life
Article
Micrometer-Scale Membrane Transition of Supported
Lipid Bilayer Membrane Reconstituted with Cytosol
of Dictyostelium discoideum
Kei Takahashi and Taro Toyota *
Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo,
Tokyo 153-8902, Japan; [email protected]
* Correspondence: [email protected]; Tel.: +81-3-5465-7634
Academic Editor: David Deamer
Received: 19 January 2017; Accepted: 2 March 2017; Published: 7 March 2017
Abstract: Background: The transformation of the supported lipid bilayer (SLB) membrane by
extracted cytosol from living resources, has recently drawn much attention. It enables us to address
the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba
locomotion, which was discussed in many previous studies, exhibits membrane deformation in the
presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a
supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted
from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining
the artificial constitution of membranes with the reconstitution using animate cytosol (without
precise purification at a molecular level), contributing to membrane deformation analysis; Results:
The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition
of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result,
pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into
the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current
findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind
to a phosphoinositide-containing SLB membrane.
Keywords: supported lipid bilayer membrane; phosphatidylinositides; cytosol; Dictyostelium discoideum
1. Introduction
Reconstitution approaches for cell motility have drawn much attention over recent decades.
Prof. van Oudenaarden’s group reported that lipid vesicles coated with the Listeria monocytogenes
virulence protein ActA, are propelled by in vitro actin polymerization [1]. Prof. Takeuchi’s group
developed self-propelled micrometer-sized polystyrene beads modified with flagella [2], and Prof.
Sonobe’s group demonstrated that an actomyosin fraction containing the sol-gel conversion of a
cytoplasmic extract moves like an amoeba [3]. These approaches have been established by using only a
fraction of the chemical reaction networks related to the cell machinery. Recently, our group established
the membrane-assisted biochemical machinery for amoeba locomotion, using a combination of cytosol
extracted from Dictyostelium discoideum and lipid film patches [4]. However, these are crude conditions,
as the amoeba cell-free extract is simply added to multilamellar lipid patches, whereas real amoeba
cells have a cellular membrane with a single lipid bilayer. Therefore, as a next step, the reconstitution
of amoeba cells using a single lipid bilayer is required.
A so-called “vesicle rupture method” using small vesicles makes it possible to constitute the
unilamellar lipid membrane on a solid surface [5–11]. This technique is based on the electrical attractive
force between the solid glass surface and the lipid, generating a supported lipid bilayer (SLB) [12].
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The membrane transformation of the SLB membrane by extracted cytosol from living resources, has
recently
drawn
Life 2017,
7, 11 much attention. For example, there are reports on the membrane transition of purified
2 of 11
phospholipid SLB membranes, induced by an injection of cytosol from Escherichia coli. Loose et al.
resources,
has recently
muchformation
attention. and
For example,
there
areproteins
reports on
the and
membrane
presented
observations
on drawn
the pattern
oscillation
of the
MinD
MinE on
transition
of purified
phospholipid
SLB membranes,model
induced
by MinD
an injection
of cytosol
from
an SLB
membrane,
and produced
a reaction-diffusion
of the
and MinE
dynamics
that
Escherichia
coli.
Loose
et
al.
presented
observations
on
the
pattern
formation
and
oscillation
of
the
accounts for this experimental observation [13].
proteins
MinD
and MinE
onforce
an SLB
membrane,
and producedthat
a reaction-diffusion
model
the MinD
In another
report,
atomic
microscopy
demonstrated
nanometer-sized
poresofwere
formed
and MinE dynamics that accounts for this experimental observation [13].
in the pure phospholipid SLB membrane, in the presence of the Escherichia coli cytosol [14]. Furthermore,
In another report, atomic force microscopy demonstrated that nanometer-sized pores were
an SLB membrane containing phosphatidylinositol-4,5-bisphosphate can lead to the self-assembly of
formed in the pure phospholipid SLB membrane, in the presence of the Escherichia coli cytosol [14].
filopodia-like structures, containing actin bundles with Cdc42, N-WASP-WIP, Toca-1, and the Arp2/3
Furthermore, an SLB membrane containing phosphatidylinositol-4,5-bisphosphate can lead to the
complex,
as well as
uniform and short
polymerized
actin
[15].
These
reports
show
that this technique,
self-assembly
of filopodia-like
structures,
containing
actin
bundles
with
Cdc42,
N-WASP-WIP,
Tocawhich
we
call
the
novel
reconstitution
method,
using
a
purified
lipid
SLB
membrane
and show
cytosol
1, and the Arp2/3 complex, as well as uniform and short polymerized actin [15]. These reports
fromthat
prokaryotic
cells
or
purified
proteins
related
to
cell
motion
or
dynamics,
is
a
powerful
tool
this technique, which we call the novel reconstitution method, using a purified lipid SLB
for measuring
the cytosol
energyfrom
and prokaryotic
kinetics of acells
membrane
transition.
Giventothat
purified
lipid SLB
membrane and
or purified
proteins related
cell the
motion
or dynamics,
membrane
is artificially
andenergy
the extracted
cytosol
reconstituted,
this current
technique
is a powerful
tool forconstituted
measuring the
and kinetics
of aismembrane
transition.
Given that
the
purified
lipid SLB membrane
artificially
constituted
and the extracted
cytosol[14],
is reconstituted,
is a novel
reconstitution
method.is Here,
based
on the Noireaux
group’s work
we develop this
a new
current technique
is using
a novelcytosol
reconstitution
method.
Here,
based on the
Noireaux
group’s work
[14],
reconstitution
method
extracted
from the
eukaryotic
amoeba
Dictyostelium
discoideum,
a new
reconstitution
method SLB
using
cytosol extracted
from
the eukaryotic
amoeba
and we
by develop
generating
a purified
phospholipid
membrane
on a glass
substrate
using the
vesicle
Dictyostelium
discoideum,
and
by
generating
a
purified
phospholipid
SLB
membrane
on
a
glass
rupture method (Figure 1). It enables us to address the question of whether the purified phospholipid
using
the vesicle
rupture
method
(Figurelocomotion,
1). It enableswhich
us to address
the question
of whether
SLB substrate
membrane.
including
lipids
related
to amoeba
was discussed
in many
previous
the purified phospholipid SLB membrane. including lipids related to amoeba locomotion, which was
studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba. In this
discussed in many previous studies, exhibits membrane deformation in the presence of cytosol
study, the SLB membrane is comprised of purified phosphatidylcholines and phosphatidylinositol
extracted from amoeba. In this study, the SLB membrane is comprised of purified
phosphates (Scheme 1), and is stained by a fluorescence probe in order to visualize the micrometer-scale
phosphatidylcholines and phosphatidylinositol phosphates (Scheme 1), and is stained by a
membrane
deformation in the presence of Dictyostelium discoideum cytosol, using a confocal laser
fluorescence probe in order to visualize the micrometer-scale membrane deformation in the presence
scanning
fluorescence
microscope.
of Dictyostelium discoideum
cytosol, using a confocal laser scanning fluorescence microscope.
