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CHANGES OF SURFACE.CARBOHYDRATES AND MEMBRANE POTENTIAL
DURING THE ENCYSTMENT PROCESS OF

BLASTOCLADIELLA EMERSONII ZOOSPORES

 

By

CHAUYING JACK JEN

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Biophysics

1980

6//é 4/57

ABSTRACT

CHANGES OF SURFACE CARBOHYDRATES AND MEMBRANE POTENTIAL
DURING THE ENCYSTMENT PROCESS OF
BLASTOCLADIELLA EMERSONII ZOOSPORES
by

Chauying Jack Jen

The unicellular, non-filamentous, lower aquatic fungus,
BZastocZadieZZa emersonii was selected as an excellent model
system for developmental studies. Changes of surface carbo-
hydrates accompanied with the encystment process were studied
by using concanavalin A as a probe. The possible role of
membrane potential in regulating the develOpmental process
was investigated.

Binding of concanavalin A to the sell surface of develo-
ping BZastocZadieZZa emersonii zoospores was explored by
fluorescence microscopy, electron microscopy and radioactive
labelling. After labelling with fluorescein isothiocyanate-
conjugated concanavalin A, the fluorescence intensity of
individual non-induced zoospores varies greatly. On the
other hand, similarly labelled zoospores, induced with K+

ions for synchronous development, revealed a more even dis-

tribution of fluorescence intensity. The zoospore surface
contained 4.3 x 107 concanavalin A binding sites per cell
which were randomly distributed and closely attached to the
surface. The affinity constants range from 7.5 x 107
M‘1 to 3.5 x 105 M", while the Scatchard plot was
typical of heterogeneous binding. Further developed cells,
round cells and germlings contained 3.0 x 107 to 2.1 x
107 concanavalin A binding sites per cell which were mostly
loosely associated patches protruding into the extracellular
region. The affinity constants decreased appreciably com-
pared with those measured at the zoospore stage. Specific
concanavalin A binding characteristics appeared to correlate
with the respective develOpmental stage of the zoospore.

Concanavalin A induced lysis of zoospores of the aquatic
fungus BZastocZadieZZa emersonii. The rapid lysis could be
completely blocked by a-methyl-mannoside. At lower tempera-
tures the cells were more tolerant towards lysis compared
with cells at room temperature. Dimeric concanavalin A
derivatives and .Lens culinaris hemagglutinin A. were less
effective, whilst other lectins tested did not induce lysis
at all. The cell lysis might be caused by the cluster forma-
tion of adjacent concanavalin A receptors.

Experiments have been performed to test the possible

role of the membrane potential in the ionic induction of

encystment in BZastocZadieZZa emersonii zoospores. A
fluorescent, voltage-sensitive dye, 3,3'-dipropylthiadicarbo-
cyanine iodide, was used to monitor changes in the membrane
potential. Only K+ ions, the standard develOping trigger--
ing agent, rapidly depolarized the membrane potential. Appli
cation of an Optimal K+ ion concentration (50 mM) to a cell
population depolarized the membrane potential most rapidly
and induced the highest percentage of cells to encyst per
unit time. The depolarization was temperature dependent and
was not affected by extracellular pH changes from 5 to 9.
Quinine practically prevented depolarization of the membrane
potential by K+ ions; however, the membrane potential was
depolarized by subsequent valinomycin application. The K"
induced response of the membrane potential was apparently
independent upon the zoospore's age, but varied dramatically
as the zoospore encysted. ,A strong correlation was estab-
lished between the K+ ion-induced depolarization of the
membrane potential and the developmental triggering by K+
ions. Specific K+ carriers were proposed to be embedded in
the lipid matrix, which exists in the gel + liquid-crystal-

line mixed lipid state.

ii.

DEDICATION

This dissertation is dedicated to my wife, daughter, and

parents. Their encouragement has made this work possible.

iii

ACKNOWLEDGEMENTS

I would like to express deepest appreciation to my major
advisor, Dr. Alfred Haug, for his guidance, encouragement,
stimulating, discussions, and long-lasting, patience through
this dissertation.

Special thanks to Dr. John L. Wang for helping me during
the time of great need. Thanks also to Dr. Estelle J.
McGroarty and Dr. Derek T. A. Lamport for helpful discussions
and endorsements. My friends in this laboratory, especially
Richard Allison, provided assistance throughout the duration
of this work and deserves recognition.

My sincere gratitude to my wife, Ya-Chu, for her love,
faith and support during my graduate years.

The financial support from United States Department of

Energy (contract number DE-AC02-76ER01338) is acknowledged.

LIST OF

iv

TABLE OF CONTENTS

FIGURES

LIST OF TABLES

CHAPTER
I.
II.

III.

GENERAL INTRODUCTION 0 O O O O O O O O O O O O O O 0 1

CHANGES IN SURFACE PROPERTIES OF DEVELOPING ZOOSPORES
BLASTOCLADIELLA EMERSONII: BINDING OF CONCANAVALIN A

 

Summary

i. Introduction. . . . . . . . . . . . . . . . .14

ii. Methods . . . . . . . . . . . . . . . . . . .15
Reagents . . . . . . . . . . . . . . . . . .15
Organism and culture conditions . . . . . . . .15
Binding of [3H]Con A to cells . . . . . . .15

Labelling of cells with FITC- Con A and
quantitative fluorescence measurements. . . 15
Ultrastructural localization Con A receptor
sites . . . . . . . . . . . . . . . . . . . 15
iii. Results . . . . . 15
Number and affinity of [3H1Con A binding
sites at the surface of BZastocZadieZZa
emersonii . . . . . . . . 15
Cells labelled with FITC- Con A . . . . . . . . 15
Ultrastructural localization of Con A receptor
, sites . . . . . . . . . . . . . . . . . . . 17
iv. Discussion . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . 20

CONCANAVALIN A-INDUCED LYSIS OF ZOOSPORES OF
BLASTOCLADIELLA EMERSONII

 

Summary

i. Introduction . . . . . . . . . . . . . . . . 22
ii. Materials and Methods . . . . . . . . . 23
iii. Results . . . . . . . . . . . . . . . . . . 23
iv. Discussion . . . . . . . . . . . . . . 25

References . . . . . . . .

 

n‘

POTASSIUM-INDUCED DEPOARIZATION OF THE TRANS-MEMBRANE
POTENTIAL IN BLASTOCLADIELLA EMERSONII ZOOSPORES
PRECEDES ENCYSTMENT

Summary

i. Introduction . . . . . . . . . . . . . .

ii. Materials and Methods. . . . . .
Reagents . . . . . . . . . . . . . . . . . .
Organism and culture conditions . . . . .
Fluorescence experiments . . . . . . . . . . .

iii. Results . . . . . . . . . . . . .
Zoospores labeled with diS- C3 -(5). . . . . .
The ion specific membrane potential depolariza-
tion.....................
Effect of K+ ion concentration . . . . . . . .
Effect of temperature . . . . . . . . . . . .
Effect of pH . . . . . . . . . . . . . . . . .
Effect of valinomycin and quinine. . . . . . .
Response of developing cells to a second KCl

pulse . . . . . . . . . . . . . . . . .
The effect of calcium/ionophore and synthetic
permeant cations. . . . . . . . . . . . . .
iv. Discussion . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . .

GENERAL REFERENCES

.29
.30
.30

'32

3O
3O

33
34
34
35
35

36
36

37
44

.57

2.2

2.3

2.4

3.1

4.2

4.3

404

4.5

vi

LIST OF FIGURES

The life cycle of BZastocZadieZZa emersonii- . . . . .
Schematic diagram of the concanavalin A tetramer . . .
The fluorescent dye 3,3'-diprOpylthiadicarbocyanine. .
The Scatchard plot of specific concanavalin A binding
to BZastocZadieZZa emersonii C8113 during encystment .
Fluorescent micrographs 0f BZastocZadieZZa emersonii
cells treated with fluorescein isothiocyanate
conjugated concanavalin A. . . . . . . . . . . . . . .
Distribution of fluorescence intensity of
BZastocZadieZZa emersonii zoospores during encystment.
Ultrastructural localization of concanavalin A binding
Sites 0f BZastocZadieZZa emersonii C8118 . . . . . . .
Absorbancy changes of zoospores of Blastocladiella
emepsonii exposed to various concentrations of
concanavalin A . . . . . . . . . . . . . . . . . . . .
The time course of fluorescence intensity at various
concentrations of voltage sensitive dye diS-C3-(S) . .
The time course of fluorescence intensity; the effect
of various salts . . . . . . . . . . . . . . . . . . .
Potassium concentration dependent fluorescence
intensity changes. . . . . . . . . . . . . . . . . . .
Temperature dependent fluorescence intensity changes
upon KCl addition. . . . . . . . . . . . . . . . . . .
PH dependent fluorescence intensity changes upon KCl

addition . . . . . . . . . . . . . . . . . . . . . . .

5
9

.ll

l6

l7

l7

18

23

46

46

46

47

. 47

 

4.6

4.7

vii
The time course of fluorescence intensity; the effect
of potassium, valinomycin and quinine. . . . . . . . . . 47
Fluorescence intensity changes upon a second potassium

pulse at the respective time of cellular development . . 47

2.1

3.1

3.2

viii

LIST OF TABLES

Association constants and total number of concanavalin

A binding sites per BZastocZadieZZa emersonii C3113- - 015
The temperature dependence of concanavalin A-induced

lYSiS 0f ZOOSPOIGS BZastocZadieZZa emersonii . . - . - -24
The effect of various lectins on BZastocZadieZZa

emersonii zoospores at 22°C. 0 O O O O O O O O O O O O O 24

CHAPTER ONE

GENERAL INTRODUCTION

-2-

An outstanding characteristic of cells is that the
relationship between their internal activities and the
external environment is regulated by a barrier. This barrier
may exist as the plasma membrane alone or be part of a more
complex cell-surface structure. Crucial activities of the
cell membrane are documented in terms of transport function,
receptor function, and mechanical function.

Studies of' receptor function. and 'mechanical function
have led to the realization not only that components of the
cell surface are mobile in the plane of the membrane but that—
this lateral mobility is subject to regulation (Singer and
Nicolson, 1972; Nicolson, 1979). Such ‘regulated lateral
mobility of membrane components has been the basis for many
hypotheses on the molecular mechanisms mediating cell
recognition, cell. growth. control, and cell transformation
(Edelman, 1976; Nicolson, 1976a, 1976b).

