Download CYTOCHALASIN B: ASPECTS OF PHAGOCYTOSIS IN NUTRIENT

J. Cell Set. 15, 403-406 (1974)
403
Printed in Great Britain
CYTOCHALASIN B: ASPECTS OF
PHAGOCYTOSIS IN NUTRIENT UPTAKE IN
TETRAH YMENA
ELSE K. HOFFMANN, L. RASMUSSEN AND E. ZEUTHEN
The Biological Institute of the Carhberg Foundation, 16 Tagensvej,
2200-Copenhagen N, Denmark
SUMMARY
Cytochalasin B (37 fig per ml) reduces the rate of food vacuole formation, i.e. the rate of
phagocytosis, in Tetrahymena pyriformis. Cytochalasin B in this concentration suppresses
multiplication rates in a nutrient medium consisting of 2 % proteose peptone, but multiplication is unaffected if this medium is supplemented with glucose and high concentrations of
nucleosides. Thus nutrients in high concentrations circumvent the necessity for phagocytosis in
Tetrahymena.
INTRODUCTION
Cytochalasin. B inhibits endocytosis in several cell types, e.g. leukocytes, macrophages and liver cells (Malawista, Gee & Bensch, 1971; Wagner, Rosenberg &
Estensen, 1971). It also inhibits food vacuole formation, i.e. phagocytosis or endocytosis, in Tetrahymena pyriformis in concentrations which have only slight effects
on cell multiplication (Nilsson, Ricketts & Zeuthen, 1973).
Particulate material is required for formation of food vacuoles in Tetrahymena.
Since particles are essential for rapid cell multiplication in 2% proteose peptone
broth, it has been proposed that uptake of some essential compounds in solution
depends on food vacuole formation or phagocytosis (Rasmussen & Kludt, 1970). So
far, the alternative possibility that nutrient uptake was stimulated by mechanical
action of the particles on the general cell surface, could not be excluded, since the
growth-restrictive medium is a sterile-filtered, 'particle-free' broth. However, it has
been found recently that T. pyriformis multiplies fast in particle-free, strongly supplemented media (Rasmussen, 1974). Therefore, .yve can now investigate the possibility
of particle-stimulated action on the cell surface using cytochalasin B as a tool for
blocking phagocytosis without interfering with cell multiplication in the supplemented broth.
METHODS
T. pyriformis, strain GL, was cultivated axenically in 10-ml portions of nutrient medium in
125-ml conical flasks closed by screw caps. The nutrient medium was based on 2 % proteose
peptone broth (Difco Laboratories) sterilized by being heated for 30 min at 120 °C in a pressure cooker and supplemented as indicated. The glucose solution was sterilized by filtration
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E. K. Hoffmann, L. Rasmussen and E. Zeuthen
(Millipore membrane filters of pore size 022 fim). The cultures were kept in a room regulated
to 28 ± 0-5 °C.
The initial cell concentration upon inoculation was 1000 cells per ml. During the experiment,
cell concentrations in the cultures were determined at intervals of 1 or 2 h as follows: representative samples were removed aseptically from the cultures, fixed with glutaraldehyde and
counted with an electronic counter calibrated against direct counts in a Sedgwick-Rafter
chamber made with the aid of a microscope. Growth curves were plotted on semi-log graph
paper and from each curve a mean generation time was computed.
In order to determine the number of food vacuoles formed per cell, culture samples of 1 ml
were removed and incubated with Indian ink for 25 min. The cells were then fixed in 2 %
glutaraldehyde, and the number of food vacuoles containing Indian ink was calculated on a
per cell basis. At least 100 cells were counted for determination of each mean value.
Cytochalasin B (5 mg per ml) was dissolved in 70 % ethanol prior to use. The mixture of
nucleosides consists of adenosine, cytidine, guanosine and uridine, and is sterilized by filtration.
RESULTS
Cytochalasin B inhibits cell multiplication in exponentially growing populations
of T. pyriformis in proteose peptone broth. Table 1 shows the effect of cytochalasin B
both on the average generation time during the period from 1 to 13 h after addition
Table 1. Effects of cytochalasin B on cell generation times and rates of phagocytosis in
Tetrahymena growing in proteose peptone broth supplemented in various ways
Gen. time
±S.E.M., h
(a) PP
PP + cytochalasin B
PP + nucleosides
PP + nucleosides +
cytochalasin B
(e) PP +glucose +
nucleosides
if) PP+glucose+
nucleosides +
cytochalasin B
(g) PP + glucose
(A) PP+glucose +
cytochalasin B
(b)
(c)
(d)
7-0 ±0-3 (n = 7)
16±2 (n = 7)
6-o±o-8 (n = 4)
8 ± 1 (n = 4)
Food vacuoles made
per cell in 25 min
7-8
I-I
7-2
09
6-2 ±0-2 (n = 7)
92
7-0 ±0-3 (n = 6)
1-2
7-5 ±0-3 (n = 4)
io-6±o-4 (n = 3)
8-9
1-7
PP: 2 % proteose peptone broth, sterilized by heating. Cytochalasin B: final concentration,
37fg P e r rrJ- Nucleosides: adenosine, cytidine, guanosine and uridine, each at a final concentration of 15 rriM. Glucose: final concentration, 25 %. n, no. of experiments.
