Protoplasma 97, 365--371 (1978) PROTOPLASMA

9 by Springer-Verlag 1978

D y n a m i c s of the E c t o p l a s m i c Walls D u r i n g Pulsation

o f P l a s m o d i a l V e i n s of Physarum polycephalum

Brief Report
A. GRI~BECKI"" and M. CIESLAWSKA

Department of Cell Biology, Nencki Institute of Experimental Biology, Warszawa, Poland

Received July 31, 1978

Accepted September 14, 1978

Summary
The thickness of ectoplasmic walls decreases at the contraction phase and increases at the
expansion phase of each pulsation cycle. In average, 29~ of material forming the walls of
expanded veins is evacuated during contraction with the endoplasm streaming.

1. Introduction
The dynamics of ectoplasmic walls, i.e., the fluctuation of their thickness in
the course of successive contraction-relaxation cycles of plasmodial veins,
remained until recently an unexplored question. The only information found
in the literature was that of WINER and Moor.r (1941) stating that the veins
pulsate "without, however, any observable change in the thickness of the
walls, and hence no change in the plasma-sol/plasma-gel relation", but it was
not supported by any experimental evidence. An empirical answer to this
question became particularly important at the moment when some authors
(e.g., BARANOWSKr 1976, GR~BrCKI and Moczo~ 1978) try to evaluate the
changing conditions of protoplasm flow through the internal channel basing
on fluctuations of the external dimensions of veins. The present study was
undertaken to measure simultaneously the fluctuations of the external and of
the internal diameter of veins pulsating in situ. In the meantime HOLSMANN
and WOHLFARTH-BoTTERMAN~ (1978) found by morphological methods, in
fixed and living material, that the walls are thinner in the contracted veins
than in the expanded ones.

2. Material and Methods

P1asmodia of Pbysarum polycephalmn were investigated in situ, the next day after being
transferred from stock cultures on 2 o non-nutritive agar gel substrate. The Petri dishes with
migrating plasmodia were placed upside-down, on the microscope stage. In this inverted
24 Protoplasma97/4

0033-183X/78/0097/0365/$ 01.40
366 A. GRF,BZCKIand M. CIE~LAWSKA

position plasmodia migrate normally, and the fact that the light beam meets first the plas-
modial strand on its way, creates optical conditions particularly favourable for dark field
observations. When the dark field illumination was produced by a non-conventlonal use of
the variable phase contrast optics (KFZ system of PZO) the endoplasm-ectoplasm interface
became visible as a well contrasted bright borderline. This effect was obtained by applying
the "20 • phase ring with the 5 • lens, or the "40 X" phase ring with the 10 • lens, the
polarizer being set to the negative contrast position. In this way not only the dynamics of
external diameter of vein could be cinematographically recorded, but also the changes of its
internal diameter (the channel's lumen). At least 500 time-lapse pictures were taken at 1 sec-
ond intervals, for each vein. The pulsation curves were produced in the course of film
projections, by means of the slit te&nique described elsewhere (CI~LAXeSKAand GI~BECKI
1978, and GI~gB~CKIand MoczoN 1978).

3. R e s u l t s

An example of typical picture presented, under the optical conditions

described above, b y a vein f r o m the network of migrating plasmodium is
given in Fig. 1. The six film frames were selected to show three successive
m a x i m a and minima of the contraction-relaxation cycles. The inner and outer
outlines of the ectoplasmic walls are distinct. The s-s line in Fig. 1 a indicates
the position of transversal slit in the screen, serving to plot the curve which
reflects the changes of vein's internal and external dimensions in the course of
pulsation. The pulsation curve of this vein is presented in Fig. 2.
It is clearly seen that the rhythmic changes of the external and internal
diameters manifest exactly the same frequency, but there is a clear difference
in amplitudes between them: the fluctuations of the internal diameter are
relatively tess pronounced than those of the external one. I t aliows to state,
as a preliminary conclusion, that the ectoplasmic wall thi&ens during ex-
pansion of the vein, and becomes thinner at the contraction phase.
The Fig. 3 presents second example of pulsation curve, recorded in another
vein. In spite of certain a s y m m e t r y of this vein, the same pattern of dynamics
of the wall m a y be seen, at least on the left side of the channel.
The internal and external diameters were measured f r o m pulsation curves
produced for several veins. The data obtained in this w a y served to construct
diagrams presenting the thi&ness of walls and the size of &annel's lumen at
the m a x i m a and minima of successive pulsation cycles. One of the diagrams
is shown in Fig. 4. The separate graphic presentation of dimensions of the

Fig. 1. Plasmodial vein shown at three successive maxima (a, c, e) and minima (b, d, f) of its
pulsation cycle. Note the bright line separating the endoplasm and the ectoplasm
Fig. 2. Pulsation curve obtained from the same vein through the slit s-s marked in Fig. 1 a.
Note the periodic changes of thickness of the walls, coinciding with the fluctuations of the
external diameter of the vein and of its lumen size. The magnification bar applies to the
Figs. 1, 2, and 3, and the time scale to the Figs. 2 and 3
Fig. 3. Another example of pulsation curve demonstrating the periodical changes of walls
thickness according in phase with the pulsation rhythm of the vein
 Dynamics of the Ectoplasmic Walls During Pulsation of Plasmodial Veins 367