Investigations of transport function have led to the
understanding that living things can generate electric
currents and potentials by regulated, asymmetric movements of
ions across membranes. Nerve fibers carry information in
sequences of action potentials which consist of brief changes
in membrane permeability towards Na+ and K+ ions (Aidley,
1971). Respiration and photo synthesis associated electric
potentials which are present in ndtochondria, chloroplasts,
and various bacteria are believed to arise primarily from the

movement of H+ according to the chemiosmotic hypothesis

(Mitchell, l969). Other important cellular events including
energy transformation, nutrient uptake, and sensory trans-
duction also depend on the magnitude and time course of the
membrane potential.

Based on two reasons, it has become increasingly evident
that the cell surface might play an important role in the
regulation of,develOpment and differentiation. First, the
surface functions in the transmission of information from the
environment to the cell body, since it is the surface which
senses the environment directly. The information can be
processed as a molecular, mechanical, or electric signal.
Cell surface adhesions and membrane potential changes may be
associated with these signals which subsequently effect
intracellular activities involved in processes of develop-
ment and differentiation. Second, the surface character-
istics vary as to fulfill the specific requirements of a
genetically determined developmental state.

The development of higher, multicellular plants and
animals involves a large number of cells and in many cases
the irreversible differentiation of specialized cell types.
As a result, develOpment in multicellular eukaryotes must
depend on mechanisms that extend beyond the usual notions of
sequential gene activation. Fbr example, development of an
embryo requires that cells know where they are and where they
should be. There must be mechanism that regulate this social

behavior of cells. In this case the cell membrane is likely

 

to serve both as the donor and as the acceptor of develop-
mental signals.

Cell interactions and functional specializations in
multicellular eukaryotes are unavoidably associated with very
complex regulatory mechanism. On the other hand, single-cell
eukaryotic microorganisms are smaller and less complex; they
provide experimental systems whose regulatory processes theo-
retically are more easily analyzed. The primary virtues of
these simple organisms are ease of culture, rapid growth at
high cell density, short and manipulatable life cycles, and
ease of producing synchronous differentiation in large cell
populations. The unicellular, non-filamentous, lower aquatic
fungus, .BZastocZadieZZa emersonii, fulfills these criteria
and serves as an excellent model system for development
studies.

The growing B. emersomli plant follows a well-defined
developmental pathway which is divided into five stages: the
zoospore, cyst (round cell) and germling, exponential growth,
zoosporangium formation, and zoospore differentiation.
Figure 1.1 illustrates the life cycle and the respective time
course of individual stages, under Optimal growth conditions
(Lovett, 1975). The subject of this dissertation deals with
cell surface changes during the cell's develOpment from the
zoospores stage to the cyst (round cell) and germling stage.

This particular system was selected for three reasons.
I) The develOpmental process can be manipulated by varying

environmental conditions. The zoospore will encyst and begin

-5-

ZOOSPORES 0 mm

   

Z OOSPORE
DISCHARGE

Z OOSPORE
DIFFERENTIATION
COMPLETE

NUCLEAR CAP
FORMATION

START OF
‘ .> RIBOSOME
“ AGGREGATION

FLAGELLA
FORMATION
AND
k ' , CLEAVAGE

7:50

Z OOSPORANGI UM

'Fig.

12-15mm

 

ZOOSPORE
'ENCYSTMENT

  
  
   
     

20‘30 mm

 

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,’ BINUCLEATE
, PLANT

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FORMED

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FORMATION

40' 50mm

  
  
 

 

 

 

LATE EXPONENTIAL
PHASE PLANT

1.1 The life cycle of BZastocZadieZZa emersonii.

germination within a few minutes following its release from
the zoosporangium, or it will remain motile for many hours
depending upon the compo- sition of the medium (8011 and
Sonneborn, 1969, 1972; Truesdell and Cantino, 1971). In the
presence of 1 mM CaC12 and the absence of monovalent
cations, the zoospore canbe maintained in a viable motile
state for extended periods. When exposed to KCl, it rapidly
rounds up and encysts. Although other factors, such as cell
density and incubation temperature, influence the encystment
process, the zoospore exhibits excellent developmental syn-
chrony under optimal conditions; nearly 100% of the zoospores
triggered will encyst within 15 minutes. II) Pronounced
morphological changes ($011 ‘35 ‘91., 1969; Truesdell and
Cantino, 1971), apparently involving cell surface altera-
tions, are associated with rapid encystment. The 'major
changes are: increase of cellular adhesiveness, decrease of
cell volume, loss of motility, loss of cytOplasmic micro-
tubles, retraction of flagellar axoneme, folding of plasma
‘membrane, fusion. of cytOplasmic vesicles with the plasma
membrane, formation of primary cyst wall, disruption of the
nuclear cap membrane, and the rearrangement of the mitochon-
drion. The encystment process is an all-or-none event; once
a zoospore is induced, it rapidly completes the process and
precedes to germinate, whether or not the conditions are
suitable for subsequent growth. III) There is no detectable

RNA or protein synthesis for cells at either the

zoospore stage or the round cell stage. This indicates that

all of the gross changes associated with the encystment
process occur in the abscence of a functioning biosynthetic
machinery. Therefore, many of the changes are likely to be
the result of local membrane alterations requiring rearrange
ments of preexisting components. Thus, the system provides a
unique Opportunity for investigating 'biOphysical' mechanisms
of the environmental trigger without interference of sub-
sequent 'biochemical' events.

In recent years surface carbohydrates have been imple-
mented to play crucial roles in cellular recognition pro-
cesses such as antigen-antibody interactions, pathogen-host
interactions, and cell-cell/cell-substrate interactions.
While cells undergo differ entiation, cell cycle propagation
and malignant transformation, surface carbohydrates also
exhibit specific changes involving their type, amount,
distribution, and mobility. Lectins, the carbohydrate-
binding proteins which are mostly isolated from plants, are
excellent surface probes because they attach tightly and re-
versibLy to specific surface carbohydrates without entering
the cells (Nicolson, 1974; Sharon and L15, 1975). Under
suitable conditions, the lectin-carbohydrate interactions can
be abolished by the specific saccharide(s) which bind(s) the
respective lectin. The specificity of inhibition is usually
taken as evidence for the presence of surface saccharide
receptors whose structure is similar to that Of the inhibi-

tory sugar(s).

Considering the develOpmental significance of the plasma
membrane, in particular that Of the surface carbohydrates,
one aspect of my research was concerned with the interactions
of concanavalin A, Con A, with cell surface receptors of B.
emersonii zoospores during encystment. The binding specifi-
city Of Con A, the most widely used lectin, is directed

towards a variety of oligosaccharides containing a-D-Man and

.<x-D-Glc residues. It is one of the few lectins that contains

no covalently bound carbohydrate. It has been shown that Con
A has 1 mole each Of Ca2+ and Mn?” per mole of proto-
mer. This lectin is composed of identical subunits (proto-
omers) arragned as tetramers in its native state. A.model of
Con A showing metal ions and saccharide-binding sites is
shown in Figure 1.2 (Becker gt al., 1976). Native Con A and
its chemical derivatives with a reduced number of subunits
(Gunther gt. al., 1973) can. be prepared for studying the
mobility Of the surface receptors. Quantitative measurements
regarding Con A-receptor binding association constants and
the number of binding sites per cell can be performed with
radioactively labeled Con A. Fluorescent and electron micro-
scopic methods, using FITC-Con A and ferritin conjugated Con
A, allow for topographical localization of Con A receoptors
on the cell surface.

In this dissertation I also report about efforts to elu-
cidate the mechanism Of the develOpmental triggering process.
There exists abundant information concerning the biosynthesis

patterns during the entire life cycle of 3- emersonii (Lovett

 

 

CONCANAVALIN A

Fig. 1.2. Schematic diagram Of the Con A tetramer.
Binding sites for Ca2+, Mn2+, B-iodophenylglucose,
and specific saccharides are indicated by Ca, Mn, I, and

S, respetively.

-10..

1975). However, the existing information does not explain
how zoospores are triggered to encyst and germinate when
exposed to KCl. First, the data points collected by other
investigators were measured no earlier than five to ten
minutes following KCl application. Second, there is no
significant change of metabolite levels within the first 10
to 15 minutes after induction. However, as previously men-
tioned, dramatic changes of cell morphology occur within a
few minutes prior to the de novo synthesis of major macro-
molecules. Consequently, clues to the triggering mechanism
are likely to be obtained if one could have access to the
early events related to rapid biOphysical alterations of the
zoospore plasma membrane subsequent to the application of
KCl.

Because the effect of electrical activity of cell
membranes on growth has been recognized (Lund 35 al., 1947;
Nuccitelli and Jaffe, 1976; Jaffee, 1976), changes in the
membrane potential may constitute a conceivable trigger. The
use of fluorescent, voltage-sensitive probes which respond
optically to transmembrane potential changes provides a non-
destructive method for the continuous monitoring of the
electrical activity (Cohen and Salzberg, 1978). Therfore, a
carbocyanine dye, 3,3'-dipropylthiadicarbocyanine was se-
lected for investigating the early changes of the membrane
potential when B. emersonii zoospores were triggered to
develop. The structure of this dye is shown in Figure 1.3

(Simes et al., 1974). This positively charged dye is taken

-11-

 

7.: amunma
mz_z<>oo2<u_oo_::>A_o~:_o...m.m

+ .

Z
is

Fig. 1.3 The fluorescent dye 3,3'~dipropy1thiadi~

carbocyanine.

-12-

up by the cells upon hyperpolarization and released upon de-
polarization. The cell-associated dye is significantly less
fluorescent than the dye in the medium and the degree of
quenching depends on the amount of dye associated with the
cells. This quenching probably results from the formation of
dye aggregates with reduced fluorescence. In conjunction
with a fluorimeter designed by the author, this dye enables
the continuous measurement of alterations of membrane
potential.

On the other hand, the electrical potential difference
between aqueous phases separated by a natural membrane has
been measured directly by suitably placed microelectrodes.
However, the general use of electrodes with micro-organisms
is restricted because of difficulty in implanting electrodes
into the minute cell body and unavoidable cellular damage.
Determination of the distribution of radioactively labeled,
membrane permeant ions enables the quantitative evaluation of
trans-membrane potential. This method does not allow contin-
uous and rapid measurement which is necessary to access early
events in develOpmental triggering.

The results of my experiments indicate that I) The Con
A receptors, glucosyl/mannosyl residues, vary during the
encystment process, II) mobile surface carbohydrates, pre-
sumably glchproteins or glycolipids, are probably involved
in maintaining the osmotic balance of zoospores, and III) the
KT ions induce the depolarization Of' membrane potential,

which precedes encystment.

CHAPTER TWO
CHANGES IN SURFACE PROPERTIES OF DEVELOPING ZOOSPORES
OF BLASTOCLADIELLA EMERSONII:

 

BINDING OF CONCANAVALIN A

-114.

