Transcript protist behavioral responses to micromolar levels

Binding characteristics of amino acids inhibiting feeding in the marine tintinnid ciliate Favella sp.
Nicole J. Huber and Gordon V. Wolfe, Dept. Biological Sciences, California State Univ. Chico
[email protected]; [email protected]
a. Amino acids bind to cells over 1100 uM, similar to concentrations
that inhibit grazing
b. Binding kinetics and specificity are similar to feeding inhibition
8,000
60%
20
40
60
80
100
mM total proline
1,200
serine
proline
Series3
1,000
800
600
4,000
120%
2,000
0
400
200
0
corrected for filter blank
40
60
80
6
8
10 12 14 16 18 20
100
60%
40%
20%
0%
min incubation
mM total serine or proline
Fig. 1: Fractional feeding inhibition in presence
of proline (top, data courtesy S.L. Strom)
parallels binding of 3H-proline and 3H-serine to
cells (bottom).
Fig. 2: [3H]-proline (20 mM) shows rapid
binding within 2 min. of addition.
unlabeled compound
Fig. 3: Feeding inhibition by different AAs (top,
data courtesy S.L. Strom) parallels inhibition of
binding of [3H]-serine (bottom).
c. Amino acids bind to live cells, not to lorica, and binding is specific
Here, we characterize cell-surface binding of the tritiated amino
acids proline and serine, and show binding is consistent with
feeding inhibition. We infer from binding characteristics and
localization on the cell surface that binding sites likely represent ion
channels, possibly involved with mechanotransduction.
2. Preliminary competitive inhibition data parallel the
feeding inhibition data, with small side chain AAs
significantly blocking binding of [3H]-proline and
serine, and larger side chain AAs having little effect on
binding.
2. Where is the binding located? Does it occur on
the lorica, cell soma, or membranelles?
3. What is the nature of the binding site?
Fig. 4: Autoradiograms of cells preserved with formalin. Left: [3H]-proline binds to live cells (top) but not lorica (bottom). Center:
[3H]-proline does not bind to fixed cells (a), nor to live cells incubated with excess cold proline (b). Right: [3H]-serine binding shows
similar patterns to live (a) and fixed (b) cells. Fluorescence images are superposition of DAPI and blue excitation.
d. Amino acids bind exclusively to cell soma, not membranelles
3. Binding localizes exclusively to cells, not lorica, and
occurs on the soma, not membranelles. Binding
requires live cells and appears specific.
Acknowledgements
We thank Suzanne Strom and Kelley Bright, Shannon Pt. Marine
Laboratory, for sharing data and cultures of Favella and prey
algae. This project is funded by NSF OCE-0325025.
Literature Cited
Radiolabel binding - Cells were incubated 0-20 min in 1-30 mCi L-[3H]
proline or serine (20-80 Ci mmol-1, Moravek Biochemicals, Sigma),
then filtered onto 5 mm polycarbonate membrane filters
(Millepore), washed with 2 mL distilled water and counted in 5 mL
ScintiVerse (Fisher). For competitive binding, cells were preincubated in 1-10 mM cold amino acids (Sigma).
Microautoradiography - Cells were incubated with 0.4-1 mM label (30 mCi
total). Treatments included live cells, cells fixed with formalin or
Lugol’s, and cells incubated with 10 mM cold AAs. Cells were
filtered, washed several times with distilled water, and filters were
placed cell-side down on slides dipped 10 sec in Kodak NTB
emulsion (43 oC). The emulsion was chilled and exposed 5-12 d at
4 oC, then developed in Dektol (diluted 1:1 with water) for 2 min.
After removing filters, cells were stained with 10 mg mL-1 DAPI,
viewed on an Olympus BH-51 under epifluorescence and
brightfield, and photographed with a Pixera Penguin digital
camera. Fluorescence composites were produced in Adobe
Photoshop.
A similar mechanism might account for Strom et al’s. (2005)
observations of inhibition of Favella feeding by micromolar AAs.
However, AA binding to ion channels on the cell soma might also lead
to changes in membrane potential, as observations of swimming
behavior in the presence of AAs show evidence of membrane
depolarization leading to avoidance reactions (Wolfe, in preparation).
We will test these ideas in future work.
1. Proline and serine, which inhibit Favella from feeding
on the dinoflagellate Heterocapsa triquetra, bind to the
cell soma of Favella in a rapid manner with low affinity
(mM saturation) and specificity. AAs with small side
chains compete for binding sites and inhibit feeding to
the greatest degree.
1. Is binding similar to feeding inhibition (same
AAs, kinetics, concentration range)?
Cultures - Ciliates were cultured on Heterocapsa triquetra, Isochrysis
galbana, and Mantoniella squamata at 16 oC in Instant Ocean. Prior to
experiments, cells starved for 12-24 hrs, then concentrated by
reverse-filtration through 20-40 mm mesh to densities of ca. 20-80
cells mL-1. Cells were enumerated by settling and counting after
fixation with acid Lugol’s.
