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A study by electron microscopy of the formation of new surface by Chaos chaos
Experimental
Cell Research
A STUDY FORMATION
43, 583-601
(1966)
BY ELECTRON MICROSCOPY OF THE OF NEW SURFACE BY CHAOS CHAOS1-” VIVIANNE
Department
583
T.
NACHMIAS
of Biology, Hauerford College, Haverford, Pa., U.S.A. Received
March
10, 1966
THE formation of new cell surface, i.e., surface membrane plus attached coat material, by animal cells is not only a fundamental process of growth but occurs as well during discontinuous events such as cell division and secretion [24, 26, 28, 291. Yet relatively little is known about the mechanisms involved at either the morphological or chemical level. Amebae are potentially useful objects for the study of new surface formation. They can be used for a variety of experimental techniques, and they form new cell surface readily under several conditions at different rates. During optimal growth it has recently been estimated [lo] that up to 40-50 per cent of the cell surface of Chaos chaos may be taken up per hour by phagocytosis. Since replacement of membrane must, under such conditions, occur at a comparable rate, one can estimate a half-life for the surface of 1 hr to 14 hr. In studies of ameboid movement, when the organism is not feeding, there has been considerable controversy about the rate of turnover of the cell membrane in short term experiments [13, 14, 16, 201. Recently, an overall estimate of rate in Amoeba proteus has been provided by Wolpert and O’Neill [30]. In starving, moving amebae labelled with fluorescent antibody to the surface, they found a halflife for the surface of about 5 hr. In a totally different situation, amebae, upon return to atmospheric pressure after exposure to high pressures, apparently form some cell surface very rapidly [17, 181 although its extent is not known. These various observations suggest that the rate of new surface formation in amebae depends on external conditions and can probably be controlled experimentally. If so, preparations which form new surface rapidly could be used for studies of the process. This report describes a method for inducing rapid formation of new surface by Chaos chaos and presents observations on the process by light and 1 The work reported here was done at the laboratory of Dr J. 31. Marshall, in the Department of Anatomy, University of Pennsylvania, Philadelphia, and was supported by Grant So. CA01957 from the U.S. Public Health Service. z These findings were briefly reported at the 5th annual meeting of the American Society for Cell Biology (J. Cell Biol. 27, 71A (1965)). Experimental
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electron microscopy. The method consists in exposing pre-chilled amebae to a dye (Alcian Blue) which is irreversibly bound to the surface coat [6, 151 and has already been used by Chapman-Andresen [8] to study surface renewal in A. proteus. After rinsing and return to room temperature the new surface is seen as clear areas while dye-treated areas are deep blue. Under these special conditions it was found that: (1) this ameba can form large amounts of new surface in a short time-a half-life of as little as 20 min is estimatedbut cannot immediately repeat the process; (2) recently formed new surface is made up of all the morphological layers of the original surface, i.e., there is no evidence for the intermediate formation of a naked plasmalemma; (3) surface coat and plasmalemma move together when displaced by new surface in these experiments as they do in the early stages of pinocytosis [4, 15, 231; (4) new surface may be derived by interpolation of ground cytoplasm into the surface rather than from a fusion of pre-existing vacuoles with the surface, as appears to occur in secretory cells [24, 26j.
MATERIAL
AND
METHODS
Culture and preparation of amebae.-Chaos chaos (Pelomyxa carolinensis) were fed daily on concentrated suspensionsof Paramecium aurelia. The details of the culture have been described [lo]. Well-fed or briefly-starved (a few hr) individuals were used. They were rounded up by gentle shaking and were rinsed in several changesof ionfree water followed by several changesof medium. The medium contained 5 x lo-* M CaCl,, 5 x 1O-5M MgSO,, 1.6 x 1OV M K,HPO,, 1.1 x lo-* M KH,PO,. They were washed twice again in 5°C medium and stored at 5°C for l-5 hr before use. Bye treatment.-From the chilled amebae roughly spherical individuals of intermediate sizes were chosen. The amebae were rinsed in cold ion-free water, exposed at 5°C to dilute (0.0125 per cent to 0.1 per cent) Alcian Blue1 solutions for 2 min with constant gentle shaking, and then rinsed in several changesof ice-cold water followed by cold medium. These steps were carried out in siliconed dishesto prevent amebae from sticking to the glasssurfaces. Individual ameabae were fixed directly in cold medium, or were transferred to a large volume of medium at room temperature and fixed after 3 to 35 min. All steps were carried out under direct observations with a dissecting microscope. The Alcian Blue dye for most of the experiments was the SGN (Hartman-Leddon Co., Philadelphia) used without purification but merely filtered before use. Some experiments were also done with the SGX (Fisher) used after recrystallization, as described by Scott, Quintarelli and Dellovo [27]. Amebae behaved similarly with both dye samples. In one experiment, amebaewhich had been allowed to warm up and resume movement were exposed to 0.001 per cent toluidine blue. 1 Alcian Blue was introduced induce pinocytosis in amebae
Experimental
Cell Research 43
for staining acid mucopolysaccharides by Chapman-Andresen [6].
[26] and was first
used to
Surface formation
in Chaos by electron microscopy
5%
Fixation.-For most experiments amebae were fixed in unbuffered osmium tetroxide (2 per cent, pH about 6), followed by an equal mixture of 2 per cent uranyl acetate and 2 per cent osmium tetroxide for IO min more. This fixation has been found to preserve the filamentous layer of the cell coat uniformly and densely [22]. Experiments were also performed using osmium tetroxide buffered with Verona1 acetate to pH 8.6 [25] or using 2 per cent glutaraldehyde (Union Carbide) buffered to pH 6 with 0.04 M potassium phosphate and followed by post-fixation with either unbuffered osmium tetroxide or the alkaline osmium tetroxide solution. After fixation for 10 min at room temperature, amebae were rinsed for one min in ion-free water, dehydrated in alcohols, and embedded in araldite. Any amebae exhibiting marked changes in form or prolonged streaming during fixation were discarded. Such effects were common during glutaraldehyde fixation, rare when osmium was used. In the polymerized resin blocks, pseudopodial or uroid (tail) regions of the embedded amebae were identified by comparison with drawings made of the specimens at the time of fixation. Sections of the regions desired were made and mounted on uncoated or collodion-carbon coated grids and were examined with an RCA EMU-3 electron microscope. Sections fixed in osmium or glutaraldehyde and osmium were stained with 2 per cent phosphotungstic acid in 95 per cent ethanol for two min. Quantitative study.-In the electron micrographs of dye-treated amebae, the surface filaments are replaced by clumps occurring at irregular intervals (Fig. 2). The average frequency of occurrence of such clumps in the photomicrographs was determined under three pairs of conditions: (1) counts of clump frequency were made along profiles of the anterior surface of an ameba which had formed a new pseudopod and compared with the frequency found near the posterior end of the same ameba (Table I, Comparison 1); (2) counts of clump frequency were made along surfaces of two amebae fixed before the formation of new surface and compared with the frequency along the tail region in two other specimens fixed 25 min after formation of new surface had begun (Table I, Comparison 2); (3) the frequency of clumps was determined along the surface of an ameba fixed before any movement had occurred and compared with the frequency along surface actually taken up in the uroid of another specimen fixed 25 min after the formation of new surface had begun (Table I, Comparison 3). In all cases negatives were made at x 7400f 7 per cent over a period of several months. For a given comparison, all negatives were made on the same day and frequently on the same negative plate (5 negatives to the plate). Enlargements of x 4 were made of the negatives, and counts were made on the prints. For the counts, only surfaces cut approximately in cross section were used. A map measure was used to determine the length of each profile of the plasmalemma; lengths were from lo-70cm. The precision of these measurements was to 1 cm or better. A total of 23 profiles was counted for Comparison 1; 27 for 2; and IO for 3. Clumps were counted, using a constant set of criteria. The reasoning was as follows: clumps seen on unfixed Alcian Blue-treated surfaces, picked up directly onto grids, vary in size but are of quite uniform density; therefore, it was assumed that very faint clumps appearing in sections must be tangential cuts through clumps. Hence, a given clump should be counted only in a given section if its density is judged to be at least half that of the majority of clumps in the print. This precaution is rough, at best, since it is a visual estimate; but Experimental
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it was taken, since it was possible that on some grids adjacent sections might be used for counts. In addition, only clumps adjacent to the underlying continuous zone of the surface coat were included; a clump separated from the surface was judged to “belong” to another area of plasmalemma and to appear isolated from it due to a tangential cut across the surface. Such clumps were rare, as might be expected, where there was a ‘I’al~r.~ I. Comparison of frequency of clumps formed by the interaction and surface coat under direrent conditions. Final
magnification
of
dye
> x 29,600.