Journal of General .Wicrobiology (l979). 115, 145—151. Printed in Great Britain 145

Changes in Surface Properties of Developing Zoospores of
Blastocladiella emersonii: Binding of Concanavalin A

By C. J. JEN AND A. HAUG

MSL’-DOE Plant Research Laboratory, Michigan State University,
East Lansing, Michigan 48824, U.S.A.

(Received 1 February 1979; revised 15 May 1979)

 

Binding of concanavalin A to the cell surface of developing Blastocladiella emersom'i'
zoospores was explored by fluorescence microscopy, electron microsCOpy and radioactive
labelling. After labelling with fluorescein isothiocyanate-conjugated concanavalin A, the
fluorescence intensity of individual non-induced zoospores varied greatly. On the other
hand, similarly labelled zoospores, induced with Kt ions for synchronous development,
revealed a more even distribution of fluorescence intensity. The zoospore surface contained
43 x 107 concanavalin A binding sites per cell which were randomly distributed and closely
attached to the surface. The aflinity constants ranged from 7-5 x [0" M-1 to 3-5 x 10‘ M4,
while the Scatchard plot was typical of heterogeneous binding. Further developed cells,
round cells and germlings contained 30 x 107 to 2-1 x 107 concanavalin A binding sites per
cell which were mostly loosely associated patches protruding into the extracellular region.
The affinity constants decreased appreciably compared with those measured at the zoospore
stage. Specific concanavalin A binding characteristics appeared to correlate with the
respective developmental stage of the zoospore.

 

INTRODUCTION

The life cycle of Blastocladiella emersom'i, an aquatic unicellular fungus, constitutes a
suitable eukaryotic model to investigate developmental processes (Lovett, 1975). The non-
growing, motile zoospore of B. emersonii exhibits pronounced morphological alterations, in
particular at the cell surface, while undergoing encystment and germination. The zoospore
loses its motility and becomes adhesive, changes extensively in fine structure, retracts the
flagellum into the main body and gradually deposits material on the cell surface for primary
cell wall formation. Subsequently, a germ tube emerges from the cell and this later turns
into a branched rhizoidal system. The entire process takes less than 30 min under favourable
conditions and precedes de novo protein and RNA synthesis (Soll & Sonneborn, 1971).

Lectins are valuable tools for probing surface properties of various cell types (Gremo
et al., 1978; Sharon & 1.15, 1975; Weeks, 1975). To Obtain some insight into developmental
alterations on the zoospore surface, concanavalin A (Con A) was employed. Recently, we
have observed that Con A binding induced lysis of B. emersonii zoospores but not of round
cells (Jen & Haug, 1979). In this paper, we report zoospore surface changes as monitored
by Con A binding parameters. We used PH]Con A, fluorescein isothiocyanate-conjugated
Con A (F ITC-Con A) and ferritin-Con A.

0022-1287/79/0000 8758 $02.00 C l979 SGM

 

-15-

146 c. J. JEN AND A. HAUG

METHODS

Reagents. Con A and a-methyl-D-mannoside were purchased from Sigma, FITC-Con A and ferritin-C on A
were from Miles-Yeda, PHlacetic anhydride was from Amersham/Searle, pronase was from Calbiochem,
and 2,5-diphenyloxazole (PPO) and 1,4-bis-[2-(5-phenoxazolyl)]benzene (POPOP) from Research Products
lntemational Corp. (Elk Grove Village, 111., U.S.A.).

Organism and culture conditions. The aquatic fungus B. emersonii was grown in standard Petri dishes at
22 °C on Difco PYG agar. Zoospores were harvested approximately 20 h after inoculation by flushing
individual Petri dishes with 2 ml sporulation solution [5 mM-morpholinopropanesulphonic acid (MOPS),
1 mM-CaCl,, pH 6-8] and collecting the zoospore suspension as the filtrate through a Whatman no. 1 filter.
To induce synchronous zoospore germination, the procedure described by 5011 et al. (1969) was followed.
The zoospore suspension was incubated for 30 min at 27 °C, then mixed with 5 vol. germination solution
(5 IBM-MOPS, 1 mM-CaCl,, 10 mM-MgClg, 50 mM-KCI, pH 6-8) at 27 °C. The cells underwent transfor-
mation from zoospores to round cells, and finally to germlings. Samples were taken from the suspension
during the developmental process and fixed immediately in 1-5 9.4, (v/ v) glutaraldehyde at 22 °C for 2 h. The
cell phenotypes at the different developmental stages could be easily distinguished by light microsCOpy.
Cells were washed three times with 0-1 M-sodium phosphate buffer, pH 7-2, containing 0-1 M-NaCl.

Binding of [‘H]Con A to cells. [’H1Con A [specific activity 1000 c.p.m. (,ug protein)‘1] was prepared using
['Hlacetic anhydride (Gunther et al., 1973). Cells (1 x 10") were incubated for 2 h with [’1-1]Con A in 1-0 ml
0-1 M-sodium phosphate buffer, pH 7-2, containing 0-1 M—NaCl and 01% (w/v) bovine serum albumin.
For control experiments, 0-1 M-a-methyl-o-mannoside was present in the reaction mixture. Cells were
collected on a Whatman GF/C glass-fibre filter and washed five times with 5 ml phosphate buffer supple-
mented as above. After drying overnight, the filters with the cells were placed in 10 ml liquid scintillation
solution (4 g PPO and 0-05 g POPOP per 1 toluene). Radioactivity was measured with a Packard T ricarb
scintillation counter (model 3390).

Labelling of cells with F] TC -Con A and quantitative fluorescence measurements. Cells (2 x 10‘) were in-
cubated in 1 ml sodium phosphate buffer, pH 7-2, containing 0-1 M-mannitol, 01% (w/v) bovine serum
albumin and 200 ,ug FITC-Con A. For controls, 01 Moat-methyl-D mannoside was substituted for mannitol.
After incubation at 22 °C for about 20 min, the cells were washed with the above reaction mixtures, but
without FITC-Con A. The cells carrying the fluorescence label were viewed under a Leitz Orthomat-W
microscope with an automatic camera. Fluorescence intensity of individual cells was determined with a
Leitz photometer attached to the microscope.

Ultrastructural localization of C on A receptor sites. Fixed cells were incubated with 1 mg ferritin-Con A ml“
at 22 °C for 30 min, then washed with phosphate buffered saline and postfixed with 2% (w/v) 050, in
phosphate buffer. The samples were washed, dehydrated and finally embedded in Spurr's resin (Spurr, 1969).
Cells incubated in the presence of 0-1 M-at-methyl-D-mannoside served as controls. Thin seetions, without
heavy metal staining, were viewed under a Siemens Elmiskop I electron microscope.

RESULTS
Number and afiinity of [3H]Con A binding sites at the surface of B. emersonii

Specific Con A binding sites are those from which the bound lectin can be removed with
at-methyl-D-mannoside as the hapten. Specific binding was determined by subtracting the
contribution of non-specific binding from the total binding. The Con A binding sites at the
zoospore surface appeared to be mostly specific. At worst, in the case of germlings, non-
specific binding could account for as much as 25 9:, of the total binding at high lectin concen-
trations. To ascertain the number and afl‘inity of Con A binding sites, cells were incubated
with various concentrations of [3H]Con A. Results are presented in a Scatchard plot (F i g. 1).
Assuming only two binding systems (Rosenthal, 1967), high and low association constants
and the total number of binding sites per cell were calculated (Table l).

The non-induced zoospore surface area was estimated as 250 ,um2 from the known size
of the cell body (Lovett, 1975), an ellipsoid (7 x 9 ,um), and the cylindrical flagellum (15 x
0-2 pm), and so the site density was calculated to be 1-7 x 105 receptors per ,umz.

During the developmental process, when zoospores round up and germinate, a gradual
decrease of Con A binding was observed. High and low association constants decreased by
factors of 6 and 1-5, respectively, comparing non-induced zoospores with those within 15 min

-16..

 

 

Con .4 binding to zoospores of B. emersonii 147
- 0-3
:- ' ‘ " 0-2
2
a .. D
g 0 1 D
‘g to —
.5 5 10 IS :0 :5
< ..
V 0‘5 1\
l

 

 

 

10 15 30 :5
[3H] Con A bound (ug ml")

U0

Fig. 1. Scatchard plot Of specific concanavalin A binding to B. emersom't' cells during encystment:
C, non-induced zoospores: C, 60% zoospores and 40% round cells. 15 min after induction;
0, 5 % zoospores, 70% round cells and 25 9;, germlings, 30 min after induction; A, 2 % zoospores.
14% round cells and 84% germlings, 45 min after induction. Cells were derived from the same
batch. Except for non-induced cells ('3). all other cells were induced to germinate. Inset: lower
part of the figure drawn to a difl'erent scale.

Table 1. Association constants and total number of concanavalin A
binding sites per B. emersonii cell

A molecular weight of 52000 was assumed for acetylated FHICon A (Gunther et al., 1973). The
values listed were calculated from data presented in Fig. 1.

High aflinity Low affinity
association association Total number of
constant constant Con A binding
(M-l) (34“) sites per cell
Non-induced 7-5 x 10’ 3-5 x 10‘ 4-3 x 107
zoospores
Cells, 15 min 1-2x107 2-3 x105 3-0><107
after induCtion
Cells. 30 min I-Ox 107 2-0 x 105 2-5 x 107
after induction
Cells, 45 min 8-0x10‘ 1-6x 105 21 x107

after induction

after induction (Table 1). The number of binding sites decreased concomitantly. At the
completion of encystment, i.e. about 45 min after induction, there were 2-1 x 10" specific
binding sites per cell.

Cells labelled with FITC -C on A

Labelled zoospores exhibited intense fluorescence, including the flagellum sheath (Fig. 2 a).
The fluorescence intensity seemed to be more pronounced around the nuclear cap and the
‘side body’ complex (for a detailed structural description, see Lovett, 1975). Following the
mixing of a zoospore suspension with germination solution, the resulting elongated cells
showed somewhat weaker fluorescence. Labelled round cells also fluoresced and most Of
them were covered with many fluorescent spots (Fig. 2b). The number and size of these
spots varied from cell to cell. Later, as cells became adhesive and tended to form clumps,
the overall fluorescence intensity decreased.

In general, fixed cells fluoresced within a few minutes after application of FITC-Con A.

-17.

148 c. J. JEN AND A. HAUG

 

Fig. 2. Fluorescence micrographs of B. emersom'i cells treated with fluorescein isothiocyanate-
conjugatcd concanavalin A: (a) non-induced zoospores; (b) round cells fixed 15 min after induction.
Ear markets represent 5 gm.

 

   
 

hthm mlumly lm\ I
H 1.. .4
1

Percentage ul'uclls in Intensity range
1‘ ' .

