@Copyright 1987 by The Hurnana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/1300-0135502.00

Hint of Polar Distribution in Calcium Channels Under PIXE Analysis HANS-DIETER REISS *'l't AND KURT TRAXEL2

'Zellenlehre, Universit~t Heidelberg, and 2Physikalisches lnstitut, Universit~t Heidelberg and Nlax Planck lnstitut for Kernphysik, D-6900 Heidelberg, FRG

ABSTRACT Pollen tubes of Lilium longiflorumwere treated for 10-30 rain with 1 0 - 5 M COC12, which binds to calcium channels in the plasma mem-

brane and blocks them. Cobalt analyses were performed with the Heidelberg proton microprobe, using 3 MeV protons, a beam current of about 200 pA, and a spot size of 3 x 5 ~m ~. X-ray spectra revealed that cobalt has much higher concentrations in the cell than in the surrounding dried medium. The line scans, taken along the longitudinal cell axis in 1-p,m steps, showed a cobalt gradient similar to the calcium gradient of the same cell. Based on our findings, we can conclude that neither do the cobalt signals come from the cell wall nor is the cobalt exclusively bound to the intracellular calcium-binding sites. Therefore, the present results suggest a polar distribution in calcium channels in the plasma membrane of pollen tubes. Index Entries: Calcium channels; cobalt; pollen tube; tip growth; PIXE analysis; micro PIXE.

INTRODUCTION Pollen tubes are widely u s e d as an experimental s t a n d a r d s y s t e m on w h i c h to investigate tip g r o w t h (polar growth) of tubular plant cells (1). The m a i n event of tip g r o w t h is the oriented exocytosis of secretory vesicles. It could be d e m o n s t r a t e d that tip g r o w t h regulation is closely correlated to the m a i n t e n a n c e of the intracellular tip-to-base calcium gradient, which can be visualized by chlorotetracycline (CTC)-fluorescence as well *Author to whom all correspondence and reprint requests should be addressed. tPresent address: Zellenlehre, Im Neuenheimer Feld 230, D-6900 Heidelberg, FRG Biological Trace Element Research

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as under PIXE analysis (2-5). It is thought that the calcium gradient is built up and maintained by the locally restricted, passive influx of external calcium ions across the plasma membrane through calcium channels (6,7). Specific blocking of calcium channels by nifedipine (8) leads to disturbance of the calcium gradient and tip growth, indicating the existence of the postulated channels (9). Similar results were obtained with cobalt ions (unpublished), which can also be used as calcium channel blockers (8), but probably have additional effects (10). Therefore, we have tested to determine whether PIXE analysis--which is capable of detecting trace elements--of cobalt-treated pollen tubes can provide information on calcium channel distribution.

METHODS Pollen tubes of Lilium longiflorum Thunb. were grown in vitro in an aqueous medium containing 10% sucrose and 10 ppm boric acid. When the tubes reached an average length of 100 t~m they were treated with 10 5M COC12 for 10-30 min. Then the tubes were chemically fixed for 10 rain with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.2), transferred onto the supporting foil (Pioloform) of the target holder, and air dried. PIXE analysis was performed with the Heidelberg proton microprobe (Max Planck Institut f(ir Kernphysik, Heidelberg, FRG), using 3 MeV protons, a beam current of about 200 pA, and a spot size of 3 • 5 I~m2 for single spectra and 2 • 40 i~m2 for line scans. For focusing the beam, a thin layer of plastic scintillator was applied to an object-flee region of the target. Rutherford scattering on a gold foil, I mg/cm 2 in thickness, served as the beam current monitor. All X-ray spectra were taken for the same number of incident protons. In the line scans, 12 energy windows allowed the simultaneous registration of the count rates for P, S, C1, KK~, KKJCaK~,, CaKe, FeK~, FeK~/CoK~, COKe Zn, and As, and of the background for every point. The latter was taken as a relative measure for sample thickness and simultaneously served for background subtraction in the element windows. To correct for the varying thicknesses of the specimens, as monitored in the background window, the count rates for every point of the scans were normalized according to:

Nc = (Nu/NB - Rj)Fj, with Nc = corrected count rate Nu = count rate as measured NB = count rate in the background window Rj is derived from an X-ray energy spectrum showing background only. It is the ratio: (content in the window of element j)/ (content in the background window) Fj is a normalization factor chosen in such a way that the total count rate in a certain region of average thickness is maintained Biological Trace Element Research

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By this procedure, additional errors stemming from the statistical errors of the background count rate are introduced. The total statistical errors are calculated and are indicated by error bars in the figures. A comprehensive description of the Heidelberg proton microprobe is given in refs. (3,11).