1: Ameba, 3 min after formation of new pseudopod. I%-equency along anterior
Comparison (smooth)
and
posterior
Ant: N = 11 post: N = 12
0.963 1.070
(convoluted) clumps/cm clumps/cm
surfaces. mean mean
Standard t -= 1
error
of mean
diff:
Comparison 2: Amebae fixed before formation of new surface (two specimens, C), with amebae fixed 25 min after formation of new surface (two specimens, W). Counts on the latter made on surface still external. Both surfaces convoluted. c: s = 15 w: X-12 Nole that amebae.
0.994 1.004
the counts
Comparison 3: Amebae formation of surface e.g., Figs. 6 and 7. c: 5=5 w: x=5
clumps/cm clumps/cm
refer
fixed (IV).
mean mean
to frequency
before Counts
1.013 clumps/cm I .050 clumps/cm
Standard f
final
error
prints
of mean
and
diff:
not to original
dimensions
of the
formation of new surface (C) with amebae fixed 24 min after on the latter made on surface actually taken up in the uroid mean mean
Difference comparing
in variabilily, however, the Iwo populations
precludes
good cross section as judged by the appearance of two dense lines in the plasmalemma. Using these two criteria for the counts, duplicate observations were good to 2-3 clumps. This variation is a measure of the difficulty in deciding whether to include or exclude a given clump on the borderline of either or both criteria. For example, in Fig. 2 the surface lying between A and A’ yields a count of 16 or 17 clumps. Fig.
l.-Normal cell, fixed with unbuffered and uranyl acetate. Note the clear distinction the continuous layer (C), and the underlying x 69,000.
osmium tetroxide followed by a mixture of osmium between the filamentous layer of the surface (F), plasmalemma (P), composed of two dense lines.
Fig. 2.-Cell treated with Alcian Blue SGN in the cold, rinsed and fixed in the same manner as Fig. 1 before movement occurred. Note that the dense clumps along the surface (G) are separated from the plasmalemma by a narrow space (S). The total count of clumps from point A to A’ along the circumference is 16 or 17 (see Methods). x 22,100. Experimental
Cell Kesearch
43
Surface formation
in Chaos by electron microscopy
587
1
G
2 Experimental
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T. Nachmias
On some of the prints used for Comparison 2, the diameters of the clumps were also estimated by measuring at the widest point and parallel to the profile of the plasmalemma. The means are shown in Table II. All the measurements except those of the perimeters were made by observers who did not know what, if any, comparison was being made. II. Mean diameters
TABLE
of clumps on prints
Wa Print no.
Mean diameter (mm)
2a la 5a 1
from comparison
2.
Cb No. of clumps
Print no.
Mean diameter (mm)
No. of clumps
42 10 33 36
6a 6a 7 7 8 11
3.25 3.6 3.5 3.3 3.5 2.9
35 10 19 14 13 22
4.6 4.7 3.9 2.9 x=121
x=123 a Weighted ’ Weighted
mean mean
(overall (overall
mean): mean):
3.14 3.04
mm/clump. mm/clump.
RESULTS Direct observations When amebae were prepared and selected as described above, and then exposed to dilute solutions of dye in the cold, they behaved in a reproducible way. They first became very sticky. If allowed to congregate, they adhered to one another and formed clumps. They also adhered strongly to glass surfaces that were not silicone treated. After rinsing, while still at 5”C, the entire surface appeared stained blue. Although no undyed areas were visible with the dissecting microscope, one end of the ameba frequently appeared to be more deeply stained; this seemed to be due to the persistence of convoluted surface. When the cold amebae were pipetted into a drop of medium at room temperature, a clear unstained pseudopod was formed in almost all specimens in 6-7 min when dilutions of 0.025 per cent dye or less were used. After higher concentrations of dye, the rate was slower, e.g., after 0.1 per cent dye amebae remained spherical for lo-15 min and cleared very slowly. Exposure to dye at either acid or neutral pH with or without added buffer resulted in the same rates of clearing. Text-Fig. 1 diagrams the appearance of an ameba at each stage of clearing (arbitrarily called “clearing”, “monopodal”, and “polypodal”). When 0.025 per cent dye was used, clearing was first seen in three min or less. Ten Experimenfal
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Surface formation
in Chaos by electron
microscopy
589
min after warming, over half the amehae had reached the monopodal stage; and by 35 min all viable amebae were polypodal. At this point the several pseudopods appeared clear while at the tail or uroid there was a single very dense spot of dye. Streaming was vigorous at this stage, and amebae reaching the polypodal stage without exception moved about normally, fed, and evenually defecated the dye.
s
c
Text-Fig. l.-Diagram of form to room temperature. x, IP, monopodal: PP, polypodal.
II’
PP
changes in Chaos chaos coated with Alcian Blue (cold) dye-coated surface; e, new surface. S, spherical;
after return C, clearing:
In one experiment, amebae were rounded up again after reaching the polypodal stage, re-chilled for 45 min and re-exposed to dye. After this second cycle, all five were still spherical 35 min after warming and produced new surface only slowly thereafter. When treated amebae at the polypodal stage were put together with untreated amebae into a 0.001 per cent solution of toluidine blue, metachromatic staining of the surfaces of both untreated and dye-treated amebae was immediate. Staining was both more intense and more variable over parts of the surface of the untreated amebae, but the new surface of the Alcian Bluetreated amebae stained as intensely as the anterior parts of the pseudopods of untreated amebae. Electron
microscope
obseruations
After fixation in unbuffered osmium tetroxide, followed by the uranyl/ osmium mixture, the surface of normal amebae appears as in Fig. 1. Note that the amorphous and filamentous layers of the surface coat first described by Brandt and Pappas [4] are clearly defined. When an ameba is fixed in the same way after treatment with dye, the surface appears as in Fig. 2. The filamentous layer is replaced by clumps of dense material about 100 rnp in diameter, occurring at irregular intervals and separated from the plasmalemma by a clear zone, whose width, 150-200 hi, corresponds to the width of the amorphous layer in Fig. 1. Sometimes this clear zone appears faintly stained, but it was never as dense as in untreated amebae. The clumping of Experimental
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the filamentous zone was found on both smooth and convoluted regions of surface of treated amebae. This effect of Alcian Blue has been reported by Hayward [15] in a study of pinocytotic uptake following treatment with this dye. Clumps of dense material are also seen when the cell coats from dyetreated ruptured amebae are picked up directly onto coated grids and examined without fixation. In Fig. 3 is shown a section of the anterior part of an ameba several min after treatment with 0.05 per cent dye. Note the scarcity of vacuoles in the underlying cytoplasm, the smooth contour of the plasmalemma, and the occurrence of several clumps which appear to have filaments passing through them. Fig. 4 shobvs a higher power view from another ameba near the base of a pseudopod fixed ‘L-3 min after warming, following treatment with 0.02.5 per cent dye. The surface here is convoluted and both clumped and filamentous surface are present. In addition there is a transitional zone (Tr) lying between clumped and filamentous surfaces. The clumps appear progressively narrower and more elongated until they grade into filaments. Fig. 5 shows another example of an area of new surface formation 3-4 min after bringing to room temperature. Since the filaments on the new surfaces appeared less dense than those on control, untreated amebae (Fig. l), some untreated amebae were fixed in the same drop of rinse medium as the dye-treated amebae. The surface filaments on such controls appeared identical with those on new surface (Fig. .5 insert). The results with other fixatives were studied less intensively but the occurrence of new surface, transitional zones, and scarcity of vacuoles in cytoplasm were all confirmed. The appearance of the outer layer varies with the fixation, and when the unbuffered osmium solutions were used, the normal surface appeared clumped [X2] so that it was difficult to distinguish old and new surface. Altogether, 14 amebae were examined for new surface. Figs. 6 and 7 are sections from the tail regions of an ameba which had been at room temperature for 25 min after dye treatment. Masses of membra-
Fig. 3.-Ameba treated with Alcian Blue 0.05 per cent and fixed several minutes after return to room temperature, while at the “clearing” stage. Note that filaments are present up to the junction with clumped surface, while some clumps appear to have filaments passing through them. Note also the scarcity of vesicular structures in the cytoplasm. x 17,000. Fig. 4.-Ameba treated with Alcian Blue 0.025 per cent, fixed 2-3 min after return to room temperature. Large areas of apparently clear surface were visible by light microscopy. This section was taken near the base of a pseudopod. Note the filamentous layer (F) like that of Fig. 5, inset, in the lower part of the photomicrograph, and the dense clumps (G), like those of Fig. 2, in the uXpf;:;;lf. Note also the occurrence of zones intermediate in morphology between F and G (Tr). > . Experimental