Scheme
1. Chemical
formula
phosphatidylcholine and
and phosphatidylinositol
phosphatidylinositol phosphates
used
in in
Scheme
1. Chemical
formula
of of
phosphatidylcholine
phosphates
used
this study.
this study.
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Figure
Figure 1.
1. Schematic
Schematic illustration
illustration of
of aa supported
supported lipid
lipid bilayer
bilayer consisting
consisting of
of purified
purified phospholipids
phospholipids and
and
cytosol
extracted
from
Dictyostelium
discoideum.
cytosol extracted from Dictyostelium
2.
2. Materials
Materials and
and Methods
Methods
2.1.
2.1. Reagents
Reagents
1-Palmitoyl-2-oleoyl-3-sn-glycero-3-phosphocholine
was was
purchased
from Wako
1-Palmitoyl-2-oleoyl-3-sn-glycero-3-phosphocholine(POPC)
(POPC)
purchased
from(Tokyo,
Wako
Japan).
1-Stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate)
(18:0–
(Tokyo, Japan).
1-Stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(10 -myo-inositol-40 ,50 -bisphosphate)
20:4
PI(4,5)P2),
1,2-hexadecanoyl-3-sn-glycerophosphatidylinositol-(3,4,5)-trisphosphate
(18:0–20:4
(18:0–20:4
PI(4,5)P2),
1,2-hexadecanoyl-3-sn-glycerophosphatidylinositol-(3,4,5)-trisphosphate
PI(3,4,5)P3),
1,2-dihexanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate)
(08:0
(18:0–20:4 PI(3,4,5)P3),
1,2-dihexanoyl-sn-glycero-3-phospho-(10 -myo-inositol-40 ,50 -bisphosphate)
0
0
0
0
PI(4,5)P2),
and
1,2-dihexanoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′,5′-trisphosphate)
(08:0
(08:0 PI(4,5)P2), and 1,2-dihexanoyl-sn-glycero-3-phospho-(1 -myo-inositol-3 ,4 ,5 -trisphosphate)
PI(3,4,5)P3)
were were
purchased
fromfrom
Avanti
Polar
Lipids
(Alabaster,
(08:0 PI(3,4,5)P3)
purchased
Avanti
Polar
Lipids
(Alabaster,AL,
AL,USA).
USA). TexasRed
TexasRed®®-1,2-1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine
dihexadecanoyl-sn-glycero-3-phosphoethanolaminetriethylammonium
triethylammoniumsalt
salt (TexasRed-DHPE)
(TexasRed-DHPE) was
was
provided
provided by
by Invitrogen
Invitrogen (Grand
(Grand Island,
Island, NY,
NY, USA).
USA). Organic
Organic solvents
solvents and
and inorganic
inorganic salts
salts were
wereprovided
provided
by
used as received.
received.
by WAKO
WAKO (Tokyo, Japan). These reagents were used
2.2. Vesicle
Vesicle Preparation
Preparation
2.2.
The phospholipids
phospholipids were
were dissolved
dissolved at
at 0.67
0.67 mM with 0.04 mol%
mol %TexasRed-DHPE,
TexasRed-DHPE,in
in300
300 μL
µL of
of aa
The
mixed
organic
solvent
(CHCl
–MeOH,
2:1,
v/v).
The
solution
was
poured
into
a
10
mL
round-bottom
mixed organic
v/v). The solution was poured into a 10 mL round-bottom
3–MeOH,
flask. The
Theorganic
organicsolvent
solventwas
wasremoved
removedfor
for55 min
min by
by aa rotary
rotary evaporator
evaporator (N-1110,
(N-1110,EYELA,
EYELA,Tokyo,
Tokyo,
flask.
Japan), equipped
equipped with
with aa vacuum
vacuum pump.
pump. The
The speed
speed of
of the
the rotation
rotation of
of the
the evaporator
evaporator was
was 180
180 rpm,
rpm, the
the
Japan),
◦ C by a water bath. After a lipid film
exhaust
rate
was
1.2
L/min,
and
the
temperature
was
set
to
40
exhaust rate was 1.2 L/min, and the temperature was set to 40 °C by a water bath. After a lipid film
was formed
formed at
at the
the bottom
bottom of
of the
the flask,
flask, the
the flask
flask was
was placed
placed in
in aa desiccator
desiccator to
to remove
remove any
any residual
residual
was
solvent from
from the
the lipid film under a reduced pressure at room temperature, for
solvent
for 17
17 h.
h.
◦ C and was gently poured into
Next, 22 mL
mL of
of phosphate-buffered
phosphate-buffered saline
saline (PBS)
(PBS) was
was heated
heated to
to 37
37 °C
Next,
and was gently poured into
◦ C for
the round-bottom flask
then
sealed
and
incubated
at 37
the
flask containing
containingthe
thelipid
lipidfilm.
film.The
Theflask
flaskwas
was
then
sealed
and
incubated
at 37
°C
2
h.
The
flask
was
shaken
by
a
vortex
mixer,
hourly,
and
for
further
dispersing,
the
sample
in
the
flask
for 2 h. The flask was shaken by a vortex mixer, hourly, and for further dispersing, the sample in the
◦
was ultrasonicated
for 1 h.
room
was kept
at 22
In22
order
undesired
flask
was ultrasonicated
forThe
1 h.
