/. Embryol. exp. Morph. Vol. 57, pp. 189-201, 1980
Printed in Great Britain © Company of Biologists Limited 1980
The effect of chymotrypsin on the development
of Dictyostelium discoideum
By DAVID C. KILPATRICK, 1 JERZY A. SCHMIDT, 2
JOHN L. STIRLING 2
JOHN PACY AND GARETH E. JONES 2
From the Departments of Biochemistry and Biology,
Queen Elizabeth College, University of London
SUMMARY
Development of the cellular slime mould Dictyostelium discoideum strain NQ, in the
presence of a-chymotrypsin (3 mg/ml) is reversibly arrested at the tight aggregate stage
(10/12 h). Pronase has a similar effect, but trypsin only retards normal development by
about five hours. Normally developing cells are susceptible to a-chymotrypsin if they are
transferred into its presence at any time up to the tight aggregate stage (10-12 h). Transfer
after this stage does not affect the appearance of fruiting body structures in the normal time
(24 h).
Electron microscopy showed the ultrastructure of a-chymotrypsin-blocked aggregates
after starvation for 24 h to be consistent with a block at 10-12 h of normal development.
Poorly developed prespore vacuoles, having thin incomplete walls and a paucity of electrondense material, are present in some cells. No angular vacuolated cells characteristic of stalk
cells are visible. Fruiting bodies formed in the presence of a a-chymotrypsin, either as minority
structures when the enzyme is added before 10-12 h of normal development, or as the
majority structures on later enzyme addition, were found to be abnormal. Normal stalks
were formed but the spores were immature. Prespore vacuoles were present, though disrupted, and the cells were not encapsulated by spore walls.
The electronegativity of intact slime mould amoebae was significantly reduced, and material
containing L-[6-3H]-fucose and [l-14C]leucine was removed from the cell surface on achymotrypsin treatment. Few plasma membrane proteins were affected, however, and staining
of polyacrylamide gels for glycopeptides using Con A-peroxide binding also showed little
change.
INTRODUCTION
Cellular interactions play a central role in the development of the cellular
slime mould Dictyostelium discoideum (Loomis, 1975). Several proteins have
been implicated in the formation of specific cell-cell adhesions, including contact sites A (Beug, Katz & Gerisch, 1973), discoidin (Rosen, Kafka, Simpson &
Barondes, 1973), and a glycoprotein of apparent molecular weight 150,000
(Gelosky, Weseman, Bakke & Lerner, 1979). The role of such proteins in
1
Author's present address: Endocrine Unit, Immunology Laboratories, The Royal
Infirmary, Edinburgh EH3 9YW U.K.
2
Author's address: Queen Elizabeth College, University of London, Campden Hill,
London W8 7AH, U.K.
13
EMB
57
190
D. C. KILPATRICK AND OTHERS
development is unclear, but mutants lacking contact sites A or functional discoidin fail to complete development (Beug et ah 1973; Ray, Shinnick & Lerner,
1979).
Cell surface proteins involved in cell-cell interactions may bs accessible to
attack by exogenously applied proteases and analysis of peptides released by
proteolytic action may give information about the nature of these surface
structures. There have been several reports of the susceptibility of D. discoidewn
membrane proteins to protease attack (Parish, Schmidlin & Miiller, 1977).
Trypsin has been shown to prevent reaggregation of dissociated slugs (Alexander,
Brackenburg & Sussman, 1975), recovery after removal of the enzyme being
cycloheximide sensitive. There is no effect on adhesion at an earlier stage and
contact sites A appear to be insensitive to this enzyme (Huesgen & Gerisch,
1975). Cellular contacts required for maturation of spore cells are pronase
sensitive (Kay, Town & Gross, 1979) and treatment of intact cells with this
enzyme alters the electrophoretic patterns of proteins and glycoproteins from
plasma membranes (Parish et al. 1977).
We have found that exogenously applied a-chymotrypsin and pronase, but
not trypsin, effectively inhibit D. discoideum development. Since a-chymotrypsin
is more specific in its enzyme action on peptides than pronase, we have concentrated on the effects of this enzyme on the behaviour of developing cells,
their ultrastructure, electrophoretic mobility and the nature of the products
removed. For comparison we also consider corresponding effects of trypsin and
pronase. Preliminary results have been described previously (Schmidt, Stirling,
Jones & Pacy, 1978).
