J. Biol. Macromol., 5(3), 61-68 (2005)

ArticleEffect of D2O on the orientation of cortical microtubules in Spirogyra cells
Kazuyoshi Iwata and Yoh Sano
Faculty of Pharmaceutical Sciences, Setsunan University
Nagaotoge-cho, Hirakata, Osaka, 573-01, Japan

Received June 5, 2005, accepted August 9, 2005
Effect of D2O on the orientation of cortical microtubules (MTs) in Spirogyra cells wasinvestigated. After MTs were depolymerized by 1 h treatment with an anti-MT reagent,
MTs were repolymerized in various concentrations of D2O diluted with H2O. As theconcentration of D2O increased, cells having transverse MTs decreased and thosehaving longitudinal MTs increased. When MTs were repolymerized in 25mM NaCl or25mM KCl with various concentrations of D2O, the effects of these salts apparentlydiffered. When cells were treated with an anti-MT reagent for 24 h, most of therepolymerized MTs in 50mM NaCl with 50%(V/V) D2O were Z-helix and were different
from MTs repolymerized in the same concentration of NaCl with H2O in theirorientation. These results suggest that physicochemical properties of water molecules
might be important for regulating MT orientation.

Key words: Spirogyra, cortical microtubules, D2O

Water can be given a function by

Therefore, it is necessary that effects oftreating it with a weak energy. Changes

such water on living organisms should bein physicochemical properties areinvestigated. We already reported thatthought to be essential for these functions.

functional water, irradiated bySome of this water, so-called functional

far-infrared light, affected the orientationwater, is used for living organisms (1).

of cortical microtubules (MTs) in

Spirogyra cells, a species of algae, in aTheabbreviations

Amiprophos-methyl;used

are: APM,APW,pond water; MT, microtubule.

artificial

short time (2). This result might be due tochange of its physicochemical properties.

62

Effect of D2O on the orientation of cortical microtubules

However, the deviation of data was large

paper (2). Culture condition was also theand the differences were not always clear.

same as described in the previous paper

To determine whether the(2). It was cultured in Ichimura mediumphysicochemical properties of water(7) under the light conditions of 16-h lightaffect the orientation of cortical MTs,

and 8-h darkness at 250C. Cells werewater which has physicochemical

used for the experiments within 2 to 7

properties quite different from control

days (growing stage) after transferring

water (H2O) should be investigated.

them from the stock culture to the fresh

D2O (heavy water) is different fromH2O in freezing point, boiling point,

Ichimura medium.
Artificial pond water (APW) (0.1mM

viscosity, density and so on (1). The

each of KCl, NaCl, CaCl2 and 5mMhydrogen bond of D2O is stronger andHepes-Tris [pH7.5]) was used as thethermal motion is more limited. The

experimental medium when cortical MTs

effects of D2O on living organisms arein Spirogyra cells were depolymerized.

complicated. In the case of mammals, theCells were treated with 3μg/ml

level of spermatogenesis was reported to

Amiprophos-methyl (APM) in order todecrease (3, 4). Recently, the effect of D2O

depolymerize the cortical MTs for 1 or 24on MTs was investigated. Tubulin, theh. After APM was removed by washing

main component of MTs, is an unstable

the cells quickly three times withprotein. It is easily depolymerized to a

distilled water, cortical MTs weremonomer. However, it was reported that

repolymerized in solutions of various

D2O had an ability to stabilize MTs (5).

constitutions for 1.5 h. D2O (99.8%,

D2O also slows free-running rhythm (6).

Merck, Germany) was used for

In this paper, we show that D2O clearlyaffected MT orientation and that theexperiments. All the procedures weredone at 250C.

physicochemical properties of watermight be important for regulating MTorientation.

Immunostaining
MTs were observed using the method ofHogetsu (8) as described in the previous
paper (2). Cells were fixed in 3.7%

Materials and methods

formaldehyde in 50mM sodium
phosphate buffer (pH7.0) for 30 min, cut

Plant materials and treatments within the same fixative with a razor blade tosolutions

allow entry of antibodies and thenSpirogyra fluviatilis Hilse was thewashed three times with 50mM sodium

same species as used in the previous

phosphate buffer. They were treated with

K. Iwata and Y. Sano

63a detergent solution (1% Triton X-100
and 0.4M mannitol in 50mM sodium
phosphate buffer [pH7.0]) for 30 min.
After removal of the detergent, they
were incubated with mouse monoclonal
antibody against chick brain α-tubulin(Sigma, T9026) diluted 1:250 inphosphate-buffered saline (PBS, pH7.3)
overnight. They were washed three times
with 50mM sodium phosphate buffer andstained for 3 h with FITC-conjugatedantibodies raised in sheep against mouse

MTs were repolymerized in various

IgG (Sigma, F2883) diluted 1:200 in PBS.

concentrations of D2O diluted with H2O

All the procedures were done at roomfor 1.5 h. When they were repolymerized

temperature.

in H2O, all the cells had transverse MTs

After three washes with 50mM sodium(Fig. 1a, 2). As the concentration of D2O

phosphate buffer, cells were mounted

increased, the ratio of transverse MTswith a solution (50% glycerol in PBS

decreased and that of longitudinal MTs

containing 0.1% p-phenylenediamine

increased (Fig. 1b, 2).

dihydrochloride). The orientation of MTs

shown that D2O affected MT orientation.

was determined using an epifluorescence

Especially, MT orientation changed

microscope. Orientation of MTs wassensitively to D2O concentrationsclassified into three categories based on
MT angles to the cell axis, longitudinal(L) for 00-100, oblique (O) for 100-800,
transverse (T) for 800-900. Oblique (O)was further classified into S-helix andZ-helix, which were called as SO and ZO,
respectively.

Results and Discussion

Spirogyra cells have transverse MTs intheir normal condition (8,9,10). After cells
were incubated in APM solution for 1 h,

Itwasclearly

64

Effect of D2O on the orientation of cortical microtubules

between 20 and 40%(V/V). The

concentration of NaCl and KCl wasconcentration of 30%(V/V) D2O wasadjusted to 25mM because MT

adopted in the next experiment (Fig. 3).

orientation changed sensitively to NaCl

MT orientation in Spirogyra cells wasconcentration around 25mM in 30%(V/V)

affected by ions (Na+, K+ etc.) (2, 9, 10).

D2O (Fig. 3). This species of Spirogyra is

Therefore, the effect of D2O on MT

Na+-sensitive (2, 9, 10). In 100mM NaCl,orientation in solutions containing NaCl

many cells died (data not shown).

or KCl was investigated. TheK. Iwata and Y. Sano65

After cells were incubated in APM

observed. When the concentration of D2O

solution for 1 h, MTs were repolymerized

was above 25%(V/V), MTs in about 90% of

in 25mM NaCl with various

cells were S-helix (Fig.5). In H2O, there

concentrations of D2O for 1.5 h. As thewas no apparent difference in MT

concentration of D2O increased, cells

orientation between NaCl and KCl (Fig.4,having transverse MTs decreased and5). These results suggest that Na+ and K+

those having longitudinal MTs increased

affect(Fig. 4). However, in the case of 25mM

different way.

KCl, quite a different phenomenon wasMT

orientation

inD2O in a

As one of the authors has reported, the66

Effect of D2O on the orientation of cortical microtubules

effect of Na+ and K+ on the initialsolution for 24 h, MTs were

crystallization process of lysozyme wasrepolymerized

similarwater or of functional water. The effect oftothis case (11). In the case ofin 50mM NaCl of control

H2O, the initial aggregation rate wasD2O was also investigated in the same

about the same in both Na+ and K+. In theconditions.

case of D2O, the initial aggregation rate

After cells were incubated in APM

was affected by the ion species and thesolution for 1 h, MTs were repolymerized

value was lower in K+ than in Na+. It isin 50mM NaCl for 1.5 h. MTs of S-helix,

probable that the repolymerization

Z-helix and longitudinal orientations

process of MTs might be affected by thesewereions in a similar way as crystallization

when MTs were repolymerized in 50mM

process of lysozyme, resulting in theNaCl with 50%(V/V) D2O, the orientation

phenomenon of the MT orientationof MTs in above 90% of cells was Z-helix

described above. It is interesting to(Fig. 6). It is known that the orientation

investigate the effect of these ions on theof MTs in Spirogyra cells changes fromrepolymerization process of MTs in H2O

S-helix, Z-helix to longitudinal, as theand D2O.

concentration of NaCl increases (2, 10).

observedintermingled. However,As already reported, functional water,

These results suggest that the effect ofirradiated by far-infrared light, affected

D2O on MT orientation is not throughthe orientation of cortical microtubules

inhibiting or promoting the effect of(MTs) in Spirogyra cells (2). In this case,NaCl.

after cells were incubated in APM

K. Iwata and Y. Sano67

molecules in the plasma membrane andaffect MT orientation. It is interesting toinvestigate the relationship between
physicochemical properties of watermolecules and MT orientation to know
the effects of water molecules on livingorganisms.

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Mizu Handobukku (in Japanese) pp.

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Effect of D2O on the orientation of cortical microtubules

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Communicated by Sumio Ishijima