Electron microscopy of synchronous exocytosis in
Paramecium cells: Quenched-flowkryofixation,
EDX, and
freeze-fracture analysis
M. Hardt, J. Hentschel, C. Braun, H. Plattner
Dept. of Biology, University of Konstanz, D-78434 Konstanz
Paramecium cells perform synchronous exocytosis of trichocysts (spindle shaped secretory
organelles docked at the cell membrane) within 80 ms, in response to aminoethyldextran, AED [l].
The Ca2+ required for exocytotic membrane fusion [l] primarily comes from “alveolar sacs” [2,3]
previously identified as subplasmalemmal Ca-stores [4]. This is superimposed by Ca2+-infhtx from
the medium [2,3]. Mobilization of cortical Ca-pools and occurrence of Ca2tdependent
membrane
fusion was analyzed by parallel EDX and fkeeze-fracture, following quenched flow-preparation
protocols which we previously have published [5-71.
Cells were supplied with varying extracellular Ca2+ concentration, [Ca2+le, i.e., 500 pM or
150 JIM (equivalent to resting levels in the untriggered cell, [Ca2+]i [3]), cryofixed by spraying into
liquid propane (123K) [5] and freeze-substituted under conditions of Ca-retention [6]. 500 nm
sections were subject to EDX, using a Zeiss/Leo EM912 Omega (80 kV, 10 yA emission current,
63 nm spot size, 3 1,500x) in the STEM mode and equipped with an Oxford system (type E.6356,
Lidrifted S&detector [30 mm2 front size, ATW2 atmospheric window], minimal sample distance,
20” take-off angle) and a Link ISIS 3.00 software.
In untriggered cells, clear Ca-signals (indicating several millimolar concentrations) are
registered only over Ca-stores (fig. l), but none in cilia, cytoplasm, mitochondria etc. When AEDstimulated in presence of [Ca2+], = 500 PM, Ca-signals increase in the cytoplasm, while depletion
of stores may be counteracted by Ca 2+e-influx. The latter component is eliminated when AEDstimulation occurs with [Ca2+], 5 50 nM. We then find store depletion by 30 to 40% within 30 to
80 ms, respectively (figs. 2, 3). Parallel quantitative fi-eeze-fracture analysis (for details, see ref [7])
reveals that this Ca2+ -signal activates membrane fusion only in part of the exocytosis sites, while
full exocytotic response requires superposition by Ca2fe-itt.tlux [7].
Similar questions concerning cortical Ca-stores now arise from a variety of secretory
systems, e.g., different gland cells, where however stores are much less defined. Our work shows
feasibility of the EM approach presented, while EELS/ES1 methodology can provide Ca
localization with improved spatial resolution [6].
Supported by Deutsche Forschungsgemeinschaft,
grant P178/11.
References
1. Plattner H. et al., Eur. J. Cell Biol., 55 (1991) 3.
2. Erxleben C. et al., J. Cell Biol., 136 (1997) 597.
3. Klauke N. and Plattner H., J. CeEZSci., 110 (1997) 975.
4. StellyN. etal., J. CellBiol., 113 (1991) 103.
5. Knoll G. et al., J. Cell Biol., 113 (1991) 1295.
6. Knoll G. et al., CeZZCalcium, 14 (1993) 173.
7. Plattner H. et al., J. Membr. Biol., 158 (1997) 197.
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FIG. 1 - Selective localization of Ca
in alveolar sacs; [Ca2+]e 5 50 nM,
unstimulated cell. STEM image of cortical cell region with the following
structuTes (l-10) from which EDX
spectra were recorded (only 3.0 to
4.4 keV range shown, point measurements at contamination dots):
1: off-cell background,
2: ciliun, 3,4: trichocyst contents (tip
region), 5,7: alveolar sacs (-50 nm
wide, apposed to cell membrane), 6:
cytosolic region, 8: fat droplet, 9:
brichocyst contents (body region),
10: mitochondrion. - 11: Ca-Ka line
scan, cytosol -> alveolar sac (AS) ->
outside.
FIG. 2 - EDX spectra from alveolar
sacs, cells (a) before or (b) after
80 ms AED-stimulation,
[Ca2+]e 5 50
nM. Note decrease of Ca-signal upon
stimulation.
0
0
80 ma
30
time after AED-trigge
FIG. 3 - [Cal in alveolar sacs decreases upon 30 or 80 ms AED-stimulation
([Ca2+]e 5 50 nM) by -30 to 40 %.
Number of evaluated alveolar sacs or
cells, N = 25, 31,32, n = 5, 6, 6,
for 0,30 and 80 ms, respectively.
Bars = S.E.M.