MATERIALS AND METHODS
Cell culture
Amoebae of D. discoideum (strain NC4) were grown in association with
E. coli (B/r) either on nutrient agar plates as described by Sussman (1966), or
in liquid culture as described by Gerisch (1960). Synchronous development was
induced by separating vegetative amoebae from bacteria by differential centrifugation (300g for 3 min) and spreading 0-2 ml of a suspension of cells (2-5 x
108/mf) on the surface of a Millipore filter (AAWP 04700) supported by an
absorbent pad saturated with a solution of buffered salts (Lower pad solution,
L.P.S., Sussman 1966). Amoebae were allowed to grow and develop in a humid
atmosphere maintained at 22 °C.
Effect of enzymes on development
The effect of a-chymotrypsin (pronase or trypsin) on development was assessed by allowing vegetative amoebae to develop on Millipore filters supported
by pads containing enzyme dissolved at 3 mg/ml in L.P.S. adjusted to pH 7-5
(modified L.P.S.). Cell populations treated in this way were observed for 24 h
and periodically thereafter to assess their developmental progress compared
Inhibition of slime mould development
191
with controls incubated at the same pH. Susceptibility of D. discoidewn at
different stages of development to a-chymotrypsin was investigated in two ways.
Cells on Millipore filters were transferred directly into fresh support pads containing enzyme (3 mg/ml), or disaggregated in modified L.P.S. pelletted by
centrifugation (700g for 2 min) and made to recapitulate developmental events
on fresh Millipore filters in the presence of the enzyme.
Activity of a-chymotrypsin in the support pads was measured using the
synthetic substrate, JV-benzoyl-tyrosine-ethyl-ester, according to the method of
Hummel (1959).
a-Chymotrypsin (type II) and bovine trypsin (type III) were purchased from
the Sigma Chemical Co., Poole, Dorset, U.K.; Pronase was purchased from
Koch-Light Labs Ltd, Colnbrook, U.K.
Electron microscopy
Appropriate samples of cells and the corresponding controls were fixed in situ
on Millipore filters with 2-5 % glutaraldehyde in 20 mM phosphate buffer,
pH 7-5, for 2 h. The cells (whether in the form of aggregates, a grex or a fruiting
body) were then transferred to test tubes, washed in buffer for 1 h and postfixed in 1 % osmium tetroxide in phosphate buffer for 1 h. Specimens were dehydrated, embedded in Spurrs resin and sections cut on a Reichert 0MU3
llltramicrotome fitted with a diamond knife. Sections were stained with Reynolds
lead citrate and viewed in an AEI EM 6B.
Cell electrophoresis
Interphase cells (after 5 h of normal development on Millipore filters) at
10r cells/ml in 20 mM sodium phosphate buffer, pH 7-5, were treated with
a-chymotrypsin, pronase or trypsin at a final concentration of 3 mg/ml. After
incubation with gentle shaking for 15 min at 25 °C, the cells were pelleted, resuspended in the above buffer and their electrophoretic mobilities determined
in a Mark I cell electrophoresis apparatus (Rank Bros., Cambridge) as described
by Bangham, Flemens, Heard & Seaman (1958).
Radioactive labelling of cells
Cells (40 ml at 107 cells/ml) were labelled with 10/*Ci of [l-14C]leucine
(62 mCi/nmole, Radiochemical Centre, Amersham, U.K.) and 50 juCi of
L-[6-3H]fucose (12-066 Ci/nmole, New England Nuclear) by shaking them in
20 mM sodium phosphate, pH 6-0 for 6 h in the presence of the isotopes.
Labelling of cells with [35S]methionine was carried out by adding the isotope to
L.P.S. (50 fid of 239-6 Ci/mmole, New England Nuclear) for 1 h.
Analysis of the products of protease digestion of intact cells
Release of fragments containing [14C]leucine and L-[3H]fucose by the action
of proteases was monitored by suspending labelled cells (107/ml) in 20 mM
13-2
192
D. C. KILPATRICK AND OTHERS
sodium phosphate pH 7-5 containing 0-5 mg/ml enzyme. Samples (1 ml) of the
suspension were taken at 10 min intervals, the cells removed by centrifugation
and the supernatant counted with 7 ml of scintillant (1 % Butyl PBD in Triton
x 100: toluene, 1:2) in a Packard Tricarb liquid scintillation spectrometer.
Products removed from labelled amoebae by a-chymotrypsin were analysed
by gel filtration on a column of Sephadex G 50 superfine (30 cm x 2-6 cm)
equilibrated in 10 mM-sodium phosphate pH 60. Samples (0-5 ml) of the elute
fractions (3 ml) were taken for dual isotope counting.