Thetemperature
room temperature
was
keptC.at
°C. to
Inremove
order to
remove
tubular vesicles
andvesicles
giant orand
large
vesicles,
which
are occasionally
duringformed
lipid film
hydration,
undesired
tubular
giant
or large
vesicles,
which areformed
occasionally
during
lipid
2
mL
of
the
sample
was
taken
from
the
vesicle
dispersion
and
passed
through
a
0.1-µm
IsoporeTM
film hydration, 2 mL of the sample was taken from the vesicle dispersion and passed through a 0.1Membrane
FilterMembrane
(Merck Millipore,
Darmstadt,
Germany),
twice. This
filtering treatment
led filtering
to small
μm
IsoporeTM
Filter (Merck
Millipore,
Darmstadt,
Germany),
twice. This
vesicles ofled
phospholipids
in PBS.
treatment
to small vesicles
of phospholipids in PBS.
2.3. Cover
Cover Glass
Glass Washing
Washing
2.3.
First, aa cover
cover glass
glass (24
(24 mm
mm ××60
60mm,
mm,No.1
No.1 (thickness
(thickness==0.12–0.17
0.12–0.17mm),
mm),MATSUNAMI,
MATSUNAMI,Tokyo,
Tokyo,
First,
Japan,)
was
set
in
a
rack
(SANSYO,
Tokyo,
Japan),
which
was
placed
in
a
glass
vessel
(SANSYO,
Japan,) was set in a rack (SANSYO, Tokyo, Japan), which was placed in a glass vessel (SANSYO,
® LS-II (Wako), diluted eight times with MilliQ
Tokyo, Japan).
Japan).AA
washing
solution
(Contaminon
®LS-II
Tokyo,
washing
solution
(Contaminon
(Wako), diluted eight times with MilliQ water
water
(Millipore,
Darmstadt,
Germany),
was
poured
glass
vessel
and
thecover
coverglass
glass was
was
(Millipore, Darmstadt, Germany), was poured into into
the the
glass
vessel
and
the
ultrasonicated in the washing solution for 1 h. After ultrasonication, the cover glass was rinsed with
distilled water (15 times) and MilliQ water (three times). Further rinsing with MilliQ water (six cycles
Life 2017, 7, 11
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ultrasonicated
in the washing solution for 1 h. After ultrasonication, the cover glass was rinsed4with
distilled water (15 times) and MilliQ water (three times). Further rinsing with MilliQ water (six cycles
of ultrasonication
ultrasonication for
for22min
minand
andfurther
furtherrinsing)
rinsing)
was
undertaken.
Second,
ethanol
poured
of
was
undertaken.
Second,
ethanol
waswas
poured
intointo
the
the
vessel
and
the
cover
glass
was
ultrasonicated
for
30
min.
After
the
ultrasonication,
the
cover
vessel and the cover glass was ultrasonicated for 30 min. After the ultrasonication, the cover glassglass
was
was rinsed
with MilliQ
water
three
times.rinsing
Further
rinsing
water
(six cycles of
rinsed
with MilliQ
water three
times.
Further
with
MilliQwith
waterMilliQ
(six cycles
of ultrasonication
ultrasonication
for
2
min
and
rinsing
×3)
was
undertaken
again.
Third,
0.1
M
NaOH(aq)
(prepared
for 2 min and rinsing ×3) was undertaken again. Third, 0.1 M NaOH(aq) (prepared by MilliQ
water)
by MilliQ
water)
was used
as a cleaning
andinthe
glass
in with
the vessel
with 0.1
M
was
used as
a cleaning
solution
and the solution
cover glass
thecover
vessel
filled
0.1 Mfilled
NaOH(aq)
was
NaOH(aq)
was
ultrasonicated
for
20
min.
After
ultrasonication,
the
cover
glass
was
rinsed
three
times
ultrasonicated for 20 min. After ultrasonication, the cover glass was rinsed three times with MilliQ
with MilliQ
water.
Further
rinsingwater
with (12
MilliQ
water
(12 cycles of ultrasonication
for 2 min
and
water.
Further
rinsing
with MilliQ
cycles
of ultrasonication
for 2 min and further
rinsing)
◦
further
rinsing) was
undertaken.
Finally,
the vessel
containing
the cover
wasCheated
140in°C
was
undertaken.
Finally,
the vessel
containing
the cover
glass was
heatedglass
at 140
for 40 at
min,
a
for
40
min,
in
a
dry
atmosphere.
dry atmosphere.
2.4. Supported Lipid Bilayer Membrane Preparation
fit aa bottom-open
bottom-open dish.
dish. After setting up a handmade
First, the cleaned cover glass was cut to fit
cleaned
cover
glass
to the
dish with
tape (Frame
chamber by
byattaching
attachingthe
the
cleaned
cover
glass
to bottom-open
the bottom-open
dishdouble-sided
with double-sided
tape
Seal, thickness;
280 μm, 280
BIO-RAD,
Hercules,Hercules,
CA, USA)CA,
(Figure
2A),
50 μL2A),
of the
dispersion
(Frame
Seal, thickness;
µm, BIO-RAD,
USA)
(Figure
50 vesicle
µL of the
vesicle
was introduced
onto the surface
cover of
glass
incubated
forincubated
30–60 minfor
at 30–60
more than
°C in
dispersion
was introduced
onto of
thethe
surface
theand
cover
glass and
min 24
at more
◦
an open
Second,
the chamber
times
by thesix
addition
of the
PBSaddition
(200 μL)oftoPBS
the
than
24 chamber.
C in an open
chamber.
Second,was
thewashed
chambersixwas
washed
times by
rims µL)
of the
glass,
which
gently
soaked
up, resulting
the removal
vesicles
(Figure
(200
to cover
the rims
of the
cover
glass,
whichitgently
soaked in
it up,
resultingof
insurplus
the removal
of surplus
2B).
vesicles
(Figure 2B).
Figure 2.
2. Schematic
Schematicillustrations
illustrationsofof
preparing
membrane
a cover
glass surface
by the
Figure
preparing
thethe
SLBSLB
membrane
on a on
cover
glass surface
by the vesicle
vesicle
rupture
method.
(A)
Schematic
of
how
to
fix
the
cleaned
cover
glass
in
the
bottom-opened
rupture method. (A) Schematic of how to fix the cleaned cover glass in the bottom-opened dish and
dishhow
andto
(B)
howthe
to SLB
rinsemembrane
the SLB membrane
for removing
thevesicles.
surplus vesicles.
(B)
rinse
for removing
the surplus
2.5. Fluorescence
Observation
2.5.