 

 

 

-
C?
If.
in

5 4-0

tr :3
9 ~13
II\

0

Fig. 3. Distribution of fluorescence intensity of B. emersonii zoospores during encystment. A total
of 75 individual zoospores were randomly scored for each curve. The fluorescence intensity of
individual cells was corrected for non-specific background fluorescence, determined as the average
intensity from 25 control cells, in the presence of at-methyl-D-mannoside and fixed at the same time
as the individual cells being studied. The ordinate shows the percentage of the individually scored
cells which fell within the intensity ranges. 2. Cells before induction; C, cells fixed 15 s after
induction; 0, cells fixed 5 min after induction: I, cells fixed 10 min after induction; A, cells fixed
15 min after induction. Inset: the median fluorescence intensity of the 75 individually scored cells
before (5) and at the respective times after induction.

Extensive washing of cells with bufl‘er did not afl'ect the fluorescence intensity appreciably.
The fluorescence intensity value from fixed, FITC-Con A-labelled cells, in the presence of
at-methyl-D-mannoside, was considered to be non-specific background fluorescence.

The fluorescence intensity of non-induced zoospores varied over a wide range (Fig. 3).
Within the first 15 min after induction, elongated cells and those ready to encyst exhibited
a fluorescence intensity which was weaker than that of non-induced cells, yet remained
virtually constant; the intensity values of the majority of cells ranged from I to about 2 mV.
Both round cells and germlings formed aggregates, and quantitative fluorescence measure-
ments of single cells became impossible.

Ultrastructural localization of C on A receptor sites
Non-induced zoospores were densely labelled with ferritin-Con A which was distributed
randomly over the cell surface and closely attached to the plasma membrane (Fig. 4a), in-
cluding the sheath of the flagellum. The label density varied somewhat from cell to cell.
In control samples, after application of x-methyl-D-mannoside, only occasional ferritin

-18—

Con A binding to zoospores of B. emersom‘t'

Fig.4. Ultrastructural: localization of concanavalin A binding sites of 8. mutant! cells. (a) Non-
induce dz ate the uniform distribution of ferritin molecules closely attached to the
outer surface of the plasma membrane. (b) Non-induced zoospores, in the presence of 0- l M-a-
methyl-o-mannoside: only occasional ferritin molecules are attached to the cell surface. (c) Round
cells; cells were induced and allowed to develop for 20 min before fixation and labelling; note
ferritin molea:les protruding into the extracellular region and forming patches. (d) Zoospor res were
label hferritin-conjugated concanavalin A; 'y-particles were released fro om zoospores
followingWI concanavalin A application and the sample was fixed after labelling; note the densely

belied 'y-particles especially along the inner kidney-shaped region. Bar markers represent 0-2 pm.

 

-19-

150 c. J. JEN AND A. HAUG

molecules were attached to the membrane surface (Fig. 4b). Immediately after induction,
zoospores had a rather irregular shape and were labelled with ferritin particles closely
attached to the cell membrane. However, the particle density appeared to diminish appreci-
ably, and it remained consistently low on later oval-shaped zoospores. But in oval zoospores
fairly large patches of ferritin-Con A were loosely associated with the cell surface and pro-
truded into the extracellular space. Later, in round cells, the number of these patches
increased to cover the peripheral extracellular region: however, the closely attached cell
surface layer of ferritin labels became almost undetectable (Fig. 4c). Some of these patches
appeared to result from non-specific binding, since they could not be removed in control
samples using at-methyl-D-mannoside. Compared with round cells, ferritin patches associated
with germlings became less pronounced and finally disappeared.

DISCUSSION

Major changes occur at the surface of B. emersom’i zoospores while undergoing encyst-
ment preceding detectable de novo synthesis of protein and RNA (Soll & Sonneborn, 1971).
In the course of encystment, surface coat material is deposited by the fusion of vesicles
(derived from y-particles) with the plasma membrane (Myers & Cantino, 1974). About 30
min after induction, the cells form germ tubes and this marks the completion of encystment.

Binding of Con A to the surface of zoospores during encystment was demonstrated by
three diflerent approaches: binding of radioactive Con A, fluorescence microscopy and
electron microscopy. During the entire course of encystment, Con A bound to the cell
surface. although the binding characteristics varied appreciably. Moreover, at a given
developmental stage, individual cells exhibited heterogeneity with respect to Con A binding
as shown by fluorescence and electron microscopic experiments.

It appears that induction of synchronous development is accompanied by rapid physical
alterations at the cell surface, perhaps the surface charge, membrane potential or fluidity.
Observed cell volume changes (T ruesdell & Cantino, 1971) are presumably not related to
variations in fluorescence intensity, as observed in our experiments, because the volume
decreases by 43 94, between 5 and 15 min following induction, whereas the fluorescence
intensity remains virtually constant.

We also found that LiCl, which cannot induce encystment (Soll & Sonneborn, 1972),
produced practically the same fluorescence intensity distribution as in KCl-treated cells.
Synchronous development could be induced by KCl pulses as short as 2 min (Soll & Sonne-
born, 1972). Therefore, our results seem to support the notion that both kinds of ion induce
similar physical alterations at the cell surface. Subsequent developmental processes are
presumably inhibited by LiCl.

During development from non-induced zoospores to round cells and germlings, Con A
labelling experiments demonstrated the presence of two distinct surface labelling patterns,
and also pronounced changes in binding affinity. This phenomenon may be interpreted in
two ways. First, the original Con A binding sites participating in the formation of the
closely attached layer may be removed during normal membrane turnover and then replaced
by newly synthesized Con A binding receptors with a lower affinity. However, in B. emersonii,
protein synthesis is not detectable during the short-lived encystment process. Thus, an
alternative possibility is that during encystment pre-existing membrane components are
modified to manufacture low aflinity Con A receptors, in the absence of protein synthesis.

When the primary cell wall material is deposited, the closely attached Con A binding
layer gradually becomes obscured. Meanwhile, the loosely associated ferritin-C on A patches
continue to form. We propose that this pattern change is related to the fusion of :I-particle
vesicles with the plasma membrane. These organelles have a high glycolipid content where
glucosyl units are abundant (Mills & Cantino, 1978; G. L. M ills. personal communication).
Furthermore, preliminary experiments (Fig. 4d) indicated that y-particles did exhibit a

 

 

-20-

U:

l

densely labelled surface of ferritin-Con A particles, following lysis of zoospores. release of
:t-particles and subsequent exposure to Con A.

It is not known whether Con A binding sites play an active role in the developmental
process described. Since Con A induces rapid zoospore lysis (Jen & Haug, l979). viable
cells cannot be labelled and properly examined within the short time available during
encystment. Nevertheless. the lysis experiments suggest that Con A receptors, in particular
those characterized by the closely attached layer of label. maintain zoospore integrity.
Preliminary electron microscopic studies demonstrate that this closely attached layer is not
susceptible to pronase digestion contrary to the loosely associated patches which can largely
be removed. The carbohydrate moiety of the closely attached layer perhaps prorects the
underlying proteins from pronase attack in the zoospore stage. Pronase-treated. non-induced
zoospores are capable of completing normal encystment, if pronase is removed prior to
induction. It appears that later developmental stages are sensitive to pronase digestion as
shown by retarded germination following pronase treatment at the round cell stage. Further
experiments are necessary to clarify the physiological role, if any, of lectin receptors during
zoospore development.

C 0n .4 binding to :oosporcs of B. cmersom’i i

We thank Dr J. L. Wang for valuable discussions and Dr J. E. Wilson for allowing the
use of the Leitz microscOpe. We are indebted to Dr E. C. Cantino for the B. emersonii
strain. This work was supported by US—DOE Contract EY-76-C-02-l338.

REFERENCES

 

GREMO, F.. KOBILER. D. & BARONDES, S. H. (1978).
Distribution of an endogenous lecrin in the
developing chick optic tectum. Journal of Cell
Biology 79. 491-499.

Gunman. G. R.. WANG. J. L.. YAHARA. 1.. CUN-
NINGHAM. B. A. & EDELMAN, G. M. (1973).
Concanavalin A derivatives with altered bio-
logical activities. Proceedings of the National
Academy of Sciences of the United States of
America 70. lOl 2-101 6.

JEN. C. J. & Have. A. (1979). Concanavalin A in-
duced lysis of zoospores of Blastocladiella
emersonii. Experimental Cell Research 120,
425—128.

Lovs'rr, J. S. (1975). Growth and differentiation of
the water mold Blastocladiella emersonii : cyto-
difl'erentiation and the role of ribonucleic acid
and match! synthesis. Bacteriological Reviews 39,
345-404.

MtLLs. G. I... at CANTINO, E. C. (1978). The lipid
composition of the Blastocladiella emersonii 7.
particle and the function of the 7-particle lipid in
chitin formation. Experimental Mycology 2, 99-
109.

MYERS. R. B. & Cats-mo. E. C. (1974). The gamma
particle. In Monographs in Developmental Biology,
vol. 8. pp. 15-20. Edited by A. Wolky. New York:
S. Karger.

Rossmnxt. H. E. (1967). A graphic method for the
determination and presentation of binding para-
meters in a complex system. Analytical Bio-
chemistry 20. 525—532.

SHARON, N. & Lts. H. (1975). Use of lectins for the
Study of membranes. In Methods in Membrane
Biology, vol. 3, pp. 147-200. Edited by E. D. Kom.
New York: Plenum Press.

SOLL. D. R. 8c SONNEBORN, D. R. (1971). Zoospore
germination in Blastocladiella emersom'i: cell
differentiation without protein synthesis? Pro-
ceedings of the National Academy of Sciences of
the United States of America 68. 459—463.

SOLL. D. R. 6:. Sonxsaoax. D. R. (1972). Zoospore
germination in Blastocladiella emersonii. IV. Ion
control over cell differentiation. Journal of Cell
Science 10. 315-333.

Sou. D. R.. Bamasac. R. at Soxxsaottx. D. R.
(1969). Zoospore germination in the water mold.
Blastocladiella emersonii. I. Measurement of
germination sequence of subcellular morpho-
logical changes. Developmental Biology 20. 183-
217.

SPURR, A. R. (1969). A low-viscosity epoxy resin
embedding medium for electron microscopy.
Journal of Ultrastructure Research 26, 31-43.

TRUESDELL, L. C. & CANTINO. E. C. (1971). The in-
duction and early events of germination in the
zoospore of Blastocladiella emersonii. Current
Topics in Development Biology 6. 1-44.

WEEKS. G. (1975). Studies of the cell surface of
Dictyostelium discoideum during differentiation:
the binding of (“’IICM A to the cell surface.
Journal of Biological Chemistry 250. 6706.6710.