Polyacrylamide gel electrophoresis
Plasma membranes from D. discoideum were prepared using the two phase
polymer system described by Brunette & Till (1971). Purified membranes were
analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate
(s.D.s.) using the method of Laemmli (1970); each sample applied to the gel
corresponding to membranes from 107 cells. Protein was visualized by fixing
and staining gels in 0-125 % Coomassie blue in 10% acetic acid and destaining
in the dye-free solvent until the background was clear. Staining for glycoprotein
was carried out as described by Parish et al. (1977).
RESULTS
The normal time course of development of the cellular slime mould, Dictyostelium discoideum, was altered in the presence of a-chymotrypsin, trypsin and
pronase. Cells allowed to develop in a-chymotrypsin or pronase did not complete
the developmental programme and were blocked at the formation of aggregates.
Few fruiting bodies, about 1 % of normal, were formed from these cells even
after several days. Trypsin had the least effect and slightly retarded the formation of fruiting bodies; mature spores developing 5 h after the controls. At
values below pH 7-5, all three proteases were less effective, as may be expected
from the pH dependence of their activities. At higher pH values abnormal
development was observed even in the control cells.
When viewed at low magnification, 24 h after initiation of starvation, aggregates formed in the presence of a-chymotrypsin had an appearance similar to
that of aggregates formed by control cells that had been allowed to develop for
10-12 h. Normally developing cells were susceptible to a-chymotrypsin in this
way if transferred onto support pads containing the enzyme at any time up to
the late aggregate stage (10-12 h). The resulting structures included both
aggregates and standing grexes. When transferred to the enzyme after this time,
development did not seem to be arrested and fruiting bodies were formed which
appeared normal when viewed under the light microscope. A similar observation
was made with cells made to recapitulate development in the presence of achymotrypsin. Such cells were only blocked if normal development was disrupted before the formation of aggregates. Exposure to a high concentration of
Inhibition of slime mould development
193
cyclic AMP (0-2 M in modified L.P.S., pH 7-5) for an 0-5 h period, 4 h into
normal development on Millipore filters had no effect on a-chymotrypsin
blockage. In contrast to their behaviour on solid supports, cells which had been
starved by shaking in phosphate buffer for up to 24 h, conditions which induce
the expression of contact sites A (Beug et al. 1973), were still sensitive to developmental blockage by a-chymotrypsin when subsequently deposited on Millipore
filters. Pulsing starving cells in shake flask culture with cyclic AMP (to a final
concentration of 5 JLIM at intervals of 6-5 min) for the first 4 h of starvation did
not overcome the subsequent effects of a-chymotrypsin.
Some a-chymotrypsin activity was lost during incubation with cells on Millipore filters; the activity of the enzyme in the support pad decreasing by 50 %
of its original activity in 8 h. This loss of activity was not observed when the
enzyme was incubated in the same buffer, but in the absence of cells. An enzyme
with activity towards the synthetic substrate, iV-benzoyl-tryosine-ethyl-ester,
appeared to be secreted by normally developing cells.
Recovery from treatment with a-chymotrypsin
The effect of a-chymotrypsin on the development of D. discoideum was found
to be reversible as treated cells washed free of enzyme developed normally
when replated on fresh Millipore filters. Direct transfer of cells on Millipore
filters from pads containing enzyme to fresh pads containing only L.P.S.
also resulted in normal development. Cells treated for 12 h before transfer
culminated at the same time as control cells (requiring a further 10-12 h)
whereas cells treated with enzyme for 24 h formed fruiting bodies within 6 h
of transfer. In the latter case, no migrating pseudoplasmodia were formed and
cell masses culminated directly from aggregates.
Ultrastructure of a-chymotrypsin-blocked cells
Electron microscopy of developmental^ inhibited cells was used to determine
whether prespore vacuoles were formed. These vacuoles are readily identified
and are characteristic of early differentiation along the spore-determining pathway. Prespore vacuoles (Fig. 1) first appear during the formation of aggregates
and increase in numbers rapidly during normal development. Cells within
aggregates formed in the presence of a-chymotrypsin displayed poorly formed
prespore vacuoles, these being characterized by thin, incomplete walls and a
paucity of electron-dense vacuole contents (Fig. 2). Some cells seemed to lack
any prespore vacuoles and later in development (in the standing grex stage)
a-chymotrypsin-blocked cells still displayed poorly formed prespore vacuoles
(Fig. 3). No angular vacuolated cells characteristic of maturing stalk cells were
found in any of the blocked cell masses. Calcafluor staining for cellulose (the
major polysaccharide end product of stalk cells) was negative.