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Surface formation
in Chaos by electron microscopy
Experimental
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nes have been taken up into the interior, most of them still associated dense surface clumps. In some areas clumps are no longer associated the membrane but are replaced by a larger dense mass in the center space (arrows, Fig. 6). In Fig. 7 it can be seen that in many places the plasmic surfaces of the plasmalemma have become so closely apposed they appear as a double line (arrows, G, Fig. 7). Quantitative
with with of a cytothat
observations
Counts were made of the frequency of clumps on the photomicrographs under three pairs of conditions, as described in the Material and Methods Section, and the results are summarized in Table I. A statistically significant difference in the frequency of clumps was not found between any of the three pairs. In Comparison 1, clump frequencies along anterior and posterior parts of the same ameba were compared. A difference might appear if new surface were formed by the sweeping back towards the tail of “old” surface coat without the concomitant movement of the plasmalemma. If that occurred, the clumps lvould have to come closer together as the surface was moved towards the tail in order to accommodate the same amount of surface coat on less than the original amount of plasmalemma. The same arguments apply to Comparisons 2 and 3 which tested the same possibility under conditions where an even greater difference would be expected. After 25 min at room temperature it was not easy to find sections even near the uroid sho\ving clumped surface; typically, masses of membrane and clumps \vere found such as are shown in Figs. 6 and 7. This finding supports the visual impression that the surface is almost entirely renewed after about half an hour under the present conditions. Table 11 shows the mean diameter of clumps from Comparison 2, and the number of clumps counted for each mean. When a weighted mean is obtained, one finds an average diameter (on the print) of 3.14 for the warm amebae (25 min later) and 3.04 for the cold amebae (before new surface formation).
Fig. 5.-Ameba treated as in Fig. 4. Again, note the intermediate zone (Tr). Note the presence of granular material just under the plasmalemma in many places. The large vesicle (II) is similar in appearance to vesicles resulting from uptake of material. The insert shows the surface of a normal cell fixed as in Fig. 1 but in medium with several cells previously fixed with Alcian Blue. This photomicrograph resembles the appearance of the surface after alkaline osmium fixation. Both x 44,100. Experimenfcd
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Surface formation
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in Chaos by electron microscopy
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DISCUSSION
Observations by light microscopy The observations show that under the present conditions Chaos chnos can form an extensive amount of surface rapidly but cannot immediately repeat the process. This implies that a well-fed ameba has a considerable pool of precursor surface material available, but that this or some essential component is used up in the burst of formation that occurs after warming. The amount of surface produced is probably something less than a complete new surface in 35 min. This is concluded from the results of the toluidine blue experiment which shows that the new surface stained only as intensely as anterior parts of pseudopods of control amebae. Such anterior parts have been found by electron microscopy to have few convolutions, while the uroid normally has many. Probably the new surface is altogether less convoluted than the original surface and thus occupies less total area. The occurrence of a latency period after one ingestion cycle is in agreement with the results of Chapman-Andresen 17, 81. She reported on a quantitative study of channel formation during pinocytosis, and found that in A. proteus, a latency period occurs after one complete pinocytotic cycle. She suggested that this delay might be due to the necessity for the resynthesis of new surface. Although those experiments and the ones reported here are not completely comparable, it appears that Chaos chaos may have a larger store of surface precursor material than A. proteus, possibly due to a larger volume/surface ratio. Experiments designed to compare the rates of surface renewel of species with different volume/surface ratios would be interesting. Observe tions by electron microscopy The new surface possessesall the morphologically distinguishable layers of untreated ameba surface. Even in the very earliest samples, 2-3 min after return to room temperature, no areas of uncoated plasmalemma were found.
Fig. B.-Section through the uroid of a cell treated with dye and allowed The tail region was stained deeply blue at this time. The microphotograph area containing masses of convoluted membrane, with dense clumps Arrows show areas where clumps are no longer attached to the surface center. x 8100. Fig. 7.-A similar view, at higher magnification. quently apposed very closely to one another between them, and clumps (G) line both sides. cellular. x 29,400. Experimental
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43
to warm up for 25 min. shows part of a large attached to each side. but are collected in the
Note that two layers of plasmalemma (arrows) so that essentially no cytoplasm This entire mass of membranous material
are freis left is intra-
Surface formation
in Chaos by electron microscopy
Experimental
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It appears, therefore, that amebae form coat material and plasmalemma at the same time under the present conditions, since the quantitative results (see below) shorn that new plasmalemma is indeed formed. Whether this is always true, even for amebae, is not clear. In leukocytes, Basic and Gasic [12] in a brief report stated that restoration of staining properties due to cell coat materials took place in 1 hr after removal of the polysaccharide layer with enzymes. In their study it was not clear whether secretion of coat material alone was involved, or formation of plasmalemma also. A similar study with amebae would be interesting in order to determine whether secretion of coat material through the plasmalemma can occur. This has been suggested [16]. In some secretory cells, new surface appears to be added by the fusion of vesicles with the surface [24, 261. If such were the case in the present experiments, one would expect an appearance in the electron micrographs like that of Mechanism A in Text-Fig. 2. If, on the other hand, material were fed into the surface in some continuous manner, in units smaller than the size of an individual clump, a result like that of Mechanism B would be expected. In fact, the results here are like those of R. The model B differs in two respects from A: the material is fed in continuously, and there is a zone of interaction between old and new surface. One cannot entirely rule out the possibility that the transition zones are due to diffusion of dye from clumped material to neighboring filamentous (new) surface despite the apparently irreversible nature of the binding [S]. If it were so, though, it would presumably be very rapid and it would be surprising that in some cases (Fig. 3) clumps occurred with filaments passing through them. The fact that vesicles are rare in the cytoplasm underlying new surface can be used to argue for or against their
Text-Fig. P.-Possible containing precursors plasmalemma). Experimkntal
mechanisms of surface
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43
for formation coat. Rottom:
of new surface. Top: Mechanism A; P, vesicles Mechanism B; P,, precursor material (coat plus
Surface formation
597
in Chaos by electron microscopy
participation; here, one must distinguish between vesicles that are like those resulting from uptake occurring when amebae are shaken and chilled, and vesicles which might be a source for new surface. Although our results suggest that ground cytoplasm is the source of the new surface, other methods, e.g., use of labelled precursor material, must be tried before a definite conclusion can be reached. In these experiments the dye-coated surface was ingested as a single large mass or a few large masses into the uroid. This uptake is morphologically quite different from the channel formation seen when amebae are treated with the dye at room temperature [6, 151. Nevertheless, the two modes of uptake probably depend on the same physiological processes, and the morphological difference can be explained on the basis of a simple model which makes three reasonable assumptions: (1) The pre-chilling process in some way reduces the differences between parts of the cell (e.g., it might allow equal distribution of potentially contractile material); (2) when the cell is treated with dye, attachments are made between the cell membrane and the cortical cytoplasm to a greater extent than under control conditions; and (3) if the attachments are made in a pre-chilled cell, they occur over much of the cell surface, since various parts of that surface are now equivalent in this respect. When movement begins, one area, weakest in attachment between membrane and cytoplasm, becomes the site for new surface formation. If, on the other hand, amebae are treated at room temperature with dye, the inducing stimulus may be supposed to be superimposed on already existing connections between plasmalemma and cortex, which may be of greater extent at the uroid. In that case, long channels could result if, as has been suggested [3], isolated points of attachment become the deepest parts of pinocytic channels, while nearby areas are pushed out as small pseudopods. Support for the existence of networks between membrane and cortical cytoplasm has been found in pre-chilled Alcian Blue-treated amebae [ 19, 211. L41so, Hayward [ 1.51 and Christiansen and Marshall [lo] have reported special granular or reticulate areas lining pinocytotic and phagocytotic channels of amebae ingesting dye or paramecia at room temperature. Further study of the state of the cytoplasm of amebae during movement depends on improved fixation methods. Wohlfarth-Botterman and his students have been working intensively on this problem and have already reported some success with new combinations [ 2 1. Quantitatiue results The quantitative study was made to see whether forming only ne\v cell coat and not plasma membrane, 39-661808