CHAPTER THREE
CONCANAVALIN A-INDUCED LYSIS OF ZOOSPORES
OF BLASTOCLADIELLA EMERSONII

-22-

mine the sizes of molecules that could be in-
troduced into permeable BHK cells. repli-
cate cultures of BHK cells were treated
with increasing concentrations of lyso-
lecithin and assayed for the ability of trypan

blue (970 D) and RNases (11000-14000 D) .

to enter the cells. In addition. retention of
lactate dehydrogenase (142000 D) activity
and viability were measured (table 2). At 50
ug/ml lysolecithin, 90% of the cells were
permeable to trypan blue and remained
viable; RNases were excluded from the
cells and all lactate dehydrogenase activity
was retained. Higher concentrations of
lysolecithin permitted RNases to enter the
cells. but viability was lost. Essentially all
lactate dehydrogenase activity remained in
the cells up to 100 pg/ml lysolecithin, al-
though the cells were permeable to RNases.
At concentrations of lysolecithin above 100
pg/ml. lactate dehydrogenase leaked out of
the cells.

These results indicate that permeability
can be controlled by adjusting the con-
centration of lysolecithin. Lysolecithin-
permeabilized cells are being used in these
and other laboratories to study processes
such as DNA replication, DNA repair,
mechanisms of drug action. and mech-
anisms of drug resistance. The results re-
ported here extend the lysolecithin penne-
abilization method to a wide range of animal
cells. This procedure renders many cell
types reversibly permeable to small mole-
cules following mild treatment. More severe
treatment irreversibly permeabilizes cells:
the degree of permeability can be controlled
by altering the lysolecithin concentration.

This work was partially supported by a biomedical
research grant to M. R. M.. a USPHS research grant
(CA-18612) to A. B. P.. and a USPHS predoctoral
traineeship (no. 5T32 CA 09031-02) to J. J. C. Jr. We
thank David Schneider for his help in preparing the
manuscript for publication.

Preliminary notes 425

References

1. Castellot. J J. Miller. M R & Pardee. A B. Proc natl
acad sci US 75 l 1978) 351.

2. Miller. M R. Castellot. J J & Pardee. A B. Bio-
chemistry 17 l 1978) 1073.

3. Kornberg. A. Meth enzymol 1(1951) 441.

4. Lowry. O H. Rosebrough. N J. Farr. A L &

Randall. R J. J biol chem 193 (1951) 265.
5. Giles. K W & Mayers. A. Nature 20611965) 93.

Received October 23. 1978
Revised version received December 5. 1978
Accepted December 7. 1978

Printed in Sweden

Copyright if: 1979 by Academic Press. Inc.
All rights of reproduction in any form reserved
0014-4827/79l070425oN5021XNO

Concanavalin A-induced lysis of zoospores of
Blastocladiella emersom'i
C. J. JEN and A. HAUG. Department of Biophysics

and MS U-DOE Plant Research Laboratory. Michigan
State University. East Lansing. MI 48824. USA

Summary. Concanavalin A (ConA) induces lysis of
zoospores of the aquatic fungus Blastocladiella emer-
sonu‘. The rapid lysis can be completely blocked by
a-methyl-D-mannoside. At lower temperatures the
cells are more tolerant towards lysis compared with
cells at room temperature. Dimeric ConA derivatives
and Lens culinaris hemagglutinin A are less effective,
whilst other lectins tested do not induce lysis at all. We
suggest that cluster formation of adjacent ConA re-
ceptors precedes lysis.

Concanavalin A (ConA) has been exten-
sively used to investigate surface properties
of various cell types because it binds spe-
cifically to carbohydrates. Research has
been carried out with cells during differen-
tiation [1], during different stages of the cell
cycle [2], and also in malignantly trans-
formed cells [3]. Lectin derivatives and na-
tive ConA have been also employed to
study the distribution and dynamic rear-
rangement of carbohydrate receptors at
membrane surfaces [4, 5]. '
Since only scattered information [6] is
available on lectin receptors located at cell
surfaces of aquatic fungi. we examined lec-
tin-binding properties to the cell surface of

Exp Cell Res [30 (I979)

-23-

426 Preliminary notes
0.7 l.
l
2
0.6 - 3
4
0.5 '-
0.4 "' 5
6

 

 

2 6 IO 14

Fig. I. Abscissa: time after mixing zoospores suspen-
sion with ConA (min); ordinate: absorbancy measured
at 550 nm wavelength.

Absorbancy of zoospores of Blastocladiella emer-
sonii exposed to various concentrations of ConA.
0.5 ml of cell suspension (1.5X10’ cells/ml) was mixed
with 1 ml water which contained (1) 100 pig/ml ConA
plus 0.05 M mannitol; (2) no ConA. no sugar: (3) 25 ug/
m1 ConA; (4) 50 ug/ml ConA; (5) 100 ug/ml ConA; (6)
75 ug/ml ConA. final cone.

Blastocladiella zoospores. Blastocladiella
emersonii displayed significant changes at
the plasma membrane surface of motile zoo-
spores while undergoing encystment, or
forming round cells. Zoospores tend to ad-
here to each other and also to the bottom of
the growth vessel; vesicles originating from
y-particles fuse with the plasma membrane
of zoospores prior to cell wall formation;
moreover, zoospores retract the flagellum
within several minutes after induction with
KC1[7].

In this article we report about experi-
ments concerning cell lysis subsequent to
ConA binding. The binding pr0perties of
this lectin and some of its derivatives, and
those of other lectins bound to the cell sur-
face, are also examined.

Materials and Methods

ConA and a-methyl-o-rnannoside were purchased
from Sigma Chemical Co.. St Louis. Mo, wheat germ

Exp Cell Res [20 ([979)

agglutinin (WGA) was obtained from Calbiochem. San
Diego. Calif. Fucose-binding protein (FBP). Rit'lrms
communis agglutinin-60 (RCA-60). and Lens t'iilirmris
hemagglutinin A (LCH-A) were obtained from Miles-
Yeda, Elkhart. Ind. All lectins were directly used
without any further purification. Chemical derivatives
of ConA. viz. acetyl-ConA. and succinyl-ConA. were
prepared [8].

Apart from minor modifications. Blastocludiellu cul-
tures and growth conditions were maintained as de-
scribed [7]. Zoospores were harvested from cultures of
the first generation grown on Difco PYG agar l 1.25 g
yeast extract. 1.25 g bactOpeptone. 3.0 g dextrose. and
20 g agar per 1 000 ml of medium). and then transferred
to tissue culture dishes. which contain PYG liquid
medium (same as above but no agar). After 12-18 h at
22°C. growth was terminated by decanting the growth
medium. At the same temperature. sporangia which
were attached to the plastic bottom were rinsed and in-
duced to sporulate by incubation with sporulation solu-
tion (10" M CaClg. 5x 10" M morpholinopropane sul-
fonic acid. pH 6.8). The released zoospores were in-
cubated with the respective lectin at a certain tempera-
ture. for 30 min. and were examined with a Zeiss
standard microscooe. model 14. Cell lysis was esti-
mated semi-quantitatively by observation of single
cells. In other experiments. after mixing the zoospore
suspension with ConA. the cell lysis kinetics was
measured by determining the absorbancy of the sus-
pension at 500 nm with a Gilford spectmphotometer.
model 2400.

Results

Cells in the zoospore stage began to lyse
within the first few minutes after addition of
ConA to the cell suspension, at 22°C. Im-
mediately preceding cell lysis. ConA-
treated cells were no longer able to swim
and commenced to swell until they were in-
capable of maintaining osmotic balance
across the plasma membrane. This swelling
process did not occur at all in the presence
of 0.1 M a-methyl-D—mannoside.

Measured at 500 nm. absorbancy changes
clearly reflected the lytic destruction of
cells. The rate of cell lysis was enhanced
upon increase of the lectin concentration
(fig. 1). At intermediate concentration
ranges, from about 60-120 pg/ml, the zoo-
spores were ruptured into small fragments
which was indicated by dramatic ab-
sorbancy changes. However, at C onA con-
centrations higher than 250 ug/ml. the cells
fragmented into larger pieces and the ab-

-211-

Preliminary notes 427

Table 1. Temperature dependence of C onA-inducea’ lysis ofzoospores of Blastocladiella

emersonii

++++. All cells lysed: +++. approx. 75% lysis; +--. about half of the cells lysed: +. less than 25% lysis:
5. cells swelled but did not lyse: :7. effects uncertain; -. no observable effect

 

 

 

ConA.ug/ml
Temp.
°C 12.5 25 50 100 250 500 1000
22 s + +++ ++-o-+ ++++ 4--+-++ ++++
16 t? 5 ++ ++++ ++++ ++-s-+ ++++
12 i? t? s 5 ++ ++++ ++++
8 - i? s s s s ++

 

The zoospore suspension was incubated with ConA for 30 min at the temperature indicated. Reaction mixture
contained 5X 10‘ cells. ConA, 5 mM MOPS buffer, 1 mM CaCl, in 1 ml solution. pH 6.8.

sorbancy curves became flatter (data not
shown). In the presence of 0.05 M mannitol.
the cells were characterized by irregular
shapes and failed to germinate after addi-
tion of ConA; under those conditions most
of the cells did not burst.

ConA-induced cell lysis of zoospores was
found to be temperature-dependent (table
1). Upon preincubation with sporulation
medium and treatment with ConA at a
lower temperature, the cells appeared to be
more tolerant towards lysis than those
treated with ConA at room temperature.
For example, at 8°C , most cells were still in-
tact although they had been exposed to
500 ug/ml ConA for 30 min. Nevertheless,

these zoospores—initially treated with
ConA at 8°C—lysed immediately when the
temperature was raised to room tempera-
ture.

The influence of other lectins upon the
zoospore surface was studied at room tem-
perature (table 2). The dimeric ConA de-
rivatives, Lens culinaris hemagglutinin A
and Ricinus communis agglutinin-6O
swelled but did not lyse the zoospores.
WGA and fucose-binding protein exerted
no lytic effect. LCH-A caused cells to swell
at concentrations higher than 250 pig/ml.
Besides native ConA, none of the other lec-
tins tested ruptured zoospores under other-
wise identical conditions.

Table 2. Eflect of various lectins on Blastocladiella zoospores. at 22°C

 

Lectin concentration. ug/ml

 

12.5 25 50

100 250 500 l 000

 

ConA

AcetyLConA

Succinyl-ConA

WGA

RCA-60

FBP

LCH-A

ConA +a-methyl-D-
mannoside (0.1 M) - - -

lllllw
Illlll+

++++ ++++ +++

+
S
"' S
S

m|m|mm+

l
m

 

Same notations as in footnote to table 1.

