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Study of the mechanism of diatom cell division by by steepslope9876

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									            Study of the mechanism of diatom cell division by means
                             of 29Si isotope tracing
                    J.- N. Audinota*, C. Guignardb, H.- N. Migeona and L. Hoffmannb.
               a
                  Laboratoire d’Analyse des Matériaux, Centre de Recherche Public – Gabriel Lippmann
                                    41, rue du Brill, L-4422 Belvaux, Luxembourg
     b
       Cellule de Recherche en Environnement et Biotechnologies, Centre de Recherche Public – Gabriel Lippmann,
                                    41, rue du Brill, L-4422 Belvaux, Luxembourg
                                             audinot@lippmann.lu

Abstract
        Diatoms are delicate unicellular organisms enclosed in a silica frustule, that is made up of two
valves. Multiplication of the diatoms occurs by ordinary mitotic cell division. During cell division each
cell produces two daughter cells, each of them keeping one of the two valves of the mother cell and
producing a new valve by absorbing the silicon present in the environment.
        The NanoSIMS 50 allows ion imaging to be performed on diatoms in order to determine the site
of fixation of silicon. The aim of this study was to observe and compare the mechanism of the
construction of the new valve after cell division. To this end, different types of diatoms have been
transferred in a culture medium enriched with 29Si and after several days, the distribution of the
different isotopes of silicon has been determined by NanoSIMS50 imaging. The construction of new
valves has been observed and the isotopic ratio has been determined.

Keywords: NanoSIMS, isotope tracing, diatoms, biological sample, silicon

1. Introduction

         The lateral resolution (around 50nm) of the NanoSIMS as well as very high transmission at
high mass resolution (80% at M/∆M =5 000) render this ion microscope a tool of choice for the study
of isotopic elements in biological tissues [1-3].
         Diatoms are delicate unicellular organisms that have a yellow-brown chloroplast that enables
them to photosynthesize. Most diatoms are photosynthetic micro-organisms, although some may live
heterotrophically. Although unicellular, they may form branched or filamentous colonies, and even be
embedded in a gelatinous envelope or tube. All diatoms are enclosed in a silica frustule, that is made up
of two valves fitted together by a connective zone called a girdle. Multiplication of the diatoms
generally occurs by ordinary mitotic cell division. During cell division each cell produces two daughter
cells, each of them keeping one of the two valves of the mother cell and producing a new valve by
absorbing the silicon present in the environment.
The aim of this study was to understand, observe and compare the mechanism of the construction of the
missing valve. To this end, different types of diatoms have been transferred in a culture medium
enriched with 29Si for several days (1-5 days). The distribution of the isotopes of silicon (28Si, 29Si and
30
   Si) has been determined by NanoSIMS50 imaging.

2. Experimental

Preparation of the 29Si-enriched culture medium
        Labelled silica (96.74% atom 29Si) was purchased from Euriso-top (Saclay, France). This
labelled silicium oxide (19.4 mg) was converted into potassium metasilicate (K2SiO3) by alkaline
melting with 300 mg KOH in a nickel crucible, then dissolved in 80 mL DI-water (18.2 MΩ.cm-1) and
neutralized with hydrochloric acid (HCl 37 % aqueous solution). The volume of the solution was
adjusted to 100 mL (concentration: 3.172 mmol/L 29Si). The enriched culture mixture was prepared by
adding 4 mL of this 29Si solution to 96 mL of standard diatom culture medium containing 0.134
mmol/L Si [4]. The final 29Si/28Si ratio after enrichment was 1.08.

Diatom growth
        Diatoms (Nitzschia palea) were obtained from the Diatom collection of the Public Research
Center – Gabriel Lippmann. An aliquot (2 mL) of the diatom suspension was put in a sterile culture
flask and 10 mL of labelled culture medium were added. During the incubation period, the culture flask
was kept at room temperature (21°C) under constant light.

Sample preparation
        The culture flask was shaken during 5 minutes to detach all diatoms and homogenize the culture
medium, then 5 mL were transferred into a 16 cm x 1.6 cm i.d. test tube and 15 mL of hydrogen
peroxide (H2O2 35 % aqueous solution) were added. The temperature of the mixture was increased to
90°C and maintained during 4 hours to ensure a complete oxidation of the organic content. After 4
hours of sedimentation at room temperature, the upper layer was discarded. To remove carbonate
residues, 5 mL of hydrochloric acid (HCl 37 % aqueous solution) were added and allowed to react
during 2 hours at room temperature. Finally, 10 mL of DI-water were added to rinse the frustules, then
the upper layer was discarded after 4 hours of sedimentation. This step was reiterated 3 times to obtain
a satisfactory removal of HCl, H2O2 and other salts initially contained in the culture medium.
The bottom layer of the resulting solution, containing prepared frustules, was deposited with a Pasteur
pipette on a copper plot heated at 60°C. After complete drying, the sample was analyzed.