The only previous study on AA binding to ciliates (Preston and
Usherwood, 1988) found high affinity (nM saturation) and specificity
for glutamate, located exclusively on Paramecium cilia. In contrast, our
findings that AAs bind to Favella with low affinity and specificity, a
high number of binding sites, and somatic location, are similar to
observations by Wood (1985, 1989) for tubocurarine, which bound
exclusively to Stentor somatic mechanoreceptors. This nicotinic
acetylcholine antagonist did not affect membrane potential, but
specifically decreased mechanoreceptor ion currents, leading to
decreased sensitivity and contraction upon mechanical stimulation.
Summary
Questions
Methods
Binding of both proline and serine occurs over a similar concentration
range as feeding inhibition (Fig. 1), with half-maximal concentration of
5-10 mM. Binding saturation occurs at 600-800 fmol cell-1, or
approximately 1011 molecules cell-1, and is rapid (Fig. 2), similar to
feeding inhibition (S. Strom, 2005, in preparation). Cold AAs,
especially those with small side chains, block binding of [3H]-proline
in a manner consistent with feeding inhibition (Fig. 3). Autoradiograms show that AAs bind to cells, and not to loricas or membranelles,
and is specific, as it is inhibited by fixation or incubation with excess
cold AA (Figs. 4, 5). Attempts to use FITC-labeled AAs to locate
binding failed (not shown). This is consistent with evidence that
dipeptides containing proline show little inhibition of feeding (S.
Strom, personal communication). The reduction of binding from any
increase of molecular size agrees with observations that small sidechain AAs maximally inhibit grazing (Fig. 3).
80%
DI
20
4
-2,000
0
0
2
100%
PRO
0
SER
0%
6,000
GLN
20%
corrected for filter blank
% of 3H-serine binding
40%
ARG
What is mechanism of this unexpected result? Few studies have
examined the effect of stimulus-receptor binding on feeding, but
there is large literature on the electrophysiological responses of
ciliates to mechanical disturbance and chemorepellents. Preston &
Usherwood (1988) found a glutamate-specific receptor on the ciliary
surfaces of Paramecium, while Köhidai et al. (1997) found evidence
for dipeptide binding Tetrahymena specific for proline and its
position. Wood (1985) showed that tubocurarine binds to
mechanoreceptors responsible for contraction in Stentor, which are
located on the cell surface (Wood, 1989).
80%
[3H]-proline binding
(DPM)
Favella sp.
Top: cells
viewed in
brightfield
and darkfield.
Bottom: SEM
image,
showing
hyaline lorica.
Cell lengths
approx. 180
mm.
% feeding inhibition
100%
fmol / cell
Tintinnid ciliates are important
consumers of algae in marine
coastal waters (Pierce & Turner,
1992). Strom et al. (2005, in
preparation) found that
micromolar amino acids (AAs)
and related organic osmolytes
reduce Favella sp. feeding on
Heterocapsa triquetra. Amino acids
did not reduce survivorship, and
inhibition was strongest with
small side chain AAs, rather than
a function of side chain
hydrophobicity or ionization.
Inhibition occurred in a doseresponsive manner from 0.2 – 20
mM, within seconds to minutes of
dosing, and could be immediately
relieved by removing AAs.
Discussion
Results
Introduction
Köhidai, L., P. Soós, and G. Csaba. 1997. Effects of dipeptides containing
the amino acid proline on the chemotaxis of Tetrahymena pyriformis.
Evolutionary conclusions on the formation of hormone receptors and
hormones. Cell Biol. Inter. 21: 341-345.
Fig. 5: Autoradiograms of cells preserved
with acid Lugol’s. Lugol’s cells in bottom
images are for comparison.
Left, above: [3H]- proline binding. Right:
[3H]-serine binding to deloricated cell.
Autoradiographic exposure is
superimposed on fluorescence image,
showing exposed silver grains in white at
somatic periphery.
Pierce, R.W. and J.T. Turner. 1992. Ecology of planktonic ciliates in
marine food webs. Rev. Aquat. Sci. 6: 139-181.
Preston, R.R., and P.N.R. Usherwood. 1988. Characterization of specific L[3H]glutamic acid binding site on cilia isolated from Paramecium
tetraurelia. J. Comp. Physiol. B 158: 345-351.
Strom, S.L., K. Bright, and G.V. Wolfe. Amino acids as signal compounds
affecting feeding by microzooplankton. Presented at American
Society for Limnology & Oceanography Meeting, Salt Lake City, UT,
February 2005.
Wood, D.C. 1985. The mechanism of tubocurarine action on
mechanoreceptor channels in the protozoan Stentor coeruleus. J. Exp.
Biol. 117: 215-235.
Wood, D.C. 1989. Localization of mechanoreceptors in the protozoan,
Stentor coeruleus. J. Comp. Physiol. A 165: 229-235.