Exp Cell Res III) l [979)

-25-

428 Preliminary nores

Discussion

Because of its size. the ConA protein will
presumably not enter the cell membrane
and inhibit metabolic activity within a short
time period after lectin binding. at the cel-
lular surface. We observed. however. that
ConA was able to induce zoospore lysis al-
most instantaneously. and this process
could be totally blocked by the application
of a-methyl-D-mannoside. Therefore. ConA
appears to affect primarily the plasma mem-
brane. Once a sufficient number of ConA
molecules is attached to the zoospore sur-
face. the plasma membrane is altered and
cannot function any more osmotically. On
the other hand. mannitol stabilizes the cell
osmOtically. although mannitol is nm a spe-
cific inhibitor for ConA binding. Prelimi-
nary experiments in our laboratory in-
dicate that round cells, which have a primi-
tive cell wall—in contrast to zoospores——
are not susceptible to ConA lysis. They are
osmotically stable and proceed to germinate
in the presence of ConA concentrations
which would totally lyse zoospores.

There exist at least two reasons for the
diminished ConA-induced cell lysis at lower
temperatures. (1) At lower temperatures
ConA binding to the cell surface might be
impaired. However. we found neither ap-
preciable differences in the number of bind-
ing sites nor in their affinity, when ConA
was attached to fixed cells either at room
temperature or at 8°C. (2) We suggest that
cell lysis requires formation of lectin recep
tor clusters which—to a certain extent-
depends on receptor mobility in the cell
membrane. The latter process is tempera-
ture dependent. The pr0posed cluster
mechanism is triggered by native ConA
which possesses four saccharide binding
sites per molecule [8], which may partici-
pate in the cross-linkage of adjacent sites.
Aggregation between neighbouring sites

Exp Cell Res ,201l970l

was also observed on maStocytoma cells.
where a linear relationship was found be-
tween cap formation and cytolytic effect of
ConA [9].

The mobility of receptor sites is reduced
at temperatures below 10-12°C. where spin
labelling experiments demonstrate discon-
tinuities in the Arrhenius plots of membrane
microviscosity vs temperature [10].
LC H-A has the same sugar specificity and
also two binding sites per molecule as the
dimeric ConA derivatives studied. Com-
pared with dimeric ConA. LCH-A pos-
sesses a 50 times smaller binding affinity
[1 l] and a rather similar lytic capacity to-
wards zoospores. This seems to indicate
that the crucial step in the formation of Ice-
tin clusters is the ability to cross-link and
not the affinity to bind.

This project was supported by the US Department of
Energy Contract EY-‘76-C-02-1338. We thank Dr E. C.
Cantino for helpful discussions and for prOViding the
original strain of Blastocladiella emersonii.

References

. Weeks. G. J biol chem 2501 1975) 6706.

. Shoham. J & Sachs. L. Exp cell res 8511974) 8.

. Noonan. K D 81: Burger. M M. J biol chem 248

11973) 4286.

4. Ash. J F & Singer. S J. Proc natl acad sci US 73
(197614575.

5. Schlessinger. J. Elson. E L. Webb. W W. Yahara.
l. Rutishauser. U at Edelman. G M. Proc natl acad
sci US 7411977) 1110.

6. Levmos. E M & Lodi. W R. Exp cell res 94 1 1975)
43 .

7. 5011. D R. Bromberg. R 8:. Sonneborn. D R. Dev
biol 2011969) 183.

8. Gunther. G R. Wang. J L. Yahara. l. Cunningham.
B A & Edelman. G M. Proc natl acad sci US 70
(1973) 1012.

9. Lustig. S. Asher. D. Fishman. P. Djaldetti. M &
Pluznik. D H. J cell biol 75 (1977) 388.

10. Leonards. K & Haug, A. Plant physiol. suppl. 61
11978) 113.

11. Stein. M D. Howard. 1 K 8t Sage. H J. Arch

biochem bi0phys 146(1971) 353.

Mid-—

Received November ‘7. 1978
Revised version received January 3. 1979
Accepted January 8. 1979

CHAPTER FOUR
POTASSIUM-INDUCED DEPOLARIZATION OF THE TRANS-MEMBRANE
POTENTIAL IN BLASTOCLADIELLA EMERSONII ZOOSPORES

 

PRECEDES ENCYSTMENT

-27-

POTASSIUM-INDUCED DEPOLARIZATION OF THE TRANS-MEMBRANE POTENTIAL

IN BLASTOCLADIELLA EMERSONII ZOOSPORES PRECEDES ENCYSTMENT.

C. J. JEN and A. HAUG

Department of Biophysics and MSU-DOE Plant Research Laboratory
Michigan State University

East Lansing, Michigan 48824

Running title: Trans-membrane potential and developmental triggering

 

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-23-

SUMMARY

Experiments have been performed to test the possible role of the membrane
potential in the ionic induction of encystment in Blastocladiella emersonii
zoospores. A fluorescent, voltage-sensitive dye, 3,3'-dipr0pylthiadicarbo-
cyanine iodide, was used to monitor changes in the membrane potential. Only

KI ions, the standard developing triggering agent, rapidly depolarized the
membrane potential. Application of an optimal K+ ion concentration (50 mM)

to a cell population depolarized the membrane potential most rapidly and induced
the highest percentage of cells to encyst per unit time. The depolarization

was temperature dependent and was not affected by extracellular pH changes

from 5 to 9. Quinine practically prevented depolarization of the membrane
potential by K+ ions; however, the membrane potential was depolarized by subsequent
valinomycin application. The K+ induced response of the membrane potential ‘

was apparently independent upon the zoospore's age, but varied dramatically as
the zoospore encysted. A strong correlation was established between the
K+'ion-induced depolarization of the membrane potential and the developmental
triggering by K+ ions. We propose that specific K+ carriers are embedded in the

lipid matrix, which exists in the gel + liquid-crystalline mixed lipid state.

 

-29-

The life cycle of Blastocladiella emersonii [l], a unicellular water mold,
provides a suitable model for studying the role of the plasma membrane in
eukaryotic cell differentiation. During encystment, the non-growing motile
zoospore of g, emersonii manifests pronounced morphological alterations,
concomitant with a loss of motility, flagellar retraction, increased ad-
hesiveness, fusion of cytoplasmic vesicles to the plasma membrane, and
primary cyst wall formation. Under favorable conditions the entire en-
cystment process requires less than 30 min after the zoospores have been
exposed to KCl. These observable morphological modifications involve ap-
parently the plaSma membrane, and precede denggyg_detectable RNA and pro-
tein synthesis. A working hypothesis has been proposed whereby local
membrane alterations result from rearrangements of pre-existing structures
[2]. However, there is little information available on early (less than a
few min) physico-biochemical membrane processes occurring prior to the
structural changes reported. In our previous work on g, emersonii
zoospores we had demonstrated a rapid change of concanavalin A binding
characteristics to the plasma membrane upon KCl addition [3, 4]. There-
fore, the present studies were undertaken to determine whether the devel-
opmental induction might be associated with initial changes of the trans-
membrane potential. To follow possible transient ion-induced responses of
the membrane potential, the fluorescent, voltage-sensitive dye 3, 3'-
dipropylthiadicarbocyanine iodide, diS-Ca-(S), was employed [5, 6, 7]. We
observed a rapid membrane potential depolarization which occurred simul-
taneously with K+ induction. At the time of the completion of this article

a partially related study was published [8] which we are going to discuss.

-30-

MATERIALS AND METHODS

Reagents. The dye diS—CB-(S) was a gift from Dr. A. Waggoner (Amherst
College, Mass.). ATP, valinomycin, quinine, 9-amino acridine were pur-
chased from Sigma; the ionophore A23187 was bought from Calbiochem;
triphenylmethyl phosphonium bromide, TPMP+ bromide, was bought from ICN,

and tetrabutylammonium chloride was purchased from Aldrich. The amphiphilic
spin probe 4-(dodecyl dimethyl ammonium)-l- oxyl-2,2,6,6-tetramethyl

piperidine bromide (CAle) was synthesized in our laboratory as described

 

[9]. All other reagents were purchased and used without any further r‘

purification.

Organism and culture conditions. g, emersonii zoospores were prepared as
described [2]. Cells were grown overnight in liquid PYG Difco medium in

100 x 20 mm tissue culture dishes (Falcon No. 3003). The medium was re-

3M C3012 and 5 x 10"3

M MOPS buffer, pH 6.7). The zoospores, released about four hours later,

moved and replaced by the sporulation solution (10-

were maintained in sporulation solution until the experiments commenced.
The cell suspension was filtered through filter paper (Schleicher and
Schfill, No. 588) to remove possible cell types other than zoospores. The
zoospore concentration of the filtrate was adjusted to S x 106 cells/ml

for the fluorescence studies.

Fluorescence experiments. The fluorimeter used for the trans-membrane
potential measurements was a modification of a phosphorosc0pe previously
described [10]. Briefly, the output of an EMI photomultiplier was passed

through a Keithley picoammeter, model 417, and recorded on a Hewlett-Packard

-31-

X-Y recorder, model 7034A. To vary the sample temperature, a holder

was constructed through which temperature-controlled water could be
circulated. Prior to the membrane potential measurements, the fluorimeter
was tested on erythrocytes [5]. The 1 mM ethanolic stock solution of the
dye was first diluted to 20 uM with sporulation solution. Typically, 25
ul of the diluted dye solution was injected into the cuvette which con-
tained 2 ml of the zoospore suspension. In the sample cuvette, the final
ethanol concentration was 0.025% (v/v). Previous control experiments had
indicated that the encystment process was not affected by these minute
alcohol concentrations. The fluorescence signal from the reaction mixture
was continuously monitored for about 10 min at excitation and emission
wavelengths of 629 and 671 nm, respectively. Above 500 nm interference
from intrinsic cellular absorption, emission, and light scattering was.
negligible. After a steady-state fluorescence intensity level had been
reached within a few min following dye injection, various agents were ap-
plied to the sample, and the fluorescence intensity change was recorded
continuously. The fluorescence intensity was measured in arbitrary units,
from which two parameters were derived, viz., AF/F (Z), and the initial
rate of the change, AF/F min-1. The fluorescence ratio, AF/F, represents
the percent intensity change with respect to the steady-state level estab-
lished prior to the addition of a specific reagent. The initial rate of
fluorescence change is the slope of AF/F following application of the re-
spective reagent. 9-aminoacridine, a fluorescent dye [ll], was used in
some experiments to monitor possible pH variations. Generally 1 uM
9-aminoacridine was applied to the sample, under similar conditions as

described above, except that the excitation and emission wavelengths

‘5'.
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the

-32-

corresponded to 396 and 460 nm, respectively. Although the intrinsic
fluorescence and scattering of the cells interfered with the measurements,
this background amounted to 10% at most, compared with the total signal

level and was corrected accordingly.