the amebae might be as has been proposed Experimental
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for other conditions [16]. If this occurred with dye-treated amebae, one would expect to see the clumps packed closely together after new surface had formed, since the same amount of coat material would have to occupy a smaller area of cell membrane. Alternatively, but less likely, one might find a change in clump size if cell coat were moved so that fusion of clumps could occur. This seems less likely since the clumps are found before fixation (see Results) and seem to be due to interaction of dye with surface rather than to the effect of fixation. The results on both points are as follows: Table I shows the means and standard error of the means for Comparisons 1 and 2 in which a statistical comparison of the populations was valid. In Comparison 3 it can be seen that the means of frequency are very close, but the variability of the measurements of clump frequencies on surfaces taken up into the uroid was greater than on surface before movement; so that the two populations could not be considered part of the same universe. Nevertheless, the means are very close. In Conditions 1 and 2, it was less than one, showing that there is no evidence that clumps are actually spaced differently after new cell surface is formed than before. There is, then, no evidence that the surface flows so as to compress the clumps together. For a calculation of how much of a change one would expect in Condition 2, see the Appendix. Similarly, Table II shows that there is no difference in the means of the diameters of the clumps taken from Comparison 2. These means are so close that no statistical calculation was done. Again see the Appendix for a calculation of the difference one would expect if clumps were fused together by flowing of surface membrane. Control
of rate of output of surface
in relation
to ameboid movement
In the present experiments, the rate of output of 50 per cent or more in 30-35 min is much greater than that estimated by Wolpert and O’Neill [20] for A. proteus after fluorescent labelling; from the rate of uptake of membrane, they calculated a rate of output of 12 per cent/hr. Before discussing the reasons for this difference, one point might be made about their estimate. It is possible that the rate of output and uptake is not the same, and, indeed, that the starving, moving ameba is putting out membrane at a faster rate than it is taking it up. The redundancy produced might then be used during periods of rapid feeding. Such a process could be missed by their method. A more important reason for a difference in the rate of output of surface in this study and that of Wolpert and O’Neill’s depends on the difference in label used. In our work a strong inducer of pinocytosis was used to label the surface. As discussed above, binding of Alcian Blue to the surface results in Experimental
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Surface formation
599
in Chaos by electron microscopy
the formation of large areas of fibrillar ma&Gal in the underlying cytoplasm and reticulate areas which reach to the plasmalemma. This is consistent with the idea that in those areas membrane is not free to move forwards over an advancing pseudopod because it is held to the cortical gel. In Wolpert and O’Neill’s study, antibody to the ameba surface was used as a label. This did not produce pinocytosis unless a 30-min exposure was used. It is possible that such a label resulted in fewer connections between surface and cytoplasm. If so, new surface could have been made more slowly because the existing membrane was free to move forwards over a new pseudopod. In fact, Jeon and Bell [lS], using a fluorescent-labelled basic protein derived from papain, reported clear areas at the surfaces of new pseudopods; and their results using amebae confined in capillaries (to prevent dye diffusion) were similar to the results reported here with Alcian Blue. A basic protein would be expected to be a good pinocytosis inducer at neutral pH [O, 211. Thegeneral prediction to be drawn from this speculative discussion is that when a strong pinocytosis inducer is used as label, rapid formation of new coat should occur in pre-chilled amebae; while a weak inducer should result in a slower formation. This prediction can be tested with a series of compounds. It is interesting to note that investigators using different conditions and techniques have concluded that surface in amebae was formed either rapidly [16, 17, 18, 221 or slowly [14, 20, 291. Some of the discrepancies in these findings may be explained if one assumes that the ameba surface may either move forwards over a new pseudopod, or may be attached to the underlying cortical gel in such a way that for a new pseudopod to form new surface must be formed, as in the present experiments. During normal movement, there might be a delicate balance between these two processes such that small changes in environmental conditions could favor one or the other. If this is so, it is hard to see how formation of cell surface could play a primary role in providing the force for cell movement [l], although it might be highly significant in determining its direction.
SUMMARY
The cell surface of the ameba Chaos chaos was “coated” with Alcian Blue by exposure of the cells to dilute dye solutions at 5°C. At this temperature the amebae are immobilized. Formation of new surface was studied after rinsing the specimens and removing them to a fresh drop of medium at room temperature. Direct observations of amebae indicated that complete turnover of surface occurred in about 35 min but that the resultant new surface was probExperimental
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ably not as convoluted as the original. Nate of formation of surface when amebae were recycled through the dye treatment was much slower. As previously reported, electron micrographs of fixed specimens showed that in dye-treated cells the surface filaments seen in untreated cells were replaced by dense clumps occurring at irregular intervals. New surface possessed all the layers of surface found in untreated cells. In sections including recently formed surface, the plasmalemma was smooth and few vesicles were seen in the underlying cytoplasm. In some sections clumps of old surface were found with surface filaments passing through them. More typically, a series of transitional profiles between typical filaments and typical clumps could be seen. Frequency counts of the clumps on photomicrographs before and after new surface formation provided no evidence for the hypothesis that there may be slippage between surface coat and underlying plasmalemma. In the cytoplasm of the uroid of amebae 15 min after return to room temperature, extensive areas of convoluted membranes bearing typical dense clumps were found. It is concluded that under the present conditions there is rapid and extensive formation of new surface which possesses all the morphological layers of the original surface. A mechanism involving interpolation of ground cytoplasm into the new surface appears to account better for the findings than one involving fusions of vesicles with the surface.
APPENDIX
1. Estimation of the change in frequency of surface clumps to be expected in Condition II if the surface coat were moved over the plasmalemma: assume that there is complete renewal of the surface in 35 min but that the new surface is only 4 as redundant as the original. After 25 min, 25135 x 4, or about l/3 of the original coat is replaced. Then the original coat must be moved so as to occupy only 2/3 of the original plasmalemma, since new coat occupies the other l/3. Because sections of equal thickness are used, the same number of clumps must occur on 2/3 of the perimeter or 3/2 times as many clumps/cm. Original number of clumps/cm = 1.01 (cold condition) 1.5 X 1.01 = 1.52 clumps/cm Found 1.05 clumps/cm 2. Similarly, if fusion of clumps had occurred, their volumes would have to be increased by the same factor, i.e., 1.5 x volume in order to fit 1.5 times the amount of clump material into the same perimeter. Assuming approxiExperimental
Cell Research
43
Surface formation mate spheres, r is necessary?
in Chaos by electron microscopy
601
413 nr3 would become 1.5 x 413 zr3. How much r’, or the new r, would have to be 1.15 X r.
of a change in
Original r = 3.04 1.15 x 3.04 = 3.49 Found 3.14 REFERENCES 1. 2. 3. 4. 5. 6. 7.
