The role of Ca2+ in stimulated bioluminescence of the dinoflagellate Lingulodinium polyedrum
Peter von Dassow* and
Michael I. Latz
Scripps Institution of Oceanography, University of California, San
Diego, La Jolla, CA 92037-0202, USA
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Fig. 1. Stimulation of luminescence by ionomycin. (A) Integrated luminescence over
a total of 200 s (filled circles) and over the first 12 s (open circles)
during and after addition of different concentrations of ionomycin or control
solution to night-phase cells of Lingulodinium polyedrum in
Hepes-buffered filtered seawater (HbFSW; N=3-4 for each stimulus
tested). Values are means ± S.D. (B—D) Calcium dependence of
luminescence stimulation by ionomycin when added to night-phase cells in
Ca-free artificial seawater (ASW; total [Ca] = <40 µmol l-1
and cheated by 1 mmol l-1 EGTA). (B) Ionomycin treatment at a final
concentration of 5 µmol l-1 plus 2xCa to return
extracellular Ca2+ to normal levels. The bar below the
x-axis marks the approximate time of injection. (C) As in (B), but
ionomycin was added without Ca2+. There was decreased stimulation
and the time course of stimulation was slower. (D) Integrated luminescence
produced by ionomycin or a control solution when added to Ca-free cells with
or without simultaneous replacement of Ca2+ (N=11 for all
stimuli except for 0.5% DMSO without Ca, for which N=5). Integrated
luminescence over a total of 200 s (filled bars) and over the first 12 s (open
bars) is shown. Values are means ± S.D.
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Fig. 2. Flow-stimulated luminescence in Lingulodinium polyedrum. (A)
Stimulation by stirring, which started at approximately 13.4 s. (B) Computed
maximum shear stress for Couette flow during and after 1 s acceleration of the
outer cylinder to final velocities of 0.8-6.4 revs s-1 (final shear
stress=0.11-0.86 N m-2). (C) Typical responses of night-phase cells
to the developing Couette flows shown in (B).
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Fig. 3. Effects of 5 µmol l-1 BAPTA-AM treatment. (A) Effect of
stirring of samples tested at different times after treatment. BAPTA-AM
treatment (open circles) decreased mechanically sensitive luminescence
(integrated over 6 s stirring; 1st 6 s MSL) compared with control-treated
samples (filled circles). The curve represents an exponential decay model fit
to the data: y=3.0x108 e-0.0933t
(r2=0.93), where y is the 6 s integrated MSL, and
t is the treatment time in min. (B) Spontaneous glow emission of
BAPTA-AM-treated (open circles) and control-treated (filled circles) samples.
(C) Response of BAPTA-AM-treated and control-treated samples to developing
Couette flow.
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Fig. 4. Effects of 5 µmol l-1 BAPTA-AM treatment on other luminescent
parameters. (A) Integrated luminescence stimulated by addition of BAPTA-AM
(open bars) or control (filled bars) solutions, and mechanically sensitive
luminescence (1st 6 s MSL) and total luminescent capacity (TLC) of the same
samples 40-50 min later (N=4 for each treatment). Values are means
± S.D. Only 1st 6 s MSL was significantly different between treatments
(t=-10.3, P<0.0001). (B) Response to 5 µmol
l-1 ionomycin after 40-50 min of BAPTA-AM or control treatment.
Total ionomycin-sensitive luminescence (TISL) integrated over 200 s and the
first 12 s of integrated ionomycin-sensitive luminescence (1st 12 s ISL) were
measured (N=4 for each treatment). Values are means ± S.D.
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Fig. 5. Effects of Ruthenium Red. (A, B) Effects of Ruthenium Red on luminescence
parameters assayed by stirring. Values are means ± S.D. (A) Effect of
Ruthenium Red on relative luminescent response. Total luminescent capacity
(TLC) was normalized to the mean of the control samples run on each date of
testing (open squares). Mechanically sensitive luminescence was integrated
over the first 6 s (1st 6 s MSL; filled circles) and over a total of 200 s
(TMSL; filled diamonds) while stirring and normalized to TLC of each sample
(N=3-10 for each treatment). (B) Effect of Ruthenium Red on maximum
intensity of mechanically sensitive luminescence (Max I) normalized to TLC for
each sample. (C) Effects of Ruthenium Red on motility and ecdysis. Cells not
swimming in suspension (on bottom; filled circles) and empty theca (open
circles) are expressed as proportions of pigmented cells (N=3-9).
Values are means ± S.D. (D) Effect of Ruthenium Red on the response to
developing Couette flow. Results are representative of five independent
samples tested for each 0 µmol l-1, 5 µmol l-1 or
50 µmol l-1 treatment.
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Fig. 6. Stimulation of luminescence by increasing [K+]. (A) Sample
records of luminescence stimulated by addition of solutions containing high
(31 mmol l-1) or low (6 mmol l-1) [K+]. The
box beneath the x-axis marks the time of injection (controlled by a
syringe pump). (B) Dose-dependence of stimulation by K+. Integrated
luminescence (over 200 s total) stimulated by addition of [K+]
solutions normalized to total luminescent capacity (K+-SL/TLC) for
each sample tested (N=4-7). Data are expressed as means ± S.D.
and are a function of [K+] in the test solution added rather than
the final [K+], because much of the stimulation occurs before
mixing of solution can be completed, especially at high [K+]
(N=4-7 for each stimulus tested).
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Fig. 7. The role of Ca2+ in K+-sensitive luminescence.
Results are expressed as 200 s integrated luminescent response to stimulation
by addition of indicated volume of low (7.5 µmol l-1) or high
(108 µmol l-1) [K+] solutions. (A) K+
stimulation following 40-50 min treatment with 5 µmol l-1
BAPTA-AM (open bar) or control solution (filled bar) (N=5-6 for each
treatment/stimulus combination tested). (B) K+-stimulation of
Ca-free cell suspensions with (filled bar) or without (open bar) simultaneous
replacement of Ca2+ in stimulating solution (N=5 for each
stimulus tested). n.d.=not determined. Values are means ± S.D.
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Fig. 8. Inhibition of K+-sensitive luminescence by Ruthenium Red. The
integrated response to [K+] over 200 s was normalized to total
luminescent capacity (K+-SL/TLC) for each sample to compensate for
modest decrease of apparent TLC after Ruthenium Red treatment (N=4-10
for each treatment/stimulus combination tested). Values are means ±
S.D. Treatment for >4 h with 50 µmol l-1 Ruthenium Red
inhibited stimulation by added solutions containing low to moderate
[K+] but not high [K+].
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© The Company of Biologists Ltd 2002