Fluorescence Microscopy
Microscopy Observation
The SLB
SLB membrane
membrane was
was observed
observed using
using aa confocal
confocal laser
laser scanning
scanning fluorescence
fluorescence microscope
microscope
The
(CSU-X1,
IX81,
Olympus,
Tokyo,
Japan),
equipped
with
a
100×
objective
lens
(N.A.
=
1.30,
working
(CSU-X1, IX81, Olympus, Tokyo, Japan), equipped with a 100× objective lens (N.A. = 1.30, working
distance
=
0.2
mm).
Red
and
green
fluorescence
images
were
obtained
by
using
the
corresponding
distance = 0.2 mm). Red and green fluorescence images were obtained by using the corresponding
band-pass filter
and dichroic
dichroic mirror
mirror units
units (excitation
(excitation 520–550
520–550 nm,
nm, emission
emission >580
>580 nm
nm and
and excitation
excitation
band-pass
filter and
470–490
nm,
emission
510–550
nm).
For
image
analysis
of
the
areas
with
pores
in
the
SLB
membrane,
470–490 nm, emission 510–550 nm). For image analysis of the areas with pores in the SLB membrane,
binarization processing
brightness was
performed on
the microscopy
microscopy images,
images, to
clarify the
the
binarization
processing of
of the
the brightness
was performed
on the
to clarify
information on
on the
the edges
edges and
of the
information
and the
the coordinates
coordinates of
the center
center of
of mass
mass using
using the
the image
image analysis
analysis software
software
ImageJ
(NIH).
We
calculated
the
area
of
the
micrometer-sized
structures
in
pixel
× pixel
The
ImageJ (NIH). We calculated the area of the micrometer-sized structures in pixel × pixel
unit.unit.
The area
2
area was
unit converted
was converted
× pixel
to2μm
, using
a graduated
glass-scale
plate.
unit
fromfrom
pixelpixel
× pixel
to µm
, using
a graduated
glass-scale
plate.
2.6. Cell Culture
AX4 cells co-expressing the Pleckstrin homology domain of cytosolic regulator of adenylyl
cyclase (PH-crac) tagged with RFP (PH-crac-RFP), and phosphatase and tensin homolog deleted from
chromosome 10 (PTEN) and tagged with GFP (PTEN-GFP), were cultured in modified HL5 medium
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2.6. Cell Culture
AX4 cells co-expressing the Pleckstrin homology domain of cytosolic regulator of adenylyl
cyclase (PH-crac) tagged with RFP (PH-crac-RFP), and phosphatase and tensin homolog deleted from
chromosome
Life 2017, 7, 11 10 (PTEN) and tagged with GFP (PTEN-GFP), were cultured in modified HL5 medium
5 of 11
containing G418 (30 µg/mL, Wako) and Hygromycin B (60 µg/mL, Calbiochem) [16], under shaking
μg/mL, Wako) and Hygromycin B (60 μg/mL, Calbiochem) [16], under shaking
at containing
155 rpm atG418
22 ◦ C(30
[17].
at 155 rpm at 22 °C [17].
2.7. Cytosol Extraction
2.7. Cytosol Extraction
Cytosolic extracts were prepared from AX4 cells, according to the protocol described
−1 , washed
Cytosolic
extracts
were
prepared
from at
AX4
cells,
according
elsewhere
elsewhere
[18,19].
The cells
were
harvested
a cell
density
of 7 to
× the
106 protocol
cells mLdescribed
two times
6
−1
[18,19].
The
cells
were
harvested
at
a
cell
density
of
7
×
10
cells
mL
,
washed
two
times
with
80 mL by
with 80 mL of phosphate buffer (PB), and re-suspended in 500 µL of PB. Cells were disrupted
of
phosphate
buffer
(PB),
and
re-suspended
in
500
μL
of
PB.
Cells
were
disrupted
by
nitrogen
nitrogen decompression using a cell disruption vessel (model 4639, Parr Instrument Co., Moline, IL,
decompression using a cell disruption vessel (model 4639, Parr Instrument Co., Moline, IL, USA) on
USA) on ice. The cell lysate was centrifuged at 16,000× g for 30 min at 4 ◦ C. The supernatant was
ice. The cell lysate was centrifuged at 16,000× g for 30 min at 4 °C. The supernatant was isolated and
isolated and centrifuged again. The resulting supernatant was transferred to a microtube and kept on
centrifuged again. The resulting supernatant was transferred to a microtube and kept on ice until just
ice until just before use.
before use.
3. Results and Discussion
3. Results and Discussion
3.1. Supported Lipid Bilayer Membrane Formation by Rupturing Vesicles on Glass
3.1. Supported Lipid Bilayer Membrane Formation by Rupturing Vesicles on Glass
In order to trace the formation of an SLB membrane from vesicles stained with 0.04 mol %
In order to trace the formation of an SLB membrane from vesicles stained with 0.04 mol%
TexasRed-DHPE, timelapse image capture using a confocal laser scanning fluorescence microscope was
TexasRed-DHPE, timelapse image capture using a confocal laser scanning fluorescence microscope
performed. The confocal system was focused on the surface of the cover glass of the chamber after 50 µL
was performed. The confocal system was focused on the surface of the cover glass of the chamber
of 0.1 mM POPC vesicle dispersion was gently injected onto the glass surface. As shown in Figure 3A,
after 50 μL of 0.1 mM POPC vesicle dispersion was gently injected onto the glass surface. As shown
theinglass
surface
was
covered
bywas
the covered
fluorescent
membrane
for membrane
the initial 600–1020
s after
the vesicle
Figure
3A, the
glass
surface
by the
fluorescent
for the initial
600–1020
s
dispersion
injection.
The
vesicle
rupture
method
for
the
formation
of
an
SLB
membrane
enables
us
after the vesicle dispersion injection. The vesicle rupture method for the formation of an SLB to
generate
heterogeneous
composed
of a lipidfilms
mixture
[5–8,13,14].
When
the vesicle
dispersion
membrane
enables us tofilms
generate
heterogeneous
composed
of a lipid
mixture
[5–8,13,14].
When of
POPC
and
2
mol
%
phosphatidylinositide
was
dropped
on
the
glass
surface,
the
fluorescent
membrane
the vesicle dispersion of POPC and 2 mol% phosphatidylinositide was dropped on the glass surface,
was
in themembrane
same manner
as POPC
inmanner
the initial
1020–1500
s after
theinitial
vesicle
dispersion
theformed
fluorescent
was formed
invesicles
the same
as POPC
vesicles
in the
1020–1500
injection.
