Mixing of Biochemical Reagents Inside of
Giant Vesicles by Centrifugation-Induced Fusion

T. Toyota,1,2,3,4 M. Matsunaga,4 M. Fujinami4
1 University of Tokyo, Komaba,
2 Research Center for Life Science as Complex Systems
3 PRESTO, JST
4 Chiba University

Giant vesicles
 Closed lipid bilayer membrane (lipid capsule)

1,2-dioleoyl-3-sn-glycero-phosphocholine

Φ 40nm〜100µm
Differential interference contrast micrograph
Cross-section of giant vesicles

ConstrucLon of biomimeLc reacLon ﬁeld for observing/measuring
biochemical reacLon network in sense of analyLcal chemistry!!

GV preparaLon (BoUom‐up type)
ElectroformaLon Angelova et al.
• Unilamellar, spherical• High encapsulaLon raLo
(by microinjecLon etc)
• Small number of GVs
Phase contrast micrographDifferential interference
contrast micrographFilm‐swelling Bangham et al.
• Mostly mulLlamellar,
nesLng, aggregated etc• Low encapsulaLon raLo
• Large number of GVs

W/O emulsion centrifugaLon method (Top‐down type)
Pautot (2003)
Water-in-oilemulsion• Single‐wall (unilamellar)
Inner leaflet• Large number of GVs• Engineering leaﬂet asymmetry

Centrifugation

Fluorescence micrographNoireaux (2004)
outer leaflet• Gene expression of Hemolysin‐GFP complex in GVs
AggregatedSpherical

Adhered20µm• Does encapsulaLon raLo of content reach 100 %?
• How is the populaLon of GVs in shape distributed?

CentrifugaLon principle

Centrifugation Principle with Stokes’ Law
Frictional force

Frictional force = 3 π d η v
d: diameter of particle,
η: viscosity of solution,
v: speed of descending particle

Centrifugal force =Centrifugal force

1π d3 (σ - ρ) r ω2
6

d: diameter of particle,σ,ρ: density of particle and solution,r, ω: rotation radius and speed

Typical recipe
W/O emulsion
TrisHCl buffered solution
containing sugars (1 M)
and fluoresceinCentrifugation

Giant vesicles

TrisHCl bufferedsolution containing
glucose (1 M)•Fluorescein‐containing GVs formed by centrifugaLon

35µm

Flow cytometric (FCM) analysis on GV shape
Flow cytometer (EPICS ALTRA)

SchemeDetector for side light scatterand fluorescence

Irradiation laser

Ar+ Laser

Detector for
forward light scatter

Forward light scatter〜 Cross-section areaSample

From website of Bechmann Coulter

Fluorescein〜 VolumeQuantitative population analysis on GV shape
K. Nishimura, T. Hosoi et al., Langmuir, 25, 10439 (2009).

Inner water region
allophycocyanin（Ex: He-Ne laser 633 nm, Em: 650-670 nm Bandpass）
Vesicular membrane
BODIPY-tagged phospholipid（Ex: Ar+ laser 488 nm, Em: 515-545 nm Bandpass）
Fluorescence intensity of vesicular membrane
Fluorescence intensity per one fluorophore= Number of fluorophore per GV: NHPCMolar Ratio of cholesterol and fluorophore to phospholipids: rchol, rHPC
Surface area of polar heads of phospholipid and cholesterol 0.65 nm2, 0.28 nm2

Lipid membrane area / µm2

Nominal lamellarity of GV
Nominal lamellarity ＝Lipid membrane area per volume
Lipid membrane area per volume of an unilamellar vesicle

Inner water region /fL

W/O emulsion centrifugaLon method

Film swelling method

Slope: 3/2

Nominal lamellarity: 2〜3

Slope: ‐‐Nominal lamellarity: 10〜20

Lipid membrane area / µm2

Almost all GVs were spherical and their nominal lamellarity was quite low (2~3).

MoLvaLon for mixing internal contents
Enzymatic Reactions in Cells
Compartmentalized by micrometer-sized closed membrane• Confinement effect• Surface activity of membrane
etcConstruction of biomimetic reaction space inside of closed lipid membrane

Subject・Space inside of GV ~ 1 fL→ depletion of substrates
・Addition of non-permeable substrates into GVs is difficult.

Our strategy:
Vesicle Fusion or Hemi-fusion for mixing internal contents

Mixing internal contents inside of GV
CentrifugaLon

SEFusion
SE

S＋
E

P＋E

S＋E

P＋
EHemi-fusion

Why does it work?
 Number density of lipid molecule in membrane Oil phase molecules
Membrane ﬂuidity for transformaLon at contacLng siteSummary High encapsulaLon raLo of content!
• Lipid conc. in oil phase• Temperature during centrifugaLon• Density diﬀerence• 10 mol% cholesterol– EncapsulaLon raLo (so far) : 63%

 Flow cytometry revealed– Almost all are spherical and have low nominal lamellarity (2 or 3).

 GVs fused/hemi‐fused for mixing internal content– Mixing of internal content of GV was realized without any additives
for fusion/hemifusion by collecLng GVs through centrifugaLon.

Acknowledgements
Analytical Chemistry Lab. in Chiba University
Mr. Tomohiro Hosoi
Prof. Koichi Oguma
Information Bioscience Lab. in Osaka University
Mr. Kazuya Nishimura

Dr. Takeshi Sunami

Prof. Hiroaki Suzuki

Prof. Tetsuya Yomo

More for W/O-EC GV…
Poster session (Tomorrow )A. Shiga (P203)
Manipulation of W/O-EC GV by optical trapping

T. Furuya (P204)
W/O-EC GV dynamics observed by reflective interference
contrast microscopy