Development of new device for manipulationof giant plasma membrane vesicles

YamagataUniversity

巨大細胞膜小胞をマニピュレートするための新規デバイスの開発

PurposeThe GPMVs prepared from HeLa cells

Giant plasma membrane vesicles(GPMVs) isolated directly from cultured cell
membranes are known for the useful model
membranes. A protocol we reported
previously produces efficient yields of large
GPMVs (up to 10 μm). Recently, we havedeveloped new device for manipulation ofthe GPMVs to observe specific GPMVs for along time by an optical microscopic. We
prepared micro-hole quartz plates withdiameters of 2 µm and succeeded inmanipulating the GPMVs on the micro-holes
by suction of the solution.

Suction

Giant Plasma Membrane
Vesicles (GPMVs)

Size distribution of GPMVs4540

Number off GPMVs (%)

Introduction○ Keishi Sato1, Takayuki Nakaya2, Rebun Sakane2, Chiyuko Sakamoto3, Takashi Okuno3(1Grad.Sch.Eng., Yamagata Univ., 2Namiki Precision Jewel Co.,Ltd, 3Fac.Sci., Yamagata Univ.)○ 佐藤 啓史1, 中谷 隆幸2, 坂根 礼文2 , 坂本 知由子3, 奥野 貴士3 (1山形大・院理工, 2並木精密宝石(株), 3山形大・理学部)

Bleb generated
from the cell

35HeLa cell

2520Methods

1510Preparation of the GPMVs
HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine
serum at 37 ℃ in the presence of 5 % CO2. After removed DMEM,5 ml of 5 µM DiO was added
to the cells and incubated for 15 min at 37 ℃. DiO Labeled cells washed three times withbuffer 1 , added buffer 2 and incubated for 30 min at 37 ℃. Then, the media was replaced
buffer 1 and incubated for 30 min at 37 ℃. Finally, GPMVs were isolated into the supernatant byvortex for 10 min. Buffer 1:100 mM NaCl, 2 mM CaCl2, 50 mM Tris-HCl pH 7.5. Buffer 2:20 mM
ZnCl2, 2 mM CaCl2, 100 mM NaCl

50Fluorescent microscopic
image of HeLa cells treatedwith formaldehyde. Bleb wasinduced from HeLa cells. The
blebs are released from thecells.

Micro-hole
quartz plates

300-5

Phase-contrast
microscopic
images of the GPMVs.
Scar bar: 50 μm

5-10

10-15

15-20 20-25

Diameter (μm)

Ref: Scott. Science, 194, 743 (1976)
Erdinc Sezgin. Nature Protocols, 7, 1042, (2012)
Sarah L. Veatch. ACS Chem. Biol., 3, 287 (2008)

Results[Micro-hole quartz plates and suction pump system for microscopic observation of GPMVs]
Micro-hole quartz plate
Diameter:2 μm
Pitch：0.04 mm
Thickness：0.22 mm
Number of holes : 396 holes

Micro-holequartz plate

Namiki Precision Jewel Co.,Ltd 11J68

Liquid inlet

Pump

SuctionSilicon rubber(Thickness:0.5 mm)
2 μm

Manometer

0.22 mm
5~6 μmScar bar:100 µm

Scar bar: 5 µm

Sectional view (side)

Suction pump unit

35 mm/ GLASS BASE DISH

[ Manipulation of styrene micro-beads ]Time-course observation of manipulated styrene micro-beads on the micro-holes
100

Manipulated styrene micro-beads (%)

SuctionpressureSuction-30 kPa-20 kPa

9080706050
40-5 kPa

30-10 kPa

20-20 kPa

10-30 kPa

0-10 kPa

0

2040

60

80

100

120

140160180200Time (s)<styrene beads>
Diameter:6.0 µm(CV:10%)
Density:1.05 g/ml
Concentration:2.0 x 10^5 particles/ml
Volume:1 ml

60 s
120 s
30 s
180 s
Fig.1a Fluorescence microscopic images of micro-beads on micro-holes.

[ Manipulation of GPMVs ]Time-course observation of manipulated GPMVs on the micro-holes
10010090908080

Manipulated GPMVs (%)-10 kPa-3 kPaManipulated GPMVs (％)

Suctionpressure70
60
504030-2 kPa

20-3 kPa

10-10 kPa

60
50
4030

1.0×10^620

5.0×10^5
1.0×10^510000-2 kPa

70

50100150200Time (s)250300350

030

6090120Time (s)150180

Fig.2c Effect of number of GPMVs in the solution onrate of GPMVs manipulation on micro-holes.

Fig.2b Effect of suction pressure on rate ofGPMVs manipulation on micro-holes.
30 s
300 s
60 s
180 s
120 s
Fig.2a Fluorescence microscopic images of micro-beads on micro-holes.

[ The GPMVs retains its structure on themicro holes ][ Manipulated GPMVs were passed through the micro-holes, at high suction pressure ]SuctionpressureDiI-5 kPa

CalceinObserved GPMVs (%)

100-3 kPa

-3 kPa-4 kPa-5 kPa90

Structure ofGPMVs was
not collapsed
on micro-holes.

6050
4000s

30 s

60 s

120 s

Fig.3a Fluorescence microscopic images of GPMVs on micro-holes.

Conclusion

180 s

DiICalcein

Phase
contrast image

Merge

8070100
Time (s)Fig.3b Observed GPMVs on
micro-holes for 180 second in theeach suction pressure.

210Fig.4 Fluorescence microscopic imagesof calcein encapsulation GPMVs on
micro-holes. Scar bar:20 µm

In this study, we developed new suction devices for manipulation of GPMVs to observe specific GPMVs. The devices were micro-hole
quartz plates and suction pump system (suction unit). The micro-styrene beads can be manipulated on the micro-holes by using thissystem and the beads can be observed by fluorescence microscope. We tried to manipulate GPMVs on the micro-holes. Then, we
found that moderate suction pressure for manipulation of GPMVs was about -3 kPa. Moreover, structure of GPMVs was not collapsed
on micro-holes. These devices enable easily manipulation of GPMVs and observation of the GPMVs for a long time by optical
microscope. We will use this system to reveal the physical properties of membrane proteins and lipids in GPMVs.

The 53rd Annual Meeting of the Biophysical Society of Japan 2015.9.15 @ Kanazawa

Fig.1b Effect of suction on manipulation rate of the styrene micro-beads.