P-66

Study of ternary complex (TC): DNA-PC liposomes-Mg2+ as base fornuclear pore assembly by relaxation time fluorescence microscopy.
［ １ ］ Organization (Institute of Cell
Biophysics, Russian Academy of Sciences,
142290, Pushchino, Moscow reg.,Russia0Project
Leader ： Vasily
Kuvichkin,(Researcher in Shizuoka Univ. under the JSPS
Invitation Fellowship (Long term) (April to Sept.
2015)
Representative at RIE：Masahito Yamazaki（Professor of Nanomaterials Research Division,
Research Institute of Electronics, Shizuoka）［２］Research ProgressThis stage of project related to choice of model
system: ternary complex located inside giant
liposome (GUV containing inside DNA and LUV
100 nm in diameter）Addition to lipids ionophore
A23187 for Mg2+ produce pores in GUV for
addition of Mg2+ or Ca2+ inside GUV［３］Results
Try to get a TC with the unwinding of the DNA
using a method of DNA induced by the fusion ofTC with liposomes with a diameter about 1 μm.
The resulting liposomes have a diameter greaterthan 1 μm may be examined in detail in the new
microscope. To investigate such TC is convenient
if they are located in a small volume (several μm3).
This can be done microcuvette, but in this case it
is inconvenient to add any substance. Easier towork with conventional cuvette volume of 300 μl,
the type used in the laboratory Prof. M. Yamazaki[1] and TC put into giant liposomes with holes forthe cations Ca or Mg, produced by ionophore
A23187. The scheme of experiment is shown inFig.1.

Fig.1. Scheme of proposed experiments: a) dsDNAand LUV inside GUV in 10 mM HEPES buffer,
ph7.5; green circles-LUV (100nm in diameter); b)
after addition of 2 mM Mg++ forming ternarycomplexes (TC-yellow circles) interact withsimilar size LUV creating LUV (up 1-5 µm indiameter); c) and d) interaction of TC with GUVwith transfer ss DNA on the surface of GUV.
Reagents and methods.
Calf thymus DNA, DNaseI was from (SigmaAldrich, USA), MgCl2*6H20, CaCl2 (Waco, Japan),
DOPC and DOPA was from Avanty polar lipids, (USA);sucrose, glucose, NaCl, obtained from Wako (Japan).
Liposomes (LUV) from DOPC/DOPA (50/50 mol%)were got with the multilamellar liposome extrusion in
0.01M HEPES buffer, pH-7.5. The GUV with the same
lipid composition was prepared according to the method,
described in [2]. Spectroscopic studies were conductedwith the help of Hitachi spectrophotometer.
Fluorescence of such probes as CF, calcein, EB wasanalyzed with the help of a spectrofluorimeter UV-1800
Shimadzu, Japan, spectra of fluorescence obtained byspectrofluorimeter Hitachi F-4500, Japan. Samples ofGUV was analyzed in fluorescence microscope Olympus(IX-70, IX-71) Tokyo, Japan. Presented data mademinimum in 3 measurements, error of measurement
was less 10%.

(３－１) Research results+ LUV) after we have added 1mM Mg 2+. As it wasshown in [5], only 20% of the added DNA is associatedwith the liposomes in the absence of Mg2+ or Ca2+cations.
The first thing that catches the eye is the fact that
GUV1 contain less liposomes with the size ofThe remaining 80% of a free DNA can form TC from2+. In this as20
40 mLUV
andinmore
multilamellarMg
liposomes
the presenceof 1mMcase, the partcompared to the control sample (where there areof the DNA previously associated with lipids may loseno additives inside GUV). In our opinion this is
this connection and also form a TC. This will
due to fact, that the additives, which are present
undoubtedly cause the LUV movement inside GUV
in the HEPES buffer, initiate formation ofand their fusion up to micron size, visible in theliposomes (LUV), some of which are captured byphase-contrast microscope (Fig3). If we add DNaseI tolarge GUV1. The increased number of
GUV1 after the LUV movement stops the LUV
multilamellar liposomes can be explained by thismovement will resume again (Fig.3).
theory. But the most interesting thing is thesubstantial activity in GUV1 and especially insidemultilamellar liposomes even when there are nodivalent cations. It is both the movement of LUV(1-5 μm) in GUV1 and their fusion (increase inthe liposome size). In our opinion it is theevidence DNA interaction with LUV and GUV1
Fig.3. Moving and fusing of LUV inside GUV1
in the absence of Mg 2+. Previously we haveafter addition of DNaseI (time between frames isshown that such interaction is possible in the case
30 sec.
TC of zwitterion liposomes (DMPC) studied by
DSC [3]. There is other evidence of such
It can be explained by the fact that the DNA forming TC
interactions [4]. Unfortunately, there is little
inside GUV1 makes a lot of links among various
information about the nature of such interactions.
liposomes. Many of these are in a strained state; theHowever, we can assume that the nature of theDNA is supercoiled and thereby compensates for theDNA interaction with LUV and GUV in theexcess of interliposome link tension. Therefore, due topresence or absence of Mg 2+ is significantly
digestion of DNA by DNaseI, strain energy is graduallydifferent. If we add only 1mM Mg2+ to GUV1, areleased; it results in stepwise LUV rotation andrapid process of restructuring from one type ofmovement inside GUV1. We believe that DNaseI
complexes to the other is begin.
penetrates GUV1 through the pores which are formed
It is followed by the movement of LUV insideby Mg2+ and LUV.
GUV, and GUV itself, and also the LUV fusion
We believe that the formation of TC result inand it finishes at 50% of the cases by burst ofsignificantly changing the curvature of the liposomes
GUV with the release of LUV, connected together
and this system is suitable for the study using relaxation
with DNA strands (Fig. 2).
time fluorescence microscopy.
References
1. Tamba Y, Terashima H, Yamazaki M.
Chem. Phys Lipids. 2011 ;164(5):351-358
2. Yamashita Y et.al., 2002, Langmuir, 18,9638-9641;
3. Zhdanov R., Volkova L., KuvichkinV.,
Petrov A., 1994, Applied Magnetic Resonance,Fig. 2. Addition with help micropipette of 1mM of Mg2+
V. 7 (1), 115-130;to GUV containing LUV+DNA inside results in moving
4. Pott T., 2003, Interface Science, 11,
and fusion of LUVs with burst of the GUV and LUV
249–257,
binding by DNAcome out.
5. Lu D., Rhodes D.G., 2002, Biochim
Now let’s try to understand what has caused theBiophys Acta,1563, (1-2), 45-52.
movement of liposomes in the GUV 1 (containing DNA