BELL, L. G. E., J. Theoret. Riol. 1, 104 (1961). BHOWNICK, D. K. and WOHLFARTH-BOTTERMAN, K. E., Exptl Cell Res. 40, 252 (1965). BRANDT, P. W., Exptl Cell Res. 15, 300 (1958). BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cyiol. 8, 675 (1960). -J. Cell Biol. 15, 55 (1962). CHAPMAN-ANDRESEN, C., Compl. rend. lab. Carlsberg 33, 73 (1962). -in J. LUDVIK, J. LOM and J. VAVRA (eds.), Progress in Protozoology. Czech. Akad. Prague,
Profozool. 8. -9. CHAPMAN-ANDRESEN,
1963. Suppl.
Sci.
11, 11 (1964).
C. and HOLTZER, H., J. Biophys. Biochem. Cytol. 8, 288 (1960). R. G. and MARSHALL, J. M., J. Cell Biol. 25, 443 (1965). 10. CHRISTIANSEN, 11. CLAUDE, A., in SYDNEY S. BREESE, Jr. (ed.), Fifth International Congress for Electron Microscopy, vol. 2, L-14. Academic Press, New York, 1962. 12. GASIC, G. and GASIC, T., Nature 196, 170 (1962). 13. GOLDACRE, R. J., Expfl Cell Res. Suppl. 8, 1 (1961). 14. GRIFFIN, J. L. and ALLEN, R. D., Exptl Cell Res. 20, 619 (1960). 15. HAYWARD, A. I<‘., Compt. rend. lab. Carlsberg 33, 535 (1963). 16. JEON, K. W. and BELL, L. G. E., Expfl Cell Res. 33, 531 (1964). 17. LANDAU, J. V., .I. Cell Biol. 24, 332 (1965). 18. LANDAU, J. V., and THIBODEAU, L., Expfl Cell Res. 27, 591 (1962). J. M. and NACHMIAS, V. T., .J. Histochem. Cytochem. 13, 92 (1965). 19. MARSHALL, 20. MAST, S. O., .J. Morphol. 41, 347 (1926). V. T., J. Cell Biol. 23, 183 (1964). 21. NACHMIAS, 22. -Exptl Cell Res. 38, 128 (1965). 23. NACHMIAS, V. T. and MARSHALL, J. M., in T. W. GOODWIN and 0. LINDBER~ (eds), Biological Structure and Function, Vol. 2, p. 606. Academic Press, New York, 1961. 24. PALADE, G. E., in T. HAYASHI (ed.), Subcellular Particles. Ronald Press, New York, 1959. 25. PAPPAS, G. D., Ann. hr. Y. Acad. Sci. 78, article 2, 448 (1959). 26. REVEL, J. P. and HAY, E. D., Z. Zellforsch. Mikroskop. Anaf. 61, 110 (1963). 27. SCOTT, J. E., QUINTARELLI, G. and DELLOVO, M. C. Hisfochem. 4, 73 (1964). 28. STEEDMAN, H. F., Quart. .J. Microscop. Sci. 91, 477 (1950). 29. WOLPERT, L., Intern. Reu. Cyfol. 10, 163 (1960). 30. WOLPERT, L. and ONEILL, C. H., Sature 196, 1261 (1962).
Experimental
Cell
Research
43
Cell Research
A STUDY FORMATION
43, 583-601
(1966)
BY ELECTRON MICROSCOPY OF THE OF NEW SURFACE BY CHAOS CHAOS1-” VIVIANNE
Department
583
T.
NACHMIAS
of Biology, Hauerford College, Haverford, Pa., U.S.A. Received
March
10, 1966
THE formation of new cell surface, i.e., surface membrane plus attached coat material, by animal cells is not only a fundamental process of growth but occurs as well during discontinuous events such as cell division and secretion [24, 26, 28, 291. Yet relatively little is known about the mechanisms involved at either the morphological or chemical level. Amebae are potentially useful objects for the study of new surface formation. They can be used for a variety of experimental techniques, and they form new cell surface readily under several conditions at different rates. During optimal growth it has recently been estimated [lo] that up to 40-50 per cent of the cell surface of Chaos chaos may be taken up per hour by phagocytosis. Since replacement of membrane must, under such conditions, occur at a comparable rate, one can estimate a half-life for the surface of 1 hr to 14 hr. In studies of ameboid movement, when the organism is not feeding, there has been considerable controversy about the rate of turnover of the cell membrane in short term experiments [13, 14, 16, 201. Recently, an overall estimate of rate in Amoeba proteus has been provided by Wolpert and O’Neill [30]. In starving, moving amebae labelled with fluorescent antibody to the surface, they found a halflife for the surface of about 5 hr. In a totally different situation, amebae, upon return to atmospheric pressure after exposure to high pressures, apparently form some cell surface very rapidly [17, 181 although its extent is not known. These various observations suggest that the rate of new surface formation in amebae depends on external conditions and can probably be controlled experimentally. If so, preparations which form new surface rapidly could be used for studies of the process. This report describes a method for inducing rapid formation of new surface by Chaos chaos and presents observations on the process by light and 1 The work reported here was done at the laboratory of Dr J. 31. Marshall, in the Department of Anatomy, University of Pennsylvania, Philadelphia, and was supported by Grant So. CA01957 from the U.S. Public Health Service. z These findings were briefly reported at the 5th annual meeting of the American Society for Cell Biology (J. Cell Biol. 27, 71A (1965)). Experimental
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electron microscopy. The method consists in exposing pre-chilled amebae to a dye (Alcian Blue) which is irreversibly bound to the surface coat [6, 151 and has already been used by Chapman-Andresen [8] to study surface renewal in A. proteus. After rinsing and return to room temperature the new surface is seen as clear areas while dye-treated areas are deep blue. Under these special conditions it was found that: (1) this ameba can form large amounts of new surface in a short time-a half-life of as little as 20 min is estimatedbut cannot immediately repeat the process; (2) recently formed new surface is made up of all the morphological layers of the original surface, i.e., there is no evidence for the intermediate formation of a naked plasmalemma; (3) surface coat and plasmalemma move together when displaced by new surface in these experiments as they do in the early stages of pinocytosis [4, 15, 231; (4) new surface may be derived by interpolation of ground cytoplasm into the surface rather than from a fusion of pre-existing vacuoles with the surface, as appears to occur in secretory cells [24, 26j.
MATERIAL
AND
METHODS
Culture and preparation of amebae.-Chaos chaos (Pelomyxa carolinensis) were fed daily on concentrated suspensionsof Paramecium aurelia. The details of the culture have been described [lo]. Well-fed or briefly-starved (a few hr) individuals were used. They were rounded up by gentle shaking and were rinsed in several changesof ionfree water followed by several changesof medium. The medium contained 5 x lo-* M CaCl,, 5 x 1O-5M MgSO,, 1.6 x 1OV M K,HPO,, 1.1 x lo-* M KH,PO,. They were washed twice again in 5°C medium and stored at 5°C for l-5 hr before use. Bye treatment.-From the chilled amebae roughly spherical individuals of intermediate sizes were chosen. The amebae were rinsed in cold ion-free water, exposed at 5°C to dilute (0.0125 per cent to 0.1 per cent) Alcian Blue1 solutions for 2 min with constant gentle shaking, and then rinsed in several changesof ice-cold water followed by cold medium. These steps were carried out in siliconed dishesto prevent amebae from sticking to the glasssurfaces. Individual ameabae were fixed directly in cold medium, or were transferred to a large volume of medium at room temperature and fixed after 3 to 35 min. All steps were carried out under direct observations with a dissecting microscope. The Alcian Blue dye for most of the experiments was the SGN (Hartman-Leddon Co., Philadelphia) used without purification but merely filtered before use. Some experiments were also done with the SGX (Fisher) used after recrystallization, as described by Scott, Quintarelli and Dellovo [27]. Amebae behaved similarly with both dye samples. In one experiment, amebaewhich had been allowed to warm up and resume movement were exposed to 0.001 per cent toluidine blue. 1 Alcian Blue was introduced induce pinocytosis in amebae
Experimental
Cell Research 43
for staining acid mucopolysaccharides by Chapman-Andresen [6].
