniques, we may eventually be
able to map individual proteins, molecule by molecule, to
determine their specific location in the cell. With the ability
to identify protein localization in such nanoscale structures,
we are confident that the next generation of cell biology
inquiry will begin. These new techniques should be applied
to study the assembly of BAR proteins and their binding
proteins, as well as the localization of phosphoinositides. At
present, we do not have a lot of examples of ordered assembly of proteins in vivo. The new techniques might unravel
unexpected ordered assembly of the BAR proteins as we
have already observed in in vitro reconstitution experiments.
3. Antonny B. Mechanisms of membrane curvature sensing. Annu Rev Biochem 80: 101–
123, 2011.
ACKNOWLEDGMENTS
S. Suetsugu and T. Takenawa contributed equally to this
work.
Address for reprint requests and other correspondence: T.
Takenawa, Graduate School of Medicine, Kobe University,
7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650 – 0017, Japan (email: takenawa@med.kobe-u.ac.jp).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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