 

 

RESULTS

$
Zoospores labelled with diS-C,-(5). The cells were labelled with dis-03-
(5) and the time course of fluorescence intensity recorded. Upon addition 3
of the dye to a cellular suspension, the fluorescence intensity immediately f“

increased, followed by a decrease (fig. 1). This behavior indicated that
the cells have an interior negative membrane potential which was responsible
for shifting the dye's equilibrium distribution towards the cytoplasmic side
or membrane-bound state as evidenced by the decrease in fluorescence in-
tensity [5]. The addition of 50 mM KCl to the reaction mixture intensified
the fluorescence rapidly (depolarization of the membrane potential) which
stabilized at a new level within a few minutes. The percentage of KCl
induced fluorescence intensity change was dependent upon the dye concen—
tration employed. Low dye concentrations displayed small changes which

were difficult to measure. Too high dye concentrations suffered from the
following disadvantages: (a) More than 5 min were necessary to attain the
initial steady-state fluorescence level, (b) cellular injury might have

been caused, and (c) the membrane potential might have been depolarized

[7]. Consequently we selected a dye concentration of 2.5 x 10‘.7 M. Under
these standard conditions, the physiological behavior of the zoospores was
apparently not impaired as judged by the usual development in the cuvette

after KCl addition. At this concentration the dye did not exert any

-33-

measurable influence on the observed K+ effect since the same final fluo-
rescence level, characteristic for the depolarized state, was reached
irrespective of the sequence of K+ or dye application. It was not neces-
sary to add any stabilizer to our reaction mixture, in contrast to experi-
ments described in a recent report [8], where 0.1 M sucrose was used to
prevent cell lysis temporarily when the membrane potential was measured [-
with the carbocyanine dye di0-C6-(3). If suspended in sporulation medium

(K+ ion-free), zoospores can be maintained in that stage for up to 20 hr.

.. '3'. I“:
.

 

The KCl induced fluorescence intensity increased but the AF/F values de- f:

creased when the cells were older than 15 hr.

The ion specific membrane potential depolarization. Although KCl was
presumably the induction agent in the standard germination solution [12],
other reagents were also tested under similar conditions (fig. 2). These
reagents could be classified into three groups according to the pattern of
membrane potential change produced. Type 1 salts, KCl, KBr, KI, and RbCl,
depolarized the membrane potential rapidly and with practically identical
efficiency. Since Rb+ may be considered as a K+ analogue, the results
suggested that the K+ cation, not the anions, was the effector. This was
further confirmed by studying the effects of two other potassium salts.

The highly permeant anion SCN- caused no significant depolarization,
whereas the impermeant anion 4-morpholinoethanesulfonate (MES-) depolarized
the membrane potential similarly as KCl. In the presence of KMES, the
cells are induced to develop; they eventually lysed, however, when KSCN

was applied instead. The type 2 compound, NH4C1, depolarized the membrane
potential similarly, but to a lesser degree. In the presence of NH4C1, the

fluorescence intensity reached a new steady-state level later than that

-34-

level reached after KCl application. Type 3 compounds, LiCl, NaCl, CaClz,
choline chloride, MgCl2 and CsCl, generated a weak and slow fluorescence

intensity increase. We also tested the effect of LiCl, a known inhibitor

for encystment [12], on K+ induced depolarization. KCl depolarized the

membrane potential irrespective of the presence of the lithium salt. The

non-ionic compound mannitol did not affect the fluorescence intensity. We ,-

therefore concluded that osmolarity changes were not responsible for alter- r

ing the membrane potential.

 

 

Effect _£“§:_ion concentration. The fluorescence intensity change depended r'
upon the K+ ion concentration (fig. 3). A detectable increase in fluo—

rescence intensity could be observed at K+ ion concentrations as low as

0.5mM. As the K+ ion concentration increased, both the fluorescence in-

tensity change and the initial rate became more pronounced. The maximal

intensity change was reached at about 25 mM, succeeded by a small decrease

for higher concentrations. The initial rate of change reached a maximum

value at about 50 mM. Further increase of the K+ ion concentration did

not enhance significantly the initial rate of change.

Effect 2f_temperature. Samples were pre-equilibrated for 5 min at the

 

desired temperature which was subsequently maintained throughout the ex-
periment. Reagents were kept at the same respective temperature. For a
temperature less than 15°C, the steady-state base line fluorescence was

reached at later times and was higher compared with that at room tempera-
ture. Therefore we carried out the following control experiments: AF/F
values were unchanged, when cells were incubated with the dye, either at

4°C for 12 min, or at 22°C for 4 min succeeded by an additional incubation

-35-

at 4°C for 8 min, or at 4°C for 4 min. Furthermore, the AF/F values of
cells incubated with the dye at 22°C for 12 min were virtually the same

as those incubated for a 4 min period. To forestall possible cell lysis
during prolonged incubation times, the reagent was injected 4 min after the
dye application, over the entire temperature range tested. Below 4°C the
K+ induced fluorescence intensity change was undetectable. Parallel to a
temperature raise, the K+ effect became more pronounced and reached a ”1
plateau at about 2843000. At temperatures higher than 35°C, the value of

AF/F diminished rapidly (fig. 4). ' .-

Effect 2; RE. The adjusted pH value of the zoospore suspension did not
vary by more than 0.2 pH units within 15 min, irrespective of the presence
or absence of 50 mM KCl. In the absence of KCl, the base line fluoresdent
level of dye-labelled cells was pH dependent and increased as the pH value
of the medium decreased. Upon K+ ion addition, however, the ratio AF/F
remained approximately constant between pH 5 to 9 (fig. 5). The initial
rate of fluorescence change remained practically constant between pH 6 and
8; beyond this range the rate was reduced. Furthermore, upon application
of K+ ions at pH 6.7, the intracellular pH stayed constant as monitored
by the pH probe 9-aminoacridine (figure not shown). These results appear
to indicate that membrane potential changes rather than pH gradient varia-

tions are associated with the process of KCl induction.

Effect 2£_valinomycin and quinine. The fluorescence intensity level of

 

cells labelled with the voltage sensitive dye changed insignificantly upon
+
application of valinomycin, either before (curve not shown) or after K

addition (fig. 6a). Quinine, an inhibitor for Ca2+-activated K+ channels

-35-

in red blood cells [13], reduced the fluorescence level slightly when added
to the dye-labelled cell suspension. In the presence of quinine, K+ ions
induced a much smaller depolarization of the membrane potential than that

in the absence of quinine. Subsequent application of valinomycin to cells,
incubated in the presence of quinine and K+ ions, caused rapid depolarization

(fig. 6b), but cells lysed eventually.

Determination of the membrane potential by the null point method [14, 15] was
unsuccessful in our case because the K+ ion-induced AF/F values remained
essentially c0nstant irrespective of the presence or absence of valinomycin.
With the synthetic cation triphenylmethyl phosphonium (TPMP+) we were able

to confirm a membrane potential of about -100 mV at the resting state [8].

Response of developing cells _t_ a second KCl pulse. We studied the fluo-

 

rescence behavior of K+ ion-induced, dye labelled cells and their response
to a second KCl pulse which was applied after removal of K+ ions from the
previous induction medium. If the monovalent cation was removed within
less than 10 min, the membrane potential returned to its original state,
i.e., to that value prior to the application of the first K+ pulse; then
the zoospore response to a second K+ pulse resembled that of the first
ionic pulse. However, round cells responded less to the second ion pulse.
By the time cells turned into germlings, about 30 min after induction, only
a weak response was elicited as reflected by a small fluorescence intensity

increase following the second pulse (fig. 7).

The effect g£.calcium/ionophore and synthetic permeant cations. Although
our results agree in principle with those reported recently [8], we attempted

to test whether zoospore encystment can be induced by membrane potential

-37-

depolarization generated by ions other than K+ ions, in particular Ca2+ions.
In the presence of the ionophore A23187 (2.5 x 10‘6M), low Ca2+ concentra-
tions (1-10 mM) could neither depolarize the membrane potential nor induce
zoospore encystment. This is in agreement with recent observations de-
scribing the lack of zoospore encystment following the application of the
ionophore A23187 at low external calcium concentrations; under these con-
ditions a Ca2+ ion efflux occurred [16]. However, at high Ca2+ concentra-
tions (> 25 mM), in the presence of the ionophore, we observed a membrane
potential depolarization similar to that induced by equivalent K+ concentra-
tions, but they were not effective in inducing synchronous encystment. This
depolarization may be caused by inhibition of a membraneebound ATPase by
Ca2+ ions, which influxed via A23187 in exchange for protons [17]. Indeed,
preliminary experiments with zoospore plasma membrane vesicles suggest that

a membrane potential can be established in the presence of ATP.

High concentrations (50-100 mM) of synthetic permeant cations (TPMP+ and
tetrabutylammonium.) depolarized the membrane potential (data not shown).
However, the cells treated with those synthetic cations lysed eventually,

possibly as a consequence of cytotoxic effects.
DISCUSSION

These results demonstrate that induction of zoospores by K+ ions is
accompanied by a rapid depolarization of the plasma membrane potential.
This partial depolarization is practically completed within the first
minute after application of the monovalent cation, and precedes observable

morphological alterations of the cell.

 

”an
-~"'

.5‘

en;

-33-

Mbreover, at later times after induction, our results correlate well with

morphological alterations observed [12, 18]: (a) A 50 mM KCl concentration

achieved optimal induction of synchronous encystment. This is consistent

with our findings that at the same concentration the rate of AF/F reached

a plateau value. (b) The fluorescence ratio AF/F became maximal within

a temperature range from 26°C to 35°C, where the synchrony of encystment

reached an optimum and was rather insensitive to temperature shifts. Below

15°C, AF/F decreased rapidly and the time span for 50% of the zoospores to

encyst increased considerably. Above 39°C, the vast majority of the cells ,5
neither germinated nor exhibited significant AF/F values. (c) Following K+
induction, both the initial rate of fluorescence change and the develop-
mental synchrony were not influenced by pH variations between pH 6 and 8.
Beyond this pH range, both kinds of parameters decreased in a parallel’
manner. (d) Zoospores, less than 15 hr old, showed similar AF/F responses
and developmental kinetics upon Rf induction. Still older cells lost
synchrony, some cellular destruction occurred, and the value of AF/F

decreased upon induction.

