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]. Life 2017, 7, 11; doi:10.3390/life7010011

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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

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Life 2017, 7, 11 of 11 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.

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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

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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.

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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.

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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.

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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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