RESULTS

X-ray EnergySpectra X-ray energy spectra were taken from the supporting foil [Fig. l(a)], the plastic scintillator [Fig. l(b)], the medium and fixative dried-in around the specimen [Fig. 1(c)], and the pollen tube [Fig. l(d)]. In contrast to untreated pollen tubes [see (3,4)], the cobalt-treated tubes showed distinct cobalt lines, proof that cobalt comes exclusively from the C o C I 2 treatment [Fig. l(d)]. The cobalt lines are relatively weak in the spectra taken from the dried-in medium in which the cobalt salt was dissolved (Fig. 1(c)]. Neither the supporting foil nor the plastic scintillator showed traces of cobalt [Fig. l(a,b)]. Calcium can be detected only in the cell [Fig. l(d)]. The arsenic signals originated from the cacodylate buffer used during fixation. J I

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Fig. 1. X-ray-energy spectra; (a) supporting foil Pioloform); (b) plastic scintillator; (c) dried-in medium and fixative; and (d) pollen tube. Biological Trace Element Research

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Line Scans Line Scans were taken along the longitudinal cell axis, with a step width of 1 ~m, starting a few microns before the tip [Fig. 2]. In short tubes the scan extended to the grain, in which the background signals increased remarkably [Fig. 3]. The diameter of the pollen grain was in the range of 100 I~m, whereas the tube had a diameter between 15 and 20 I~m. Therefore, we normalized all data to the thickness of the specimen [Fig. 4]. Figures 3(b) and 4(e) show calcium scans before and after correction for the varying thickness. The scans reveal very different distribution patterns of the elements measured simultaneously [Fig. 4(a-f)]. The distribution patterns are comparable to those of untreated tubes, as demonstrated recently (5). In contrast to other elements, cobalt paralleled calcium in distribution [Figs. 4(e,f),5]. Both gradients reached a maximum value of about 6-7 I~m behind the tip, but the correspondence of both gradients was not absolute: e.g., the increase in calcium in Fig. 5(a) was steeper than that of cobalt in Fig. 5(b). Because potassium and iron were equally distributed [Fig. 4(c,d)], for calcium and cobalt distributions, the K~ lines could be used, leading to better statistics than the K~ lines. The shape of the elemental distributions was independent of the time of treatment with CoCl2.

Fig. 2. Light micrograph of a pollen tube after line scanning. The scan distance gets visible by the lines caused by radiation damage and/or carbon deposition. Biological Trace Element Research

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PIXE Analysis of Calcium Channels

(a)

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Fig. 3. Elemental distributions along the longitudinal cell axis of one cell, starting some microns before the tip and reaching up into the pollen grain. (a) background signals, indicating the varying thickness of the specimen; (b) distribution of calcium, including the background (thickness) fluctuations [compare with Fig. 4(e)].

DISCUSSION An interpretation of the different distributions in elements and their possible significance in pollen tubes was given recently (5). It must be reiterated that, at the moment, with the technique used only the distribution of structurally b o u n d elements and not that of free ions can be determined. Hence, potassium, which mainly exists as a free ion in the pollen tube, revealed an equal distribution [for discussion see (5)]. Therefore, the artificially caused cobalt distribution--no cobalt could be detected in control cells (4)--indicated any structure that was capable of binding this element. With regard to the strong correspondence between both the cobalt and calcium distribution, the following interpretation comes into question: Cobalt will be b o u n d to the same cellular binding sites as calcium; cobalt will be b o u n d to other structures that are enriched in t h e pollen tube tip region; or both possibilities. In any case, we can exclude from the gradient-like distribution in cobalt that cobalt is bound in higher amounts to the cell wall. Although ultrastructural studies of cobalt-treated pollen tubes revealed a distinct effect on the structure of the cell wall, which was incorporated during the time of treatment (Reiss, unpublished), the PIXE data were not d e p e n d e n t on the time of cobalt treatment. A binding of cobalt to the same binding sites as for calcium can explain the distribution gradient and the correspondence between cobalt and calcium distribution. Cobalt does not seem to penetrate through the plasma membrane, as s h o w n by the different effects of extracellularly and intracellularly applied cobalt in isolated frog spinal cord (12), but we cannot exclude such a penetration during fixation. Cobalt does not displace calcium in higher amounts from its binding sites because the count-rates for calcium are in the same magnitude as in control tubes, in which identical m e a s u r e m e n t conditions were used (4). It is also possible that many poBiological Trace Element Research