Blocked cells in all developmental stages display other signs of a-chymotrypsin treatment. Generally the cells appear less closely associated with each
194
D. C. KILPATRICK AND OTHERS
other within both aggregates and grexes, and large intercellular gaps are commonly observed. Treated material appears generally in good condition with no
signs of gross damage (such as blebs); areas of clear cytoplasm can be observed
in some cells (Fig. 2).
A small proportion (1-2%) of the aggregates formed in the presence of
a-chymotrypsin develop into fruiting bodies that are apparently normal under
the optical microscope. Electron microsopy reveals that while the stalks of
these structures appear normal the spore cells have remained immature (Figs
4, 5). Prespore vacuoles were still present and the cells had not been encapsulated
in spore coats. No mature spores were ever seen in these fruiting bodies. Similar
abnormal fruiting bodies were also observed under the electron microscope
when normally developing cells were allowed to form tight aggregates before
transfer to a-chymotrypsin.
Electrophoresis of protease-treated cells
It is possible that the inhibition of development by the addition of exogenous proteases may be due to an increase in net cell-surface-charge density
resulting in inhibition of cell contact. To investigate this possibility, cell electrophoresis of protease-treated interphase amoebae was performed and the results
shown in Table 1. A slight but significant reduction suggesting a loss of cell
surface electronegativity occurred on a-chymotrypsin treatment. In comparison,
trypsin brought about a reduction (not significant at the 5 % level) and pronase
a significant increase in cell surface electronegativity.
Analysis of material removed from intact cells by cc-chymotrypsin
When intact cells labelled with [14C]leucine and [6-3H]fucose were treated
with a-chymotrypsin (0-5 mg/ml), material containing both these labels was
solubilized. In contrast, when a similar experiment was performed with [35S]methionine-labelled cells, there was no removal of labelled material. Pronase
was effective in removing [14C]leucine and L-[6-3H]fucose containing material from cells but trypsin removed only [14C]leucine-labelled material and
there was no solubilization of L-[6-3H]fucose. In all cases maximal release of
label was achieved within 10 min of the addition of the enzyme [Fig. 6].
FIGURES 1-3
Fig. 1. Untreated cell from an early grex showing numerous prespore vacuoles. The
vacuole wall is well structured and packed with electron-dense material. In this case
the cells are closely associated with each other, x 35000.
Fig. 2. Cell from late aggregate (10-12 h) in presence of a-chymotrypsin. Malformed
prespore vacuoles are evident, x 15000.
Fig. 3. Early grex in presence of a-chymotrypsin. Large intercellular spacing and
poorly developed prespore vacuoles can be seen, x 15000.
Inhibition of slime mould development
195
196
D. C. KILPATRICK AND OTHERS
Inhibition of slime mould development
197
Table 1. Effect of proteolytic enzymes on electrophoretic
mobility of amoebae
Enzyme treatment
Mobility (mm2 V"1 s-1)
Significance
None
0-78 ±005
P < 0001
Chymotrypsin
0-66 ± 0 1
0- 1 < P < 0-25
Trypsin
0-75 ±007
P < 0001
Pronase
0-96 + 007
Interphase amoebae which had undergone 5 h development were used for cell electrophoresis as described in the Methods. Results are expressed as the mean of 20 determinations
±the standard deviation. The significance of the difference between the protease-treated
cells and the control are expressed as probabilities from Student's / tests.
Of the label incorporated into whole cells, 9-4% of L-[6-3H]fucose and 2-7%
of [14C]leucine were specifically removed by a-chymotrypsin. Analysis of these
fragments by chromatography on Sephadex G50 (Fig. 7) showed most of the
material removed to be of low molecular weight, between 1500 (the lower
fractionation limit of the gel) and 10000. Some radioactivity was also associated
with a molecular weight around 23000 and co-eluted with a-chymotrypsin.
Much of the radioactive material in the void volume was not specifically removed and was also observed in supernatants from labelled control cells.
Polyacrylamide gel electrophoresis of cell membranes
Membranes derived from aggregation competent cells treated with 0-5 mg/ml
a-chymotrypsin showed only a few minor changes in the protein profile as
revealed by Coomassie-blue staining of polyacrylamide gels. Con A-peroxidase
staining for glycoprotein showed possibly one component to be affected. Pronase and trypsin both have a much greater effect on membrane proteins than
chymotrypsin. To a lesser extent, pronase and trypsin were also more effective
at removing membrane glycoproteins than chymotrypsin.