of the inhibitor, and on the rate of food vacuole formation. The generation time in the
control group as shown in Table 1 (line a) is 7-0 h, and after addition of cytochalasin B
it is increased to 16 h (b). Furthermore, the number of food vacuoles made in a
standard interval of 25 min is reduced from 7-8 t o r i per cell. If the nutrient medium
is supplemented with nucleosides, a generation time of 6-o h is obtained in the absence
of cytochalasin B (c) whereas it is 8 h in its presence (d). Thus the inhibitory action of
Phagocytosis in Tetrahymena
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cytochalasin B on cell multiplication is substantially reduced in presence of high
concentrations of nucleosides. However, if glucose is also added (e), growth inhibition
is abolished (/). Although cytochalasin B under these conditions reduces food vacuole
formation from 92 to 1-2 per cell it does not affect the generation times significantly
(P > 0-995). Moreover, 2-5% glucose alone reduces the growth inhibition resulting
from the presence of cytochalasin B ((&) and (h)).
Inhibition of food vacuole formation is maximal within iomin after addition of
inhibitor.
DISCUSSION
Nilsson et al. (1973) found that cytochalasin B inhibited food vacuole formation in
T. pyriformis and that it had only a slight effect on cell division in concentrations of
less than 37 /tg per ml. They observed that the cells in the nutrient medium were of
less than normal size after exposures to the drug lasting for 24 h. This led them to
suggest that cytochalasin B in this concentration interferes with food uptake in
Tetrahymena.
We too, have observed that addition of cytochalasin B leads to significantly reduced
rates of food vacuole formation, and, in an unsupplemented peptone broth, also of cell
multiplication. Similar inhibitions are observed when T. pyriformis is suspended in
particle-free, sterile-filtered proteose peptone broth (Rasmussen & Kludt, 1970). The
similarity extends even further, since glucose and high concentrations of nucleosides
(not synthesized de novo by Tetrahymena (Kidder & Dewey, 1951)) can overcome the
need for food vacuole formation both in the presence of cytochalasin B (present paper)
and in the particle-free proteose peptone broth (Rasmussen, 1974). Summing up,
phagocytosis can be significantly reduced in 2 ways, either by addition of cytochalasin B, or by removal of an essential prerequisite, such' as particulate material,
in the broth. In both cases the resulting starvation can be overcome by addition of
nucleosides and glucose. Since the phagocytotic rate in the presence of cytochalasin B
is not increased by addition of nucleosides or glucose these results suggest that the
cells are capable of obtaining nutrients via an alternative pathway.
We have noted that food vacuole formation is reduced both in cells suspended in
sterile-filtered, particle-free media and in cells inhibited by cytochalasin B. It would
be tempting to conclude that the food vacuoles are equipped with routes of entry
necessary for rapid uptake of nucleosides and glucose from the proteose peptone
broth. However, it is possible that particle-induced uptake of dissolved nutrients
makes use of other pathways for entry into the cell than food vacuoles, and that these
are blocked by cytochalasin as well. This drug inhibits certain types of pinocytosis
(Allison, Davies & de Petris, 1971) and active transport of thymidine in cells (Everhart, 1973). In view of this latter result it is worth noting that the need for high
concentrations of nucleosides in the presence of cytochalasin B does not reflect a
requirement for thymidine in our case, since addition of thymidine (1-5 nw) plus
glucose did not release the inhibition better than glucose alone.
It is our hope that applications of two approaches as different as removal of particles
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E. K. Hoffmann, L. Rasmussen and E. Zeuthen
from the media and addition of cytochalasin B will enable us to throw light on structures and functions of mechanisms responsible for uptake of solutes in Tetrahymena.
We thank Mrs B. Dohn for technical assistance and Mrs E. Palludan for secretarial help in
preparation of the typescript. We also thank Dr S. B. Carter, ICI, Pharmaceuticals Division,
Alderley Park, Macclesfield, Cheshire, UK, for the donation of the samples of cytochalasin B.
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(Received 29 November 1973)