[26] and was first
used to
Surface formation
in Chaos by electron microscopy
5%
Fixation.-For most experiments amebae were fixed in unbuffered osmium tetroxide (2 per cent, pH about 6), followed by an equal mixture of 2 per cent uranyl acetate and 2 per cent osmium tetroxide for IO min more. This fixation has been found to preserve the filamentous layer of the cell coat uniformly and densely [22]. Experiments were also performed using osmium tetroxide buffered with Verona1 acetate to pH 8.6 [25] or using 2 per cent glutaraldehyde (Union Carbide) buffered to pH 6 with 0.04 M potassium phosphate and followed by post-fixation with either unbuffered osmium tetroxide or the alkaline osmium tetroxide solution. After fixation for 10 min at room temperature, amebae were rinsed for one min in ion-free water, dehydrated in alcohols, and embedded in araldite. Any amebae exhibiting marked changes in form or prolonged streaming during fixation were discarded. Such effects were common during glutaraldehyde fixation, rare when osmium was used. In the polymerized resin blocks, pseudopodial or uroid (tail) regions of the embedded amebae were identified by comparison with drawings made of the specimens at the time of fixation. Sections of the regions desired were made and mounted on uncoated or collodion-carbon coated grids and were examined with an RCA EMU-3 electron microscope. Sections fixed in osmium or glutaraldehyde and osmium were stained with 2 per cent phosphotungstic acid in 95 per cent ethanol for two min. Quantitative study.-In the electron micrographs of dye-treated amebae, the surface filaments are replaced by clumps occurring at irregular intervals (Fig. 2). The average frequency of occurrence of such clumps in the photomicrographs was determined under three pairs of conditions: (1) counts of clump frequency were made along profiles of the anterior surface of an ameba which had formed a new pseudopod and compared with the frequency found near the posterior end of the same ameba (Table I, Comparison 1); (2) counts of clump frequency were made along surfaces of two amebae fixed before the formation of new surface and compared with the frequency along the tail region in two other specimens fixed 25 min after formation of new surface had begun (Table I, Comparison 2); (3) the frequency of clumps was determined along the surface of an ameba fixed before any movement had occurred and compared with the frequency along surface actually taken up in the uroid of another specimen fixed 25 min after the formation of new surface had begun (Table I, Comparison 3). In all cases negatives were made at x 7400f 7 per cent over a period of several months. For a given comparison, all negatives were made on the same day and frequently on the same negative plate (5 negatives to the plate). Enlargements of x 4 were made of the negatives, and counts were made on the prints. For the counts, only surfaces cut approximately in cross section were used. A map measure was used to determine the length of each profile of the plasmalemma; lengths were from lo-70cm. The precision of these measurements was to 1 cm or better. A total of 23 profiles was counted for Comparison 1; 27 for 2; and IO for 3. Clumps were counted, using a constant set of criteria. The reasoning was as follows: clumps seen on unfixed Alcian Blue-treated surfaces, picked up directly onto grids, vary in size but are of quite uniform density; therefore, it was assumed that very faint clumps appearing in sections must be tangential cuts through clumps. Hence, a given clump should be counted only in a given section if its density is judged to be at least half that of the majority of clumps in the print. This precaution is rough, at best, since it is a visual estimate; but Experimental
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it was taken, since it was possible that on some grids adjacent sections might be used for counts. In addition, only clumps adjacent to the underlying continuous zone of the surface coat were included; a clump separated from the surface was judged to “belong” to another area of plasmalemma and to appear isolated from it due to a tangential cut across the surface. Such clumps were rare, as might be expected, where there was a ‘I’al~r.~ I. Comparison of frequency of clumps formed by the interaction and surface coat under direrent conditions. Final
magnification
of
dye
> x 29,600.
1: Ameba, 3 min after formation of new pseudopod. I%-equency along anterior
Comparison (smooth)
and
posterior
Ant: N = 11 post: N = 12
0.963 1.070
(convoluted) clumps/cm clumps/cm
surfaces. mean mean
Standard t -= 1
error
of mean
diff:
Comparison 2: Amebae fixed before formation of new surface (two specimens, C), with amebae fixed 25 min after formation of new surface (two specimens, W). Counts on the latter made on surface still external. Both surfaces convoluted. c: s = 15 w: X-12 Nole that amebae.
0.994 1.004
the counts
Comparison 3: Amebae formation of surface e.g., Figs. 6 and 7. c: 5=5 w: x=5
clumps/cm clumps/cm
refer
fixed (IV).
mean mean
to frequency
before Counts
1.013 clumps/cm I .050 clumps/cm
Standard f
final
error
prints
of mean
and
diff:
not to original
dimensions
of the
formation of new surface (C) with amebae fixed 24 min after on the latter made on surface actually taken up in the uroid mean mean
Difference comparing
in variabilily, however, the Iwo populations
precludes
good cross section as judged by the appearance of two dense lines in the plasmalemma. Using these two criteria for the counts, duplicate observations were good to 2-3 clumps. This variation is a measure of the difficulty in deciding whether to include or exclude a given clump on the borderline of either or both criteria. For example, in Fig. 2 the surface lying between A and A’ yields a count of 16 or 17 clumps. Fig.
l.-Normal cell, fixed with unbuffered and uranyl acetate. Note the clear distinction the continuous layer (C), and the underlying x 69,000.
osmium tetroxide followed by a mixture of osmium between the filamentous layer of the surface (F), plasmalemma (P), composed of two dense lines.
Fig. 2.-Cell treated with Alcian Blue SGN in the cold, rinsed and fixed in the same manner as Fig. 1 before movement occurred. Note that the dense clumps along the surface (G) are separated from the plasmalemma by a narrow space (S). The total count of clumps from point A to A’ along the circumference is 16 or 17 (see Methods). x 22,100. Experimental
Cell Kesearch
43
Surface formation
in Chaos by electron microscopy
587
1
G
2 Experimental
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43
t588
Vivianne
T. Nachmias
On some of the prints used for Comparison 2, the diameters of the clumps were also estimated by measuring at the widest point and parallel to the profile of the plasmalemma. The means are shown in Table II. All the measurements except those of the perimeters were made by observers who did not know what, if any, comparison was being made. II. Mean diameters
TABLE
of clumps on prints
Wa Print no.
Mean diameter (mm)
2a la 5a 1
from comparison
2.
Cb No. of clumps
Print no.
Mean diameter (mm)
No. of clumps
42 10 33 36
6a 6a 7 7 8 11
3.25 3.6 3.5 3.3 3.5 2.9
35 10 19 14 13 22
4.6 4.7 3.9 2.9 x=121
x=123 a Weighted ’ Weighted
mean mean
(overall (overall
mean): mean):
3.14 3.04
mm/clump. mm/clump.