Figs. 1-3

24*
368 A. GRIiB~CKIand M. CIESLAWSKA

walls and of the lumen visualizes better the differences between the wall's
thickness at the contracted and at the expanded state of the vein.
The quantitative data collected in this manner were used to estimate the
volume changes of the wall material, as well as the changes of space left
free for the flowing protoplasm inside the central channel, during an average
contraction-relaxation cycle. The calculations were limited to the upper part
of vein, which is more regular in shape than the vein's basis and may be
assumed to be approximately hemi-cylindrical. The mean values of dimensions

wall lumen wall

ilili!iiiii!iii!iiiiiii;iiiililililililiiiii!ililililill
iiiiiiiiiiiiiilNiiiiill i:: :: ": :::::::~: i:iH ~!i~iiiiii!
:!: : : : : : !: i: :: : : : 9

|~HH~iHiliH|~
Niiiiiiiiiiiiiiiili!iiiiiii!iliiiiiiiiili!iiiiiiilililililililil
iiiiiiiiiiilliiiiiiiiiii
!iiiilililili!ilil!iii!i!iiiiliiii!iiiililiiii!lililililil
iiiiiiiii!!iiii!iiiii!il
!iiii!iiiiililiiiiii
!!Hiii!iiiliiiiiiiil iiiiilili!i!]ii!i!ili!i!iii!ili!iiiiiill
ii!iiiiiiiiliiiiiliiii!i
i!ili!ililiii!ililili!i!ili!ililili!i!i!i!i!i!i!i!i!ililil
iiiiiiiiiii!iiiililHi!l
!ii!ililiiiiiiiii!i
iilliiilHHiii!!!i iiii!ilililili;iiiili!i!iii!i!ili
!iiiiiii!iiiiiiiliiiiill
!ili!ililili!i!i!i!ili!ili!i!ilililili!ililili
i!iiillii!iiiiiiiiiiiii!
iiiiiii!iiiiiiiil iiiilililili;iiiiiiiili!ili! iiiiiiiiiiiiiH!i
iiiiiiiiiiii!iiiiii
~ i!i;ii!i!ililililili!ilililililililili!i!ililili!i!
iiiiiiii!iiiiiiiiii
iiiiiiiiiiiiiii
iiiiiiiiiiiiiiii !ilili!iiiiiii!iiiiiiililililililil
iiiiiiiiiiiiiiiiiiii
iiiHiiiiiiiliiiiii!!!ilili!i!ililili!iiiii!iiiiiiiiiliiili!ili!i!i
iiii!iiiiiiiiiil iliiiiiiilili!i!i!i!i!ililili i!iiiiiiiiiiill
50 ~ m

Fig. 4. Diagrammatic presentation of the thickness of vein's walls and of the lumen diameter
of the endoplasmic channel, at the maxima and at the minima of six successive pulsation
cycles

were calculated from the data concerning all the pulsation cycles recorded
in all the veins studied. The mean external and internal dimensions of ex-
panded and contracted vein, are schematically shown in cross section in Fig. 5.
In the same diagram the information is given to what extent the wall material
and the volume of flowing protoplasm are reduced during an average con-
traction.

4. D i s c u s s i o n
The present results contradict the opinion of WINEr, and MOORE (1941) that
the thickness of walls of plasmodial veins remains unchanged during the
contraction-relaxation cycle. On the contrary, the quantitative data obtained
 Dynamics of the Ectoplasmic Walls During Pulsation of Plasmodial Veins 369

in this study by investigating a number of successive pulsation cycles of the

same vein, are consistent with the conclusions drawn by H/2LSMANN and
WOHLFAi~TH-BoTTEt~MANN(1978) from visual estimations of wall thickness in
different veins fixed at different stages of their contraction-relaxation cycles.
In our material, the contracting veins were found to lose ca. 300/0 of their
volume. There is no doubt that the material transferred, at the contraction
phase, from the investigated site toward other regions of plasmodium, origi-
nated as well from the walls as from the stream. The loss in the wall material
is only slightly inferior (ca. 29%) to the loss of the flowing protoplasm
volume (ca. 35~ The morphological and cinematographic results of
I-IOtsMANN and WOHLI~ar,TI~-BoTTEV,Mt~NN (1978) explain the mechanism of
this phenomenon: during contraction a considerable amount of fluid proto-