DISCUSSION
We have shown that a-chymotrypsin can cause a developmental block when
slime mould cells are allowed to develop in its presence. This blockage seems to
be at a specific point in development and occurs at the stage when tight aggregates have formed. Developmental events leading up to the formation of tight
FIGURES 4 AND 5
Fig. 4. Untreated fruiting body showing mature spores with well-developed spore
coat and no prespore vacuoles. x 20000.
Fig. 5. Treated fruiting body in which the spores have remained immature; the
prespore vacuoles not having released their spore coat material, x 12000.
198
D. C. KILPATRICK AND OTHERS
120
-
(fl)
/
^
80
-
5
40
/
i
i
10
20
Time (min)
Fig. 6. Kinetics of the release of material containing L-[6-3H]fucose (O) and
[l-14C]leucine ( • ) by (a) a-chymotrypsin, (b) pronase and (c) trypsin from the
surface of intact cells. Non enzymic release of radioactive label was determined in
a control sample and deducted from the other values. For (b) and (c) the same cells
were used; (a) was carried out on a separate occasion when the cells were labelled
with a three-fold greater concentration of isotopes, and double the volume of
cell suspension was used for each value.
aggregates appeared to be normal and, as we have already reported (Schmidt
et al. 1978), EDTA-insensitive cohesion was unimpaired.
Relatively high concentrations of a-chymotrypsin (greater than 1-5 mg/ml)
are required to effect this developmental block which nevertheless seems to be
specific since neither bovine serum albumin, chymotrypsinogen A or trypsin at
the same concentration had appreciable influence on the course of development.
Activity of a-chymotrypsin was lost on incubation of the enzyme with developing slime mould cells but we do not know whether this is due to uptake
199
Inhibition of slime mould development
Excluded volume
Included volume
50 r-
- i 2-5
40
o
X
30
1-5 d
-6
c
=
-0
io g
u
2.
10
r
0-5
20
Fraction number
|
^
40
Fig. 7. Gel filtration on Sephadex G50 superfine of the products removed by
a-chymotrypsin from the surface of labelled cells; ( # ) L-[6-3H]fucose and ( • )
[14C]leucine-labelled material. The elution profile of non-enzymically removed
material (not shown) accounted for most of the material eluted with the void volume.
of the enzyme by the cells, extracellular degradation of the enzyme, the production of an a-chymotrypsin inhibitor, or a combination of these. A high
starting concentration of the enzyme might be necessary to ensure an effective
concentration at some later and critical stage in development. It is notable that
D. discoidewn cells secrete an enzyme with activity similar to that of chymotrypsin but we have not assessed whether this has any developmental function.
From the ultrastructure of cells allowed to develop in the presence of <xchymotrypsin we conclude that the enzyme can block development at two stages.
The first and most profound effect being before or at the start of stalk-spore
differentiation, the second affecting the maturation of spore cells.
It is evident that a-chymotrypsin (and the other proteases) are able to act on
the surface structures of the intact cells. The electronegativity of slime mould
cells is significantly reduced on a-chymotrypsin treatment thus favouring nonspecific electrostatic attraction. In contrast, a large increase in electronegativity
on pronase treatment may alter interaction between components and account
for its effect on development. Yabuno (1970) has made a similar observation.
a-Chymotrypsin had no dramatic effect on the pattern of plasma membrane
polypeptides and glycosylated polypeptides separated on SDS polyacrylamide
gels. Trypsin and pronase on the other hand had generalized effects on polypeptides, but glycosylated polypeptides seemed more resistant to their attack
and we speculate that one function of the carbohydrate groups of these membrane glycoproteins may be protection against proteolytic degradation. Both
200
D. C. KILPATRICK AND OTHERS
a-chymotrypsin and pronase released low molecular weight material labelled
with both [14C]leucine and L-[3H]fucose, but trypsin removed only [14C]leucinelabelled peptides. This specificity is interesting in view of the fact that trypsin
was almost ineffective in altering the course of development and may point
to the involvement of a fucosylated glycoprotein in the step affected by pronase
and a-chymotrypsin.
As yet we have been unable to identify the precise nature of the earlier funtions
blocked by a-chymotrypsin though we have discounted any significant interference with the adhesive properties of these cells (Schmidt et al. 1978). However, it is possible that other contacts between cells are necessary for postaggregative development and that these may be sensitive to a-chymotrypsin.