RESULTS Direct observations When amebae were prepared and selected as described above, and then exposed to dilute solutions of dye in the cold, they behaved in a reproducible way. They first became very sticky. If allowed to congregate, they adhered to one another and formed clumps. They also adhered strongly to glass surfaces that were not silicone treated. After rinsing, while still at 5”C, the entire surface appeared stained blue. Although no undyed areas were visible with the dissecting microscope, one end of the ameba frequently appeared to be more deeply stained; this seemed to be due to the persistence of convoluted surface. When the cold amebae were pipetted into a drop of medium at room temperature, a clear unstained pseudopod was formed in almost all specimens in 6-7 min when dilutions of 0.025 per cent dye or less were used. After higher concentrations of dye, the rate was slower, e.g., after 0.1 per cent dye amebae remained spherical for lo-15 min and cleared very slowly. Exposure to dye at either acid or neutral pH with or without added buffer resulted in the same rates of clearing. Text-Fig. 1 diagrams the appearance of an ameba at each stage of clearing (arbitrarily called “clearing”, “monopodal”, and “polypodal”). When 0.025 per cent dye was used, clearing was first seen in three min or less. Ten Experimenfal
Cell Research
43
Surface formation
in Chaos by electron
microscopy
589
min after warming, over half the amehae had reached the monopodal stage; and by 35 min all viable amebae were polypodal. At this point the several pseudopods appeared clear while at the tail or uroid there was a single very dense spot of dye. Streaming was vigorous at this stage, and amebae reaching the polypodal stage without exception moved about normally, fed, and evenually defecated the dye.
s
c
Text-Fig. l.-Diagram of form to room temperature. x, IP, monopodal: PP, polypodal.
II’
PP
changes in Chaos chaos coated with Alcian Blue (cold) dye-coated surface; e, new surface. S, spherical;
after return C, clearing:
In one experiment, amebae were rounded up again after reaching the polypodal stage, re-chilled for 45 min and re-exposed to dye. After this second cycle, all five were still spherical 35 min after warming and produced new surface only slowly thereafter. When treated amebae at the polypodal stage were put together with untreated amebae into a 0.001 per cent solution of toluidine blue, metachromatic staining of the surfaces of both untreated and dye-treated amebae was immediate. Staining was both more intense and more variable over parts of the surface of the untreated amebae, but the new surface of the Alcian Bluetreated amebae stained as intensely as the anterior parts of the pseudopods of untreated amebae. Electron
microscope
obseruations
After fixation in unbuffered osmium tetroxide, followed by the uranyl/ osmium mixture, the surface of normal amebae appears as in Fig. 1. Note that the amorphous and filamentous layers of the surface coat first described by Brandt and Pappas [4] are clearly defined. When an ameba is fixed in the same way after treatment with dye, the surface appears as in Fig. 2. The filamentous layer is replaced by clumps of dense material about 100 rnp in diameter, occurring at irregular intervals and separated from the plasmalemma by a clear zone, whose width, 150-200 hi, corresponds to the width of the amorphous layer in Fig. 1. Sometimes this clear zone appears faintly stained, but it was never as dense as in untreated amebae. The clumping of Experimental
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the filamentous zone was found on both smooth and convoluted regions of surface of treated amebae. This effect of Alcian Blue has been reported by Hayward [15] in a study of pinocytotic uptake following treatment with this dye. Clumps of dense material are also seen when the cell coats from dyetreated ruptured amebae are picked up directly onto coated grids and examined without fixation. In Fig. 3 is shown a section of the anterior part of an ameba several min after treatment with 0.05 per cent dye. Note the scarcity of vacuoles in the underlying cytoplasm, the smooth contour of the plasmalemma, and the occurrence of several clumps which appear to have filaments passing through them. Fig. 4 shobvs a higher power view from another ameba near the base of a pseudopod fixed ‘L-3 min after warming, following treatment with 0.02.5 per cent dye. The surface here is convoluted and both clumped and filamentous surface are present. In addition there is a transitional zone (Tr) lying between clumped and filamentous surfaces. The clumps appear progressively narrower and more elongated until they grade into filaments. Fig. 5 shows another example of an area of new surface formation 3-4 min after bringing to room temperature. Since the filaments on the new surfaces appeared less dense than those on control, untreated amebae (Fig. l), some untreated amebae were fixed in the same drop of rinse medium as the dye-treated amebae. The surface filaments on such controls appeared identical with those on new surface (Fig. .5 insert). The results with other fixatives were studied less intensively but the occurrence of new surface, transitional zones, and scarcity of vacuoles in cytoplasm were all confirmed. The appearance of the outer layer varies with the fixation, and when the unbuffered osmium solutions were used, the normal surface appeared clumped [X2] so that it was difficult to distinguish old and new surface. Altogether, 14 amebae were examined for new surface. Figs. 6 and 7 are sections from the tail regions of an ameba which had been at room temperature for 25 min after dye treatment. Masses of membra-
Fig. 3.-Ameba treated with Alcian Blue 0.05 per cent and fixed several minutes after return to room temperature, while at the “clearing” stage. Note that filaments are present up to the junction with clumped surface, while some clumps appear to have filaments passing through them. Note also the scarcity of vesicular structures in the cytoplasm. x 17,000. Fig. 4.-Ameba treated with Alcian Blue 0.025 per cent, fixed 2-3 min after return to room temperature. Large areas of apparently clear surface were visible by light microscopy. This section was taken near the base of a pseudopod. Note the filamentous layer (F) like that of Fig. 5, inset, in the lower part of the photomicrograph, and the dense clumps (G), like those of Fig. 2, in the uXpf;:;;lf. Note also the occurrence of zones intermediate in morphology between F and G (Tr). > . Experimental
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Research
43
Surface formation
in Chaos by electron microscopy
Experimental
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Vivianne
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nes have been taken up into the interior, most of them still associated dense surface clumps. In some areas clumps are no longer associated the membrane but are replaced by a larger dense mass in the center space (arrows, Fig. 6). In Fig. 7 it can be seen that in many places the plasmic surfaces of the plasmalemma have become so closely apposed they appear as a double line (arrows, G, Fig. 7). Quantitative
with with of a cytothat
observations
Counts were made of the frequency of clumps on the photomicrographs under three pairs of conditions, as described in the Material and Methods Section, and the results are summarized in Table I. A statistically significant difference in the frequency of clumps was not found between any of the three pairs. In Comparison 1, clump frequencies along anterior and posterior parts of the same ameba were compared. A difference might appear if new surface were formed by the sweeping back towards the tail of “old” surface coat without the concomitant movement of the plasmalemma. If that occurred, the clumps lvould have to come closer together as the surface was moved towards the tail in order to accommodate the same amount of surface coat on less than the original amount of plasmalemma. The same arguments apply to Comparisons 2 and 3 which tested the same possibility under conditions where an even greater difference would be expected. After 25 min at room temperature it was not easy to find sections even near the uroid sho\ving clumped surface; typically, masses of membrane and clumps \vere found such as are shown in Figs. 6 and 7. This finding supports the visual impression that the surface is almost entirely renewed after about half an hour under the present conditions. Table 11 shows the mean diameter of clumps from Comparison 2, and the number of clumps counted for each mean. When a weighted mean is obtained, one finds an average diameter (on the print) of 3.14 for the warm amebae (25 min later) and 3.04 for the cold amebae (before new surface formation).
Fig. 5.-Ameba treated as in Fig. 4. Again, note the intermediate zone (Tr). Note the presence of granular material just under the plasmalemma in many places. The large vesicle (II) is similar in appearance to vesicles resulting from uptake of material. The insert shows the surface of a normal cell fixed as in Fig. 1 but in medium with several cells previously fixed with Alcian Blue. This photomicrograph resembles the appearance of the surface after alkaline osmium fixation. Both x 44,100. Experimenfcd
Cell
Research
43
Surface formation
593
in Chaos by electron microscopy
Experimental
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43
,594
Vivianne
T. Nachmias
DISCUSSION
Observations by light microscopy The observations show that under the present conditions Chaos chnos can form an extensive amount of surface rapidly but cannot immediately repeat the process. This implies that a well-fed ameba has a considerable pool of precursor surface material available, but that this or some essential component is used up in the burst of formation that occurs after warming. The amount of surface produced is probably something less than a complete new surface in 35 min. This is concluded from the results of the toluidine blue experiment which shows that the new surface stained only as intensely as anterior parts of pseudopods of control amebae. Such anterior parts have been found by electron microscopy to have few convolutions, while the uroid normally has many. Probably the new surface is altogether less convoluted than the original surface and thus occupies less total area. The occurrence of a latency period after one ingestion cycle is in agreement with the results of Chapman-Andresen 17, 81. She reported on a quantitative study of channel formation during pinocytosis, and found that in A. proteus, a latency period occurs after one complete pinocytotic cycle. She suggested that this delay might be due to the necessity for the resynthesis of new surface. Although those experiments and the ones reported here are not completely comparable, it appears that Chaos chaos may have a larger store of surface precursor material than A. proteus, possibly due to a larger volume/surface ratio. Experiments designed to compare the rates of surface renewel of species with different volume/surface ratios would be interesting. Observe tions by electron microscopy The new surface possessesall the morphologically distinguishable layers of untreated ameba surface. Even in the very earliest samples, 2-3 min after return to room temperature, no areas of uncoated plasmalemma were found.