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Fig. 4. Background (thickness) corrected data from the same cell as shown in Fig. 3. tential calcium binding sites are yet free and will be saturated only by an extensive addition of calcium (13) or other divalent cations as cobalt in the present study. Some details of the present results contradict the assumption that cobalt binds exclusively to calcium-binding sites. In the

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Fig. 5. Comparison between calcium and cobalt distribution measured in a long pollen tube (corrected data). measurements, the extension of both gradients often are not absolutely parallel, e.g., in one case shown here the cobalt signals increase one step (1 I~m) after the increase of calcium in the same cell. Here the structure in question is the plasma membrane, which is very small (about 10-12 nm) in comparison to the proton beam used (2-3 I~m), so that it is a rare event that the beam hits the plasma membrane before the intracellular structures, resulting in a scan, as shown in Fig. 4(e,f). In the plasma membrane there are calcium channels through which calcium passively enters the cell; they will be blocked by cobalt (14,8). The existence of calcium channels in pollen tubes could be proved by inhibitor experiments (9). Their involvement in the polar entry of calcium into the growing pollen tube tip was discussed for a long time [for review see (7)], and, therefore, a polar distribution was expected. Although, of course, additional work must be done to ensure the polar distribution of calcium channels in the pollen tube, the present results can be taken as a first suggestion for such a polar distribution, expecting that the cobalt blocked calcium channels are among the detected cobalt-binding structures.

SUMMARY Pollen tubes of L. longiflorum were treated for 10-30 min with 10 5M

CoCI2, which blocks calcium channels, then chemically fixed and analyzed with the Heidelberg proton microprobe. X-ray spectra revealed that the cobalt concentration in the cell was higher than in the surrounding dried-in medium. The line scans, demonstrating the elemental distributions along the longitudinal cell axis, showed a cobalt distribution similar to the calcium distribution, both with the highest content at the pollen tube tip. From the results it could be concluded that the cobalt distribution hints to a polar distribution in calcium channels, located in the plasma membrane of the pollen tube.

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ACKNOWLEDGMENTS The authors thank Prof. E. Schnepf and Drs. W. Herth and Ch. B~ihrle for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft.

REFERENCES 1. A. Sievers and E. Schnepf, in Cell Biology Monographs, vol. 8, O. Kiermayer, ed., Springer, Wien, New York, 1981, pp. 265-299. 2. H. -D. Reiss and W. Herth, Protoplasma 97, 373 (1978). 3. F. Bosch, A. E1 Goresy, W. Herth, B. Martin, R. Nobiling, B. Povh, H. -D. Reiss, and K. Traxel, Nucl. Sci. Appl. 1, 33 (1980). 4. H. -D. Reiss, W. Herth, E. Schnepf, and R. Nobiling, Protoplasma 115, 153 (1983). 5. H. -D. Reiss, G. W. Grime, M. Q. Li, F. Takacs, and F. Watt, Protoplasma 126, 147 (1985). 6. H. -D. Reiss, W. Herth, and R. Nobiling, Planta 163, 84 (1985). 7. M. H. Weisenseel and R. M. Kicherer, in Cell Biology Monographs, vol. 8, O. Kiermayer, ed., Springer, Wien, New York, 1981, pp. 379-399. 8. H. Reuter, Nature 301, 569 (1983). 9. H. -D. Reiss and W. Herth, J. Cell Sci. 76, 247 (1985). 10. M. Tepfer and I. E. P. Taylor, Can. J. Bot. 59, 1522 (1981). 11. B. Martin and R. Nobiling, in Applied Charged Particle Optics, A. Septier, ed., Academic, New York, NY, 1980, pp. 321-346. 12. B. Buchert-Rau and U. Sonnhof, Pfl~gers Arch. 394, 1 (1982). 13. W. Herth, H.-D. Reiss, B. Hertler, R. Bauer, K. Traxel, and Ch. Ender, J. Ultrastruct. Res. 93, 71 (1985). 14. M. Kohlhardt, B. Bauer, H. Krause, and A. Fleckenstein, Pfliigers Arch. 338, 115 (1973).

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