Once made these contacts would appear to have a lasting effect since normal
cells having formed tight aggregates are then resistant to the early blocking
effect of a-chymotrypsin. It would follow that contacts are not made effectively
in cells shaken in suspension since under these conditions cells do not develop
resistance to a-chymotrypsin even after 24 h. Another possibility is that postaggregative cAMP signalling (Town & Gross, 1978) may be blocked.
The effect of a-chymotrypsin in blocking development at the maturation of
spore cells may well be explained by its effect on the Y interaction described by
Kay et al. (1979). From their work it would appear that the Y interaction is
mediated by a pronase-sensitive membrane component.
We are grateful to the SRC for a project grant and for studentships to David C. Kilpatrick
and Jerzy A Schmidt and to the Central Research Fund of the University of London for
providing equipment.
REFERENCES
ALEXANDER, S., BRACKENBURY, R. & SUSSMAN, M. (1975). Tryptic destruction of Aggregative competence in Dictyostelium discoideum and subsequent recovery. Nature, Lond.
254, 698-699.
BANGHAM, A. D., FLEMENS, R., HEARD, D. H. & SEAMAN, G. V. F. (1958). An apparatus for
microelectrophoresis of small particles. Nature, Lond. 182, 642-644.
BEUG, H., KATZ, F. E. & GERISCH, G. (1973). Dynamics of antigenic membrane sites relating
to cell aggregation in Dictyostelium. J. Cell Biol. 56, 647-658.
BRUNETTE, D. M. & TILL, J. E. (1971). A rapid method for the isolation of L-cell surface
membranes using an aqueous two phase polymer system. /. Membrane Biol. 5, 215-224.
GELOSKY, J. E., WESTMAN, J., BAKKE, A. & LERNER, R. A. (1979). Identification of a cell
surface glycoprotein involved in cell aggregation in Dictyostelium discoideum. Cell 18,
391-398.
GERISCH, G. (1960). Zellfunktionen und Zellfunktionswechsel in der entwicklung von
Dicytostelium discoideum. I. Zellagglutination und induction der fruchtkorperpularitat.
Wilhelm Roux Arch. Entw Mech. Org. 152, 632-654.
HUESGEN, A. & GERISCH, G. (1975). Solubilized contact sites A from cell membranes of
Dictyostelium discoideum. FEBS Lett., Amsterdam, 56, 46-49.
HUMMEL, B. C. W. (1959). A modified spectrophotometric determination of chymotrypsin,
trypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399.
KAY, R. R., TOWN, C. D. & GROSS, J. D. (1979). Cell differentiation in Dictyostelium discoideum. Differentiation, 12, 7-14.
Inhibition of slime mould development
201
U. K. (1970). Cleavage of structural proteins during the assembly of the head of
Bacteriophage T4. Nature, Lond. 227, 680-685.
LOOMIS, W. F. (1975). Dictyostelium discoideum; A Developmental System; New York:
Academic Press.
LAEMMLI,
PARISH, R. W., SCHMIDLIN, S. & MULLER, U. (1977). The effects of proteases on proteins and
glycoproteins of Dictyostelium discoideum plasma membranes. Expl Cell Res. 110, 267-276.
J., SHINNICK, T. & LERNER, R. (1979). A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum.
Nature, Lond. 279, 215-221.
ROSEN, S. D., KAFKA, J. A., SIMPSON, D. L. & BARONDES, S. H. (1973). Developmental^
regulated carbohydrate binding protein in Dictyostelium discoideum. Proc. natn. Acad.
Sci. U.S.A. 70, 2554-2557.
SCHMIDT, J. A., STIRLING, J. L., JONES, G. E. & PACY, J. (1978). A chymotrypsin sensitive
step in the development of Dictyostelium discoideum. Nature, Lond. 214, 400-401.
SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mould
development. In Methods in Cell Physiology, vol. 2. (ed. D. Prescott), pp. 397-410. New
York: Academic Press.
TOWN, C. & GROSS, J. (1978). The role of cyclic nucleotides and cell agglomeration in postaggregative enzyme synthesis in Dictyostelium discoideum. Devi Biol. 63, 412-420.
YABUNO, K. (1970). Changes in electronegativity of the cell surface during the development
of the cellular slime mould, Dictyostelium discoideum. Devi, Growth & Differ. 12, 229-239.
RAY,
(Received 4 December 1979, revised 21 January 1980)