Protist Behavioral Responses to Micromolar Levels of ...

Protist Behavioral Responses to Micromolar Levels of ...

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]

Binding of both proline and serine occurs over a similar
concentration range as feeding inhibition (Fig. 1), with halfmaximal concentration of 5-10 M. 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). Autoradio-grams
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).

b. Binding kinetics and specificity are similar to
feeding inhibition

100%

8,000

60%

60

80

100

M total proline
1,200

serine
proline
Series3

1,000
800
600

4,000
2,000

0

corrected for filter blank
20

40

60

80

2

4

6

8 10 12 14 16 18 20

-2,000

0
0

100%

0

400
200

120%

100

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).

60%
40%
20%
0%

min incubation

M total serine or proline

80%

Fig. 2: [3H]-proline (20 M) shows
rapid binding within 2 min. of
addition.

PRO

40

SER

20

GLN

0

ARG

0%

6,000
(DPM)

20%

corrected for filter blank

DI

40%

% of 3H-serine binding

80%

[3H]-prolinebinding

% feeding inhibition

a. Amino acids bind to cells
over 1-100 uM, similar to
concentrations that inhibit
grazing

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
Favella sp.
not reduce survivorship, and
Top: cells
viewed in
inhibition was strongest with
brightfield
small side chain AAs, rather
and
than a function of side chain
darkfield.
Bottom:
hydrophobicity or ionization.
SEM image,
Inhibition occurred in a doseshowing
responsive manner from 0.2
hyaline
lorica. Cell
20 M, within seconds to
lengths
minutes of dosing, and could
approx. 180
be immediately relieved by
m.
removing
AAs.
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 Khidai 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).

Discussion

Results

Introduction

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.

Summary

1. Is binding similar to feeding inhibition
(same AAs, kinetics, concentration
range)?

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?

Cultures - Ciliates were cultured on Heterocapsa triquetra,
Isochrysis galbana, and Mantoniella squamata at 16 oC in
Instant Ocean. Prior to experiments, cells starved for 1224 hrs, then concentrated by reverse-filtration through
20-40 m mesh to densities of ca. 20-80 cells mL-1. Cells
were enumerated by settling and counting after fixation
with acid Lugols.
Radiolabel binding - Cells were incubated 0-20 min in 1-30
Ci L-[3H] proline or serine (20-80 Ci mmol-1, Moravek
Biochemicals, Sigma), then filtered onto 5 m
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 M
label (30 Ci total). Treatments included live cells, cells
fixed with formalin or Lugols, 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 g 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 als. (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
(M saturation) and specificity. AAs with
small side chains compete for binding sites
and inhibit feeding to the greatest degree.

Questions

Methods

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.

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

Fig. 5: Autoradiograms of cells
preserved with acid Lugols. Lugols
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.

Khidai, L., P. Sos, 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.
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: 345351.
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.

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