Fig. 5. Diagram summarizing the quantitative results of this study. The upper part of vein,
assumed to be hemi-cylindrical, is shown in cross section, at the stages of its maximal expan-
sion (left) and maximal contraction (right). In both situations the total dimensions of the vein,
the thickness of its walls, and the size of its lumen, correspond to the mean values obtained
from analysis of nearly 50 studied cycles of contraction-relaxation. The figures show the
relative decrease of the ectoplasm and the endoplasm volumes, and of the total volume of
vein, in the course of an average contraction

plasm is squeezed, in form of streamlets, from the cavities present inside the
ectoplasmic walls of veins, and it joins the main stream in the central channel.
Such pockets inside the walls were earlier noticed by STEWART and STEWART
(1959) and by JA~IN (1964). The periodical evacuation of protoplasm from
them was also seen in the present material.
HOLSMANN and WOHLFt~I~TH-BoTTer,~aNN (1978) distinguish two types of
veins differing by the thickness of their walls (and by their position in plas-
modium). In the thin veins with relatively very thick walls the lumen of the
channel may not change at all during pulsation, all the observable contraction
effects being limited to the walls. In the present study the distinction between
these two types of veins was not made, however, a few cases of veins were
found in which the decrease of wall thickness during contraction was so
important that the lumen of channel was even slightly increasing, instead to
diminish, at the minima of pulsation cycles. But in the great majority of cases
the external and the internal diameters decrease in phase, although the
amplitudes of their changes are usually not proportional one in respect to the
370 A. Gt~3rclii and M, C*E~LAXeSKA

other. Therefore, it should be concluded that, in general, the measurements

of the external diameter of a vein may serve to an approximative evaluation
of the rhythmic changes of the conditions of flow through the internal channel
(BARANOWS~I 1976, GR~I3ECKI and MoczoIq 1978), but they do not permit to
describe them in the precise quantitative terms.
Without the decrease of walls thickness the ectoplasm-endoplasm ratio in
piasmodial veins would drastically increase during contraction (in spite of
the geometrically erroneous statement of WIN~I~ and MooR~ 1941 cited
above). It was found in the present study that, in fact, this increase is almost
insignificant: 78.50/0 of volume is occupied by the ectoplasm in the expanded
state of veins, and 800/0 at their contracted condition. This calculation is,
of course, based on the presumption that the circulation of protoplasm be-
tween the central channel and the cavities inside the walls is related to the
ectoplasm-endoplasm interconversion cycle.
The fact that during contraction the ectoplasmic walls lose in average 290/0
of their volume, and that this materiai is evacuated to other regions in the
form of endoplasm, is obviously related to the problem of the "sol-gel cycle"
in Physarurn. It has been already postulated by JmtN (1964) that "active
contraction is necessarily followed by solation". More recently WOH~FAr,TI~-
BOTTERMAIqN and FLEISCHER (1976) and ISENBERO and WOtJLVARTi-I-BoTTER-
MANN (1976) demonstrated the assembly-disassembly cycle of actin filaments
in Pbysarurn what is consistent with the view that a part of the ectoplasmic
material is converted to the more fluid endoplasmic state during each con-
traction. It is certainly related to the decrease in volume of the walls as
described in this study. The present results seem to indicate that in plas-
modium the ectoplasm-endoplasm interconversion takes place not only at
the growing front and in the withdrawing ends of the terminal veins, but also
all along the veins system, in a cyclic manner strictly following the con-
traction-relaxation phases.

References
BARANOWSKI,Z., 1976: Three-dimensional analysis of movement in Physarum polycepbalurn
plasmodia. Cytobiologie 13, 118--131.
CI~LAXVSKA, M., GR~CKI, A., I978: Contraction-expansion rhythms simultaneously ob-
served in two sites of Pbysarurnpolycephalum plasmodium. Acta Protozool. 17, in press.
GR~B~CKI, A., MoczoN, M., 1978: Correlation of contractile activity and of streaming
direction between branching veins of Physarum polycephalum plasmodium. Protoplasma
97, 153--164.
HOLSMANN,iNT.,WOHLFARTH-BOTTERMANN,K. E., 1978: Spatio-temporal relationships between
protoplasmic streaming and contraction activities in plasmodial veins of Pbysarum poly-
cephalum. Cytobiologie 17, 317--334.
ISrNB~RG, G., WO~L~AI~T~-BoTT~I~MANN,K.E., 1976: Transformation of cytoplasmic actin.
Importance for the organisation of the contractile gel reticulum and the contraction-
relaxation cycle of cytoplasmic actomyosin. Ceil Tiss. Res. 173, 495--528.
 Dynamics of the Ectoplasmic Walls During Pulsation of Plasmodial Veins 371

JAHN, T.L., 1964: Protoplasmic flow in the mycetozoan, Physarurn. II. The mechanism of
flow; a re-evaluation of the contraction-hydraulic theory and of the diffusion drag
hypothesis. Biorheology 2, 133--152.
STEWART, P, A., STEWART,B.T., 1959: Protoplasmic movement in slime mold plasmodia: the
diffusion drag force hypothesis. Exp. Cell Res. 17, 44--58.
WINER, B.J., MOORE, A.R., 1941: Reactions of the plasmodium Physarum polycephalum to
physico-chemical changes in the environment. Biodynamica 3, 323--345.
WOHLFARTH-BoTT~RMANN,K.E., FLEISCHER,M., 1976: Cyclic aggregation patterns of cyto-
plasmic F-actin coordinated with oscillating tension force generation. Cell Tiss. Res. 165,
327--344.