In order
remove the
surplus In
vesicles,
theremove
chamber
washed
morethe
than
12 times
with
s after the
vesicletodispersion
injection.
order to
thewas
surplus
vesicles,
chamber
was
PBS
(Figure
2B).than 12 times with PBS (Figure 2B).
washed
more
Figure 3.
Figure
3. Cont.
Cont.
Life 2017, 7, 11
Life 2017, 7, 11
6 of 11
6 of 11
Figure 3. A POPC SLB membrane formed by the vesicle ruptured method. (A) Time-course change of
Figure
3. A POPC SLB membrane formed by the vesicle ruptured method. (A) Time-course change
fluorescence microscopy images of the glass surface during POPC vesicles rupture. The size of each
of fluorescence microscopy images of the glass surface during POPC vesicles rupture. The size of
observation space is 75.5 μm × 75.5 μm; (B) The mean fluorescence intensity of red fluorescence image
each observation space is 75.5 µm × 75.5 µm; (B) The mean fluorescence intensity of red fluorescence
was plotted along the z position; (C) The cross-section reconstructed fluorescence images of the POPC
image was plotted along the z position; (C) The cross-section reconstructed fluorescence images of
SLB membrane. obtained by confocal laser scanning fluorescence microscopy with red fluorescence
the POPC SLB membrane. obtained by confocal laser scanning fluorescence microscopy with red
filter units. The images were captured 30 min after the injection of the vesicle solution. The SLB
fluorescence filter units. The images were captured 30 min after the injection of the vesicle solution.
membrane was stained with TexasRed-DHPE.
The SLB membrane was stained with TexasRed-DHPE.
After washing the chamber, which was incubated for 30 min after the vesicle dispersion
2 area)
After washing
the chamber,
which
was incubated
30 min after
the vesicle
injection,
the fluorescence
intensity
profile
(the mean for
fluorescence
intensity
of an dispersion
81 × 81 μminjection,
2
the z-position
in red
modes
(excitation
520–550 nm,intensity
emissionof
> 580
nm)
that aalong
singlethe
thealong
fluorescence
intensity
profile
(the
mean fluorescence
an 81
× revealed
81 µm area)
peak with
fullmodes
width (excitation
at half maximum,
of nm,
less than
1 μm,>was
at the zthat
position
of the
surface
z-position
in ared
520–550
emission
580 located
nm) revealed
a single
peak
with a
of
the
cover
glass
(Figure
3B,C).
Thus,
the
vesicles
in
the
dispersion
were
transformed
to
the
SLB
full width at half maximum, of less than 1 µm, was located at the z position of the surface of the cover
membrane
on the Thus,
glass substrate,
viain
rupturing
in the PBS
[20,21].
We alsoto
observed
POPC-based
glass
(Figure 3B,C).
the vesicles
the dispersion
were
transformed
the SLBthe
membrane
on the
SLB
membrane,
including
the
phosphatidylinositol
phosphates,
which
revealed
a
single
peak
with
a
glass substrate, via rupturing in the PBS [20,21]. We also observed the POPC-based SLB membrane,
full
width
at
half
maximum,
of
less
than
1
μm,
similar
to
the
SLB
membrane
for
only
POPC.
including the phosphatidylinositol phosphates, which revealed a single peak with a full width at half
maximum, of less than 1 µm, similar to the SLB membrane for only POPC.
3.2. Pore Formation on the SLB Membrane after Injection of Cytosol Extract
3.2. PoreCytosol
Formation
on the SLB
after discoideum
Injection of (the
Cytosol
Extract
extracted
fromMembrane
Dictyostelium
PH-crac-RFP/PTEN-GFP
co-expressing
strain)
was
injected
onto
the
SLB
membrane
formed
in
the
chamber.
As
shown
in
Figure
4A and
Cytosol extracted from Dictyostelium discoideum (the PH-crac-RFP/PTEN-GFP co-expressing
Video S1, pore formation in the POPC SLB membrane was observed in the initial 444–552 s after the
strain) was injected onto the SLB membrane formed in the chamber. As shown in Figure 4A and Video
injection of cytosol. Then, every pore swelled in the x-y plane, almost at the same time. As a negative
S1, pore formation in the POPC SLB membrane was observed in the initial 444–552 s after the injection
control reference in terms of the ionic strength change of the buffer and protein degeneration, pore
of cytosol. Then, every pore swelled in the x-y plane, almost at the same time. As a negative control
formation did not occur in the POPC SLB membrane after the injection of previously boiled cytosol
reference in terms of the ionic strength change of the buffer and protein degeneration, pore formation
(110 °C, over 1 h). Figure 4B shows the time-course change of the standard deviation of
red
◦ C, over
didfluorescence
not occur inintensity
the POPC
after
the
of previously
cytosol
(110
2) of
(anSLB
areamembrane
of 81 × 81 μm
theinjection
POPC SLB
membrane.boiled
The effect
of the
injection
1 h).
Figure 4B
the time-course
change
of the standard
deviation
of red60
fluorescence
intensity
of cytosol,
i.e.,shows
the increase
of the standard
deviation,
was observed
in the initial
s, possibly because
2
(anthe
area
of 81 × 81intensity
µm ) ofincreased
the POPC
membrane.
The effect
of the At
injection
cytosol,
i.e., the
fluorescence
bySLB
adsorption
of fluorescent
proteins.
aroundof
240
s, brilliantly
increase
of
the
standard
deviation,
was
observed
in
the
initial
60
s,
possibly
because
the
fluorescence
fluorescent spots disappeared, affording the transient change of S.D. They plausibly correspond to
intensity
increased
by adsorption
proteins.layers
At around
240 s, brilliantly
fluorescent
spots
aggregate
residues
of vesicles of
or fluorescent
localized multiple
of membranes
attached
to the SLB
disappeared,
the transient
change
ofarrow
S.D. They
plausibly
correspond
to the
aggregate
residues
membrane affording
and detached
to the cytosol.
The
depicted
in Figure
4B shows
transition
of the of
vesicles
or localized
multiple
attachedtended
to thetoSLB
membrane
and
detached
SLB membrane.