Fig. B.-Section through the uroid of a cell treated with dye and allowed The tail region was stained deeply blue at this time. The microphotograph area containing masses of convoluted membrane, with dense clumps Arrows show areas where clumps are no longer attached to the surface center. x 8100. Fig. 7.-A similar view, at higher magnification. quently apposed very closely to one another between them, and clumps (G) line both sides. cellular. x 29,400. Experimental
Cell Research
43
to warm up for 25 min. shows part of a large attached to each side. but are collected in the
Note that two layers of plasmalemma (arrows) so that essentially no cytoplasm This entire mass of membranous material
are freis left is intra-
Surface formation
in Chaos by electron microscopy
Experimental
595
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43
596
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T. Nachmias
It appears, therefore, that amebae form coat material and plasmalemma at the same time under the present conditions, since the quantitative results (see below) shorn that new plasmalemma is indeed formed. Whether this is always true, even for amebae, is not clear. In leukocytes, Basic and Gasic [12] in a brief report stated that restoration of staining properties due to cell coat materials took place in 1 hr after removal of the polysaccharide layer with enzymes. In their study it was not clear whether secretion of coat material alone was involved, or formation of plasmalemma also. A similar study with amebae would be interesting in order to determine whether secretion of coat material through the plasmalemma can occur. This has been suggested [16]. In some secretory cells, new surface appears to be added by the fusion of vesicles with the surface [24, 261. If such were the case in the present experiments, one would expect an appearance in the electron micrographs like that of Mechanism A in Text-Fig. 2. If, on the other hand, material were fed into the surface in some continuous manner, in units smaller than the size of an individual clump, a result like that of Mechanism B would be expected. In fact, the results here are like those of R. The model B differs in two respects from A: the material is fed in continuously, and there is a zone of interaction between old and new surface. One cannot entirely rule out the possibility that the transition zones are due to diffusion of dye from clumped material to neighboring filamentous (new) surface despite the apparently irreversible nature of the binding [S]. If it were so, though, it would presumably be very rapid and it would be surprising that in some cases (Fig. 3) clumps occurred with filaments passing through them. The fact that vesicles are rare in the cytoplasm underlying new surface can be used to argue for or against their
Text-Fig. P.-Possible containing precursors plasmalemma). Experimkntal
mechanisms of surface
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for formation coat. Rottom:
of new surface. Top: Mechanism A; P, vesicles Mechanism B; P,, precursor material (coat plus
Surface formation
597
in Chaos by electron microscopy
participation; here, one must distinguish between vesicles that are like those resulting from uptake occurring when amebae are shaken and chilled, and vesicles which might be a source for new surface. Although our results suggest that ground cytoplasm is the source of the new surface, other methods, e.g., use of labelled precursor material, must be tried before a definite conclusion can be reached. In these experiments the dye-coated surface was ingested as a single large mass or a few large masses into the uroid. This uptake is morphologically quite different from the channel formation seen when amebae are treated with the dye at room temperature [6, 151. Nevertheless, the two modes of uptake probably depend on the same physiological processes, and the morphological difference can be explained on the basis of a simple model which makes three reasonable assumptions: (1) The pre-chilling process in some way reduces the differences between parts of the cell (e.g., it might allow equal distribution of potentially contractile material); (2) when the cell is treated with dye, attachments are made between the cell membrane and the cortical cytoplasm to a greater extent than under control conditions; and (3) if the attachments are made in a pre-chilled cell, they occur over much of the cell surface, since various parts of that surface are now equivalent in this respect. When movement begins, one area, weakest in attachment between membrane and cytoplasm, becomes the site for new surface formation. If, on the other hand, amebae are treated at room temperature with dye, the inducing stimulus may be supposed to be superimposed on already existing connections between plasmalemma and cortex, which may be of greater extent at the uroid. In that case, long channels could result if, as has been suggested [3], isolated points of attachment become the deepest parts of pinocytic channels, while nearby areas are pushed out as small pseudopods. Support for the existence of networks between membrane and cortical cytoplasm has been found in pre-chilled Alcian Blue-treated amebae [ 19, 211. L41so, Hayward [ 1.51 and Christiansen and Marshall [lo] have reported special granular or reticulate areas lining pinocytotic and phagocytotic channels of amebae ingesting dye or paramecia at room temperature. Further study of the state of the cytoplasm of amebae during movement depends on improved fixation methods. Wohlfarth-Botterman and his students have been working intensively on this problem and have already reported some success with new combinations [ 2 1. Quantitatiue results The quantitative study was made to see whether forming only ne\v cell coat and not plasma membrane, 39-661808
the amebae might be as has been proposed Experimental
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598
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for other conditions [16]. If this occurred with dye-treated amebae, one would expect to see the clumps packed closely together after new surface had formed, since the same amount of coat material would have to occupy a smaller area of cell membrane. Alternatively, but less likely, one might find a change in clump size if cell coat were moved so that fusion of clumps could occur. This seems less likely since the clumps are found before fixation (see Results) and seem to be due to interaction of dye with surface rather than to the effect of fixation. The results on both points are as follows: Table I shows the means and standard error of the means for Comparisons 1 and 2 in which a statistical comparison of the populations was valid. In Comparison 3 it can be seen that the means of frequency are very close, but the variability of the measurements of clump frequencies on surfaces taken up into the uroid was greater than on surface before movement; so that the two populations could not be considered part of the same universe. Nevertheless, the means are very close. In Conditions 1 and 2, it was less than one, showing that there is no evidence that clumps are actually spaced differently after new cell surface is formed than before. There is, then, no evidence that the surface flows so as to compress the clumps together. For a calculation of how much of a change one would expect in Condition 2, see the Appendix. Similarly, Table II shows that there is no difference in the means of the diameters of the clumps taken from Comparison 2. These means are so close that no statistical calculation was done. Again see the Appendix for a calculation of the difference one would expect if clumps were fused together by flowing of surface membrane. Control
of rate of output of surface
in relation
to ameboid movement
In the present experiments, the rate of output of 50 per cent or more in 30-35 min is much greater than that estimated by Wolpert and O’Neill [20] for A. proteus after fluorescent labelling; from the rate of uptake of membrane, they calculated a rate of output of 12 per cent/hr. Before discussing the reasons for this difference, one point might be made about their estimate. It is possible that the rate of output and uptake is not the same, and, indeed, that the starving, moving ameba is putting out membrane at a faster rate than it is taking it up. The redundancy produced might then be used during periods of rapid feeding. Such a process could be missed by their method. A more important reason for a difference in the rate of output of surface in this study and that of Wolpert and O’Neill’s depends on the difference in label used. In our work a strong inducer of pinocytosis was used to label the surface. As discussed above, binding of Alcian Blue to the surface results in Experimental
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Surface formation
599
in Chaos by electron microscopy
the formation of large areas of fibrillar ma&Gal in the underlying cytoplasm and reticulate areas which reach to the plasmalemma. This is consistent with the idea that in those areas membrane is not free to move forwards over an advancing pseudopod because it is held to the cortical gel. In Wolpert and O’Neill’s