These physical and developmental responses can perhaps be elicited by
compounds other than K+ ions. Under certain conditions the encystment

process can be induced by reagents such as sulfonic acid azo dyes, MgClz,

CaCl NaCl, CsCl, and choline chloride [8, 12, 19]. The sulfonic acid azo

2’
dyes inhibit growth in the early germling stage. Whether these azo dyes

affect the membrane potential is unknown. Both MgCl and CaCl initiate

2 2
encystment but with less synchrony as KCl does, germ tube formation is
inhibited. These divalent cations modify the membrane potential much less

than K+ ions (fig. 2). However, NaCl, CsCl, and choline chloride generated

-39-

minor potential changes, and induced encystment but with less synchrony as
compared to that induced by K+ ions, under our experimental conditions.

On the other hand, it has been reported [12] that these three monovalent
cations induce encystment similarly as compared with K+ ions. Presently

we do not know the reason for that discrepancy.

Although both LiCl and KCl screen the surface charge (Jen and Haug, un-
published results) and affect concanavalin A binding to the cell surface
similarly [4], these salts display pronounced differences in regard to the
physiology of encystment and membrane depolarization. LiCl is a known
inhibitor; KCl is an inducer of encystment [12]. LiCl depolarizes the
trans-membrane potential slightly in contrast to KCl. Since LiCl did not
prevent the K+ induced depolarization (in contrast to quinine), we conclude

that LiCl acts at a later time in blocking the developmental process.

One of these subsequent processes might involve biochemical signals. In-
deed, the germination process was reported to depend upon the intracellular
cyclic AMP levels [20]. In spite of the fact that both cyclic AMP and
adenine induced germination [20], our preliminary experiments indicate that
neither compound caused a depolarization of the membrane potential. It is
therefore conceivable that membrane potential depolarization can be by-
passed in triggering the developmental process, provided that the secondary

biochemical signals can be manipulated directly.

If one takes into account the individual heterogeneity within a cell

population, then the K+ concentration-dependent changes of the membrane
+

potential (fig. 3) may be interpreted in at least two ways. First, K

ions primarily affect the rate of individual cell transformation, whereby

:-40-

individual cells may respond differently to the respective monovalent
cation concentration. This assumption might be consistent with the fact
that lectin binding characteristics of the cell surface vary appreciably
from cell to cell. However, upon treatment with K+ ions, the individual
heterogeneity is significantly reduced which results in a rather defined
synchrony of the cell population [4]. Moreover, heterogeneous membrane
potential responses to lectins were reported recently in lymphocytes [21].
Consequently we favor the following second interpretation as to the Ki
effect. Application of an optimal K+ ion concentration to a cell popu-
lation induced the highest percentage of cells to encyst per unit time,
hence the largest rate of fluorescence intensity change. Our results are
also in accord with the observation that cells, which undergo rapid

germination, apparently do so at the same rate [12].

As to the mechanism of K+ ion depolarization, we propose the following
hypothesis: Specific K] carriers‘ which permit passage of K+ ions across
the membrane are embedded in the mixed lipid state of the plasma membrane. The
K+ carriers are presumably proteins, perhaps different from those Ca2+
activated K+ channels reported elsewhere [13]. This hypothesis is sup-
ported by the following observations. l.) The developmental induction
process and the transmembrane potential depolarization are effected by
specific ions. In preliminary electron paramagnetic resonance experiments
with the amphiphilic surface charge sensitive spin probe CAle, we ruled
out the possibility of specific K+ surface charge screening effects (Jen
and Haug, unpublished). 2.) Without quinine, the K+ ion-induced depolar-
ization was not measurably affected by the presence or absence of valino-

+
mycin. Consequently the membrane may contain already existing K carriers.

 

-41-

This is further supported by the fact that quinine application resulted in
' +
a virtual block of K passage across the membrane. From transport studies

the existence of similar channels had been inferred in Neurospora and

 

animal cells [13, 22]. Moreover, B, emersonii zoospores accumulate K+
ions from the medium [8]. 3.) The existence of a gel + liquid crystalline
mixed lipid state between 40 and 35°C had been deduced from spin labelling
experiments on intact zoospores and isolated plasma membrane vesicles [23].
4.) The growth range of B, emersonii [18] is limited to that of the mixed
lipid state. 5.) The temperature-dependent changes of AF/F exhibit

discontinuities at 4°C and 35°C (fig. 4).

The K+ carrier structure is perhaps maintained by the specific bulk
lipid state, or a specific lipid.domain structure, or a specific physical
‘state of boundary lipids around the channel protein. Below 4°C, the B,
emersonii zoospore membrane is in the rigid lipid state and above 35°C
the membrane is in the liquid crystalline state. Both states are pre-
sumably not conducive to the proper carrier functioning. A somewhat
similar situation has been found for the outer membrane of B, ggli_which
contains pore proteins. This bacterium can only grow in a temperature
range where the cell can maintain the outer membrane lipids in a mixed

lipid state [24].

From the effect of a second K+ ion exposure (fig. 7) on the membrane poten-
tial in developing cells we conclude that a functional K? carrier-can only
exist during the zoospore stage. The disappearance of this carrier apparently
coincides with the commencement of amino acid incorporation in later devel—
opmental states [25]. Moreover, zoospores exposed to a K+ pulse for about

2 min, then resuspended in K+ -free sporulation medium, developed with the

-42-

same synchrony as those continuously exposed to K+ [12].Also in amphibian
oocytes the K+ channel in the plasma membrane can be turned off after a
certain time coinciding with long term membrane potential depolarization

and meiotic divisioanG]. These carriers may process environmental information
for cellular differentiation. Once the information has been transmitted,

the cells are triggered to develop and the carriers are no longer needed.

 

-43-

ACKNOWLEDGEMENT

We are indebted to Dr. A. S. Waggoner for a gift of the dye. We are
grateful for helpful discussions by Dr. J. L. Wang. The original strain
of the organism was provided by Dr. E. C. Cantino. We also gratefully
acknowledge the work by Mr. C. R. Caldwell in preparing the spin probe
CAle. This research was supported by Department of Energy contract No.

DE-AC02-76ER01338.

 

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14. Hoffman, J F & Laris, P C, J physiol 239 (1974) 315.
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acta 436 (1976) 475.
16. Comes, S L, Mennucci, L & Maia, J C C, Devel biol 77 (1980) 157.
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18. 8011, D R & Sonneborn D R, Devel biol 20 (1969) 218.
19. Truesdell, L C & Cantino, E C, Curr top devel biol 6 (1971) l.
20. Comes, S L, Mennucci, L & Maia, J C C, Cell differen 9 (1980) 169.
21. Shapiro, H M, Natale, P J & Kamentsky, L A, Proc natl acad sci USA
76 (1979) 5728.
22. Slayman, C L, Membrane transport in plants (eds Zimmerman, U &

-44-

Dainty, J) p 107 Springer—Verlag, Berlin (1974).

-45-

Leonards, K S & Haug, A, Biochim biophys acta (1980) in press.

Janoff, A S, Haug, A & McGroarty, E J, Biochim biophys acta 555
(1979) 56.

5011, D R & Sonneborn, D R, Proc natl acad sci USA 68 (1971) 459.

Merrill, G A & Ziegler, D, Devel biol 74 (1980) 216.

.7 .
5U

was
and

\-
{0

-45-

LEGENDS

Fig. 1. AbAcLAAa = time (min); Oldinate = fluorescence intensity

(arbitrary units)

Time course of fluorescence intensity at various concentrations

of the voltage sensitive dye diS-C3-(5). Prior to dye addition the
reaction mixture contained 5 x 106 zoospores/ml, 5 mM MOPS (pH6.7), and

1 mM CaClZ. The dye and KCl (SOmM) were added as indicated. Fluorescence
was recorded at 671 nm, with excitation at 622 nm. Experiments were

. 0
carried out at 22 C.

Fig. 2. Abécidba = time (min); Ohdinate = fluorescence intensity

(arbitrary units)

Time course of fluorescence intensity; the effect of various salts. 4 min
after injection of the dye (2.5 x 10-7M) various salts (SOmM) were added
to the same reaction mixture as given in fig. 1. Curve 1: KCl, KBr, KI,
and RbCl; curve 2: NH4C1; curve 3: NaCl, CsCl, choline chloride, CaClZ,
and MgClz. The symbols are defined as, F: the steady state fluorescence
intensity base line level before KCl addition; AF: the change of

fluorescence level following KCl addition; AF/F min—1: the initial slope

of the fluorescence change upon KCl addition.

Fig. 3. AbbcLAAa = K+ concentration (mM); ondLnaze = £851 AF/F (0/0) (0);

night initial rate (o/o min -1) (i3)

+ . -
K concentration dependent fluorescence inten51ty changes. The same

experimental conditions were maintained as in fig. 2.

_47-

Fig. 4. AbACiAAa = temperature (°C); ondinate = AF/F (%)

Temperature dependent fluorescence intensity changes upon KCl addition
(50mM). The same conditions were used as in fig. 2, except that the

reaction temperature was adjusted to the respective temperature.

Fig. 5. Abbcidéa 8 pH; ondinazc = (2251) AF/F (%) (a); (night) relative

baseline fluorescence intensity E = F(pH)/F6 7 ( U)

pH dependent fluorescence intensity changes upon KCl addition (50mM). The
same conditions were maintained as indicated in fig. 2, except that the
extracellular pH was adjusted to the desired value by adding 0.1N HCl or

NaOH to the reaction mixture.

Fig. 6. Abbcioda = time (min); ondznaze = fluorescence intensity

(arbitrary units).

The time course of fluorescence intensity; the effect of K+ (50mM),
valinomycin (luM) and quinine (lmM). Various reagents were added as
indicated. The same experimental conditions as in fig. 1 were maintained

prior to the application of reagents.

Fig. 7. AbAwéa a time (min); oxidx'na/te = (11252:) AF/F (0/0) (H);

(ughx) CpM (x1o‘3) (Du-U)

Flourescence intensity changes upon a second K] pulse (50mM) at the
respective time of cellular development. Zoospores were first induced by

a 50 mM KCl pulse at room temperature (t=0), and then resuspended into

-43-

KCl—free sporulation solution before fluorescence experiments were
performed. Abscissa indicated the time for development after initial KCl
induction, including 2 min wash and 4 min dye equilibration. The right
ordinate ([3) represents the cpm for the incorporation of 3H-leucin into
TCA precipitable material, (data adapted from [21]). Symbols for the
major cell types at the respective developmental time are indicated on

the top of the figure: o—, zoospore; 0, round cell;(fia, germling.

-u9-

 

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a

 

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3.:

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Figure 4.2

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Figure 4.4

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Figure 4.5

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Figure 4.6

-55-

 

 

 

 

 

 

Figure 4.7

10.

11.

12.
13.

14.
15.

16.

GENERAL REFERENCES

Aidley, D. J. (1971) 'The Physiology of Excitable
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E‘?‘

 

 

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