From
400 s tolayers
600 s,of
themembranes
standard deviation
increase,
whereas
the
mean of to
fluorescence
intensity
gradually
decreased,
duethe
to transition
bleaching. of
This
an increase
in the
thethe
cytosol.
The arrow
depicted
in Figure
4B shows
thesuggests
SLB membrane.
From
400 s
the fluorescence
intensity
ofto
theincrease,
SLB membrane
due
tomean
pore formation
(dark spotsintensity
on the
to variation
600 s, theofstandard
deviation
tended
whereas
the
of the fluorescence
fluorescent
image). Figure
shows theThis
profile
of the an
mean
fluorescence
intensity of
mode
gradually
decreased,
due to4C
bleaching.
suggests
increase
in the variation
ofthe
thegreen
fluorescence
along
the
z
position.
Before
the
injection
of
cytosol,
the
fluorescence
intensity
of
the
SLB
membrane
intensity of the SLB membrane due to pore formation (dark spots on the fluorescent image). Figure 4C
was the
negligible.
The
peak
was
found at theintensity
glass surface,
a certain
value
the fluorescence
shows
profile of
the
mean
fluorescence
of thewhereas
green mode
along
the zofposition.
Before the
Life 2017, 7, 11
7 of 11
injection of cytosol, the fluorescence intensity of the SLB membrane was negligible. The peak was
foundLife
at2017,
the 7,glass
surface, whereas a certain value of the fluorescence intensity was detected
over the
11
7 of 11
glass surface. This result indicates that the green fluorescent molecule, PTEN-GFP, was localized to
intensity
detected over
glass
surface.ofThis
result We
indicates
that the
green
fluorescent
the POPC
SLBwas
membrane
afterthe
the
injection
cytosol.
examined
the
cytosol
of themolecule,
wild AX4 cells
PTEN-GFP,
localized
to pore
the POPC
SLB membrane
after the
injection
of cytosol.
examined the
for injection
andwas
observed
the
formation
of the POPC
SLB
membrane
on aWe
micrometer
scale, in
cytosol
of
the
wild
AX4
cells
for
injection
and
observed
the
pore
formation
of
the
POPC
SLB
the presence of cytosol. To examine the temperature effect, this experiment was performed at low
membrane on a micrometer scale, in the presence of cytosol. To examine the temperature effect, this
room temperature (21 ◦ C) (note that all other experiments were carried out at 24–26 ◦ C). The pore
experiment was performed at low room temperature (21 °C) (note that all other experiments were
formation
inout
theatPOPC
SLBThe
membrane
was observed
inSLB
the membrane
initial 1632–3000
s after
injection of
carried
24–26 °C).
pore formation
in the POPC
was observed
in the
the initial
cytosol
(Figure
S1).
The
decrease
in
the
temperature
caused
a
delay
in
pore
formation
in
the POPC
1632–3000 s after the injection of cytosol (Figure S1). The decrease in the temperature caused a delay
SLB membrane
below
the
cytosol.
in pore formation in the POPC SLB membrane below the cytosol.
Figure 4. Pore formation in POPC SLB membrane after injection of cytosolic extract from PH-crac-
Figure 4. Pore formation in POPC SLB membrane after injection of cytosolic extract from
RFP/PTEN-GFP co-expressing cells. (A) Time-course change of microscopy images of a POPC SLB
PH-crac-RFP/PTEN-GFP co-expressing cells. (A) Time-course change of microscopy images of a POPC
membrane, captured by confocal laser scanning microscopy. The pores in the SLB membrane were
SLB membrane,
captured
by 444–552
confocals after
lasercytosol
scanning
microscopy.
pores
in the
SLB membrane
formed during
the initial
injection
(24–30 s) The
The size
of each
observation
space were
formed
during
the
initial
444–552
s
after
cytosol
injection
(24–30
s)
The
size
of
each
observation
space is
is 75.5 μm × 75.5 μm; (B) Time course change of the standard deviation of fluorescence intensity of
75.5 µm
75.5 µm; images,
(B) Time
courseby
change
of the standard
of fluorescence
intensity
the ×
microscopy
analyzed
red fluorescence
images; deviation
(C) The mean
fluorescence intensity
of of the
green fluorescence
(PTEN-GFP)
was plotted
along(C)
theThe
z position.
The SLB membrane
was
microscopy
images, analyzed
by redimage
fluorescence
images;
mean fluorescence
intensity
of green
stained
with
TexasRed-DHPE.
fluorescence (PTEN-GFP) image was plotted along the z position. The SLB membrane was stained
with TexasRed-DHPE.
The results of pore formation indicated that the cytosol extracted from Dictyostelium discoideum
has the potential to induce membrane transitions of the SLB membrane composed of only POPC. In
The
pore formation
indicated
that the cytosol
extracted
from elsewhere.
Dictyostelium
discoideum has
fact,results
the SLBof
membrane
deformation
on a nanometer
scale has
been reported
For instance,
the potential
to
induce
membrane
transitions
of
the
SLB
membrane
composed
of
only
POPC.
the atomic force microscopy observation revealed that the formation of nanometer-sized pores in theIn fact,
1,2-dimyristoyl-sn-glycero-3-phosphocholine
SLB membrane
caused
by a polyL-lysine injection
the SLB
membrane deformation on a nanometer
scale haswas
been
reported
elsewhere.
For instance,
[22]. Escherichia
coli cytosol expressing
the revealed
pore-forming
protein of
α-hemolysin,
induced pores
the atomic
force microscopy
observation
thatmembrane
the formation
nanometer-sized
formation in POPC SLB membranes on a nanometer scale, observed by AFM [14]. These studies
in thepore
1,2-dimyristoyl-sn-glycero-3-phosphocholine
SLB membrane was caused by a poly-L-lysine
imply that proteins or cytosol cause a SLB membrane of phospholipid to form pores. Figure 4A,B
injection [22]. Escherichia coli cytosol expressing the pore-forming membrane protein α-hemolysin,
shows that cytosol extracted from Dictyostelium discoideum also has the potential to deform
induced pore formation in POPC SLB membranes on a nanometer scale, observed by AFM [14].
These studies imply that proteins or cytosol cause a SLB membrane of phospholipid to form pores.
Life 2017, 7, 11
8 of 11
Figure 4A,B shows that cytosol extracted from Dictyostelium discoideum also has the potential to deform
Life 2017, 7,We
11 established a micrometer-sized experimental model that demonstrated membrane
8 of 11
membranes.
deformation using amoeba cytosol.
membranes. We established a micrometer-sized experimental model that demonstrated membrane
deformation using amoeba cytosol.
3.3. Disturbance of Pore Formation in the SLB Membrane by Phosphatidylinositides
3.3. Disturbance
of Pore
Formation in the
SLB Membrane
by Phosphatidylinositides
Figure
5A shows
the time-course
change
of fluorescence
microscopy images of an SLB membrane
containingFigure
POPC5A
and
2 molthe
% 18:0–20:4
the injection
of cytosol.
The of
SLB
shows
time-coursePI(4,5)P2,
change ofafter
fluorescence
microscopy
images
anmembrane
SLB
membrane
containing in
POPC
and 2 mol%
18:0–20:4
after the
injectionand
of cytosol.
The expanded
SLB
exhibited
pore formation
the initial
720–900
s afterPI(4,5)P2,
the injection
of cytosol
the pores
exhibited
pore formation
the initial 720–900
s afterand
the injection
cytosol andPI(3,4,5)P3
the pores did
in themembrane
x-y plane.
In contrast,
the SLBinmembrane
of POPC
2 mol %of 18:0–20:4
expanded in the x-y plane. In contrast, the SLB membrane of POPC and 2 mol % 18:0–20:4 PI(3,4,5)P3
not change after the injection of cytosol at 24–30 s (Figure S2). No pores developed in the SLB
did not change after the injection of cytosol at 24–30 s (Figure S2). No pores developed in the SLB
membrane, but instead, a slight lipid aggregation was observed. The PTEN-GFP localization in both
membrane, but instead, a slight lipid aggregation was observed. The PTEN-GFP localization in both
SLB membranes
containing
phosphatidylinositides
wasconfirmed
confirmed
green
mode
profile
SLB membranes
containing
phosphatidylinositides was
by by
the the
green
mode
profile
of the of the
fluorescence
intensity
along
the
z-position
after
the
injection
of
cytosol
(Figure
S3).
Figure
5B shows
fluorescence intensity along the z-position after the injection of cytosol (Figure S3). Figure 5B shows
the time-course
change
of
the
standard
deviation
of
red
fluorescence
intensity
for
the
SLB
membrane
the time-course change of the standard deviation of red fluorescence intensity for the SLB membrane
composed
of POPC/18:0–20:4
PI(4,5)P2.
The
arrowpoints
pointsto
tothe
thepore
pore formation
formation in
composed
of POPC/18:0–20:4
PI(4,5)P2.
The
arrow
inthe
thePOPC/18:0–
POPC/18:0–20:4
20:4SLB
PI(4,5)P2
SLB membrane,
occurring
froms 720
s to 900
s. The
standarddeviation
deviation tended
PI(4,5)P2
membrane,
occurring
from 720
to 900
s. The
standard
tendedtotoincrease
increase in a
in
a
similar
manner
to
that
of
the
POPC
SLB
membrane.
Moreover,
the
distribution
of
the
minimum
similar manner to that of the POPC SLB membrane. Moreover, the distribution of the minimum value
value of distance (the center of mass) between the centroids of pores in the captured images of the
of distance (the center of mass) between the centroids of pores in the captured images of the POPC
POPC SLB membrane and the SLB membrane of POPC/18:0–20:4 PI(4,5)P2, was evaluated by image
SLB membrane
and
the SLB membrane
POPC/18:0–20:4
PI(4,5)P2,
wasnearest
evaluated
by image
analysis.
analysis. The
distribution
in both casesofwas
in the range of 3.3–7.3
μm. The
neighbor
distance
The distribution
both cases
was
in the
of 3.3–7.3
neighbor
distance
between
between thein
centroids
of the
pores
didrange
not change
whenµm.
the The
SLB nearest
membrane
contained
18:0–20:4
the centroids
PI(4,5)P2.of the pores did not change when the SLB membrane contained 18:0–20:4 PI(4,5)P2.
Figure 5. POPC/18:0–20:4 PI(4,5)P2 SLB membrane transition after the injection of cytosol extract from
Figure 5. POPC/18:0–20:4 PI(4,5)P2 SLB membrane transition after the injection of cytosol extract from
PH-crac-RFP/PTEN-GFP co-expressing cells and the pore size distribution of an SLB membrane, in
PH-crac-RFP/PTEN-GFP co-expressing cells and the pore size distribution of an SLB membrane, in each
each condition containing phosphatidylinositides. (A) Microscopic observation of the time-course
condition
containing
phosphatidylinositides.
Microscopic
observation
of the time-course
change of
change
of the POPC/18:0–20:4
PI(4,5)P2 SLB(A)
membrane.
We captured
the pictures
by confocal laser
the POPC/18:0–20:4
PI(4,5)P2
SLB membrane.
We
by confocal
laser
scanning
scanning microscopy.
The conformation
changes
of captured
the pores inthe
thepictures
SLB membrane
occurred
during
microscopy.
The
conformation
of the pores
membrane
occurred
during
the initial
720–900
s after the changes
cytosol injection
(24–30 in
s). the
The SLB
size of
each observation
space
is 75.5 the
μm initial
× 75.5
μm; the
(B) Time
course
change(24–30
of the standard
deviation
of intensity
analyzed
byisred
fluorescence
720–900
s after
cytosol
injection
s). The size
of each
observation
space
75.5
µm × 75.5 µm;
images
of POPC/18:0–20:4
Distribution
of the mean
pore areas
measured
in the SLB
(B) Time
course
change of the PI(4,5)P2;
standard(C)
deviation
of intensity
analyzed
by red
fluorescence
images of
membrane
in
each
condition.
The
SLB
membrane
was
stained
with
TexasRed-DHPE.
POPC/18:0–20:4 PI(4,5)P2; (C) Distribution of the mean pore areas measured in the SLB membrane in
each condition. The SLB membrane was stained with TexasRed-DHPE.
Life 2017, 7, 11
9 of 11
The length of the acyl chain of phospholipids correlates with the fluidity of the bilayer membrane.
Hence, the 08:0 PI(4,5)P2 and 08:0 PI(3,4,5)P3, which have shorter acyl chains than 18:0–20:4 PI(4,5)P2
and 18:0–20:4 PI(3,4,5)P3, were also examined when mixed into the POPC SLB membrane. Figure S4A
shows the fluorescence microscopy images of the SLB membrane containing POPC and 2 mol %
08:0 PI(4,5)P2 before, and 30 min after, the injection of cytosol. The pores were formed in the SLB
membrane and expanded in the x-y plane. When using 2 mol % 08:0 PI(3,4,5)P3, pore formation was
also observed after the injection of cytosol (Figure S4B). The areas of pores were measured using the
threshold for adjusting the binarization to the fluorescence microscopy image.
Figure S4C shows the binarization of the POPC SLB membrane with pores 20 min after the
injection, whereas Figure 5C depicts the diagram of the areas of the pores in each SLB membrane,
observed 25 min after the cytosol injection. The pore size in the POPC SLB membrane was larger than
that of any other SLB membrane. This analysis implies that the SLB membranes based on the POPC
including phosphatidylinositides, tended to disturb pore formation or expansion. The POPC/18:0–20:4
PI(3,4,5)P3 SLB membranes, especially, exhibited no pore formation after the cytosol injection.
Therefore, the potential of cytosol to deform membranes in a reconstitution system is controlled
by phosphoinositides.
Note that the pores formed in the POPC SLB membrane expanded to a micrometer size.
Because PTEN is a well-known membrane-binding protein, specifically localizing to 18:0–20:4
PI(4,5)P2 [23], it is important to examine the cytosol injection to the SLB membrane including
phosphoinositides, to approach the mechanism of pore formation. Moreover, pore formation was
sensitive to temperature (Figure S1). Two reasons why the temperature affected the initial timing of the
pore formation can be envisioned: (i) the membrane fluidity and (ii) the activity of membrane-binding
proteins. The membrane fluidity becomes low as the temperature decreases, especially when the
membrane transition from a liquid crystal phase (fluid) to a gel phase (solid-like) occurs [24]. However,
POPC has a phase transition temperature of about 0 ◦ C; whereas, the temperature of the experimental
conditions mentioned above was much higher than 0 ◦ C. Hence, the activity of membrane-binding
proteins is possibly a determinant of the pore formation in the SLB membrane observed here.
In the presence of cytosol, the SLB membranes also contained tubular giant vesicles (tGV).
In spite of the same chemical conditions as shown in Figure 4, tGVs were formed in the POPC SLB
membrane on the surface of the cover glass, without the bottom-open dish (Figure S5, Video S2).
In this condition, the pores which formed in the SLB membrane were smaller than those observed
in Figure 4A, at 30 min after the injection of cytosol. The bottom-open dish used for the chamber
appears to resist the slight distorting force of the cover glass surface. Thus, the distortion-free surface
may only allow pore formation [25]. The formation time of the pores and tGVs, as well as their
sizes, are similar, not to phagocytosis, because phagocytosis-inducing foreign particles or cells are
absent in the current experiment, but to macropinocytosis of amoeba. The model describing the
regulation of macropinocytosis in Dictyostelium discoideum depicts the requirement of the activity
of RasS, PIK1, PIK2, and PKB, to complete the formation of a macropinosome on a micrometer
scale, for several tens of seconds [26–28]. Recently, some reports using Dictyostelium discoideum
demonstrated that macropinocytosis is regulated by a network of Ras family members [29] or the level
of phosphatidylinositol 3,4,5-trisphosphate [30,31] in vivo. This model implies that cytosol extracted
from Dictyostelium discoideum affects the pure lipid SLB membrane, resulting in pore formation, which
is inhibited by phosphatidylinositides, and the formation of tGVs.
4. Conclusions
In this report, SLB membranes composed of POPC and phosphatidylinositides were constructed
through the vesicle rupture method. The injection of cytosol extracted from Dictyostelium discoideum
onto the SLB membrane induced pore formation in the SLB membrane. Pore formation was inhibited
by phosphatidylinositide. These results imply that cytosol has the potential to induce membrane
transitions on a micrometer scale and that phosphatidylinositide tunes membrane deformation.
Life 2017, 7, 11
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We established a reconstitution method using amoeba cytosol for revealing micrometer-sized
membrane deformations. This technique is a powerful in vitro tool for the investigation of membrane
deformation in terms of cell motion and intercellular motility.
Supplementary Materials: The following are available online at http://www.mdpi.com/2075-1729/7/1/11/s1,
Figure S1: Pore formation on the POPC SLB membrane after the injection of the cytosol at low room temperature,
Figure S2: Microscopic observation of the time-course change of POPC/18:0–20:4 PI(3,4,5)P3 SLB membrane,
Figure S3: Mean fluorescence intensity profile of green fluorescence (PTEN-GFP) image plotted along the z position
of the POPC/18:0–20:4 PI(4,5)P2 SLB membrane, Figure S4: POPC/08:0 PI(4,5)P2 and POPC/08:0 PI(3,4,5)P3 SLB
membrane transition after the injection of the cytosol extract of PH-crac-RFP/PTEN-GFP co-expressing cells and
pore size distribution of SLB membrane in each condition, Figure S5: Time-course change of the fluorescence
images of the POPC SLB membrane on the flat slide glass (not using bottom-open dish) after the injection of the
cytosol, Video S1: Video of the pore formation on the POPC SLB membrane after the injection of the cytosol extract
of PH-crac-RFP/PTEN-GFP co-expressing cells (Figure 4A), Video S2: Movie of the tubular vesicle generation on
the POPC SLB membrane on the flat slide glass (not using bottom-open dish) after the injection of the cytosol
extract of PH-crac-RFP/PTEN-GFP co-expressing cells (Figure S5).
Acknowledgments: The authors acknowledge Satoshi Sawai (The University of Tokyo) and Nao Shimada
(The University of Tokyo) for culturing cells, assisting with experiments, and delivering fruitful discussions.
This work was supported by the Platform for Dynamic Approaches to Living System (K.T. and T.T.) and
Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Robotics” (T.T., No. 24104004), from the
Ministry of Education, Culture, Sports, Science and Technology, Japan.
Author Contributions: K.T. conceived and coordinated the study and K.T. and T.T. wrote the paper; K.T. designed,
performed, and analyzed the experiments. All authors reviewed the results and approved the final version of
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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