SIMS analysis
       The distribution of the isotopes of silicon (28Si, 29Si and 30Si) has been obtained in the
multicollection mode with parallel detection of 16O- and 12C2- at a mass resolution of 3500 (to minimize
28 1
  Si H contribution on 29Si). Maps have been acquired under standard analytical conditions, a Cs+ ion
primary beam with impact energy of 16keV and a current of 0.8 pA. All images have been acquired in
256 x 256 pixels with a counting time of 20 ms by pixel.

3. Results

        The control diatoms grown in the standard culture medium were studied first. In this sample the
isotopic images 29Si and 28Si have been recorded (figures 1a and 1b); the ratio image 29Si/28Si (figure
1c) shows an homogeneous distribution across the diatoms. The discrepancy between the “as measured
ratio” of 0.07 and the natural ratio of 0.05 is attributed primarily to the different gains of the two
detectors and, possibly, to some 28Si1H contribution on 29Si.
        Ion images of diatoms from the culture medium enriched with 29Si are shown on figures 2 and
3. Diatoms, at different moments of their life cycle, can be observed on figure 2: valves with an
isotopic ratio 29Si/28Si close to 0.05 characteristic of the mother diatom valve and a diatom valve with a
high concentration of 29Si (figure 2b, white arrow), characteristic of a newly formed valve. The ratio
between the 29Si-image and the 28Si-image is shown in figure 2d. It can be concluded that the with 29Si
enriched valve is a newly formed valve and that to construct this new valve, the silicon of the 29Si-
enriched culture medium has been absorbed during the multiplication.
Figure 1 : 28Si (left) and 29Si (center) distribution in control diatoms (image field 30 x 30µm2, linear
                                                (a)                                         (b)
scale). Right: ratio image 28/29 using a logarithmic scale.




                                                                                                     Valve enriched
                                                                                                     29
                                                                                                      Si/28Si ≈1

                                             (a)                                            (b)




                                             (c)                                            (d)

Figure 2: 28Si (a), 29Si (b), 30Si(c) and isotopic ratio 29Si/28Si (d) distribution in diatoms after culture in
                 medium enriched in 29Si (image field 30 x 30µm2, logarithmic scale).
                                    (a)                                                                   (b)                        (c)




                                                    1.5
                                                    1.4                                                                  29Si/28Si
                                                                                                                         30Si/28Si
                                                    1.3
                                                                      Labelled medium 29Si/28Si=1.1
                                                    1.2
                                                    1.1                                                  29   28
                                                                                         Labelled medium Si/ Si = 1
                                                     1
                                                    0.9
                                   Isotopic ratio




                                                    0.8
                                                    0.7
                                                    0.6
                                                    0.5                             28 28
                                                                         Natural 29Si/Si Si=0.05
                                                                                29
                                                                         Natural Si/      = 0.05
                                                    0.4
                                                                                        Natural 30Si/28Si=0.03
                                                                                                 30   28
                                                                                          Natural Si/ Si = 0.05
                                                    0.3
                                                    0.2
                                                    0.1
                                                     0
                                                          1.0   2.0      3.0          4.0          5.0             6.0       7.0
                                                                                      d (µm)


   Figure 3 : 28Si (a) and 29Si (b) maps, isotopic ratio 29Si/28Si (c) (log scale) and line-scan 29/28 (as
               depicted on image a) in diatoms after culture in medium enriched with 29Si.

        Different analyses have been realized on the same diatoms solution. In some cases, an
inhomogeneous distribution in 29Si has been found in some valves (figure 3). In this analyzed area,
valves of diatoms are more or less enriched in 29Si (figures 3a and 3b), as is also shown in the image of
the isotopic ratio 29Si/28Si (figure 3c). This representation highlights the inhomogeneous distribution in
29
   Si.

Conclusion

         These preliminary measurements have confirmed the multiplication of diatoms by ordinary
mitotic cell division. Furthermore, the lateral resolution provided by the SIMS experiments permits to
record heterogeneous distributions of the silicium 29/28 ratio inside one single valve. Together with
improved accuracy of the isotopic ratio measurements this will permit, in future works, to investigate in
details the site of fixation of silicon (growth mechanism) up to the construction of the complete missing
valve.

Acknowledgements
This work received the financial support of the Fonds National de Recherche de Luxembourg in the
framework of project FNR/01/02/01b. F. Rimet is thanked for providing the diatom culture.

References
[1] F. Hillion, B. Daigne, F. Girard, G. Slodzian, in: Proceedings of the SIMS IX, Yokohama, 1993, p. 254.
[2] N. Grignon, J.J. Vidmar, F. Hillion, B. Jaillard, in: Proceedings of the SIMS XII, Brussels, 1999, p. 903.
[3] J.- L. Guerquin-Kern, T.- Di Wu, C. Quintana and A. Croisy, accepted for publication in Biochimica and Biophysica
Acta (2005).
[4] R.R.L. Guillard and C. L. Lorenzen. J. Phycol., 8 : 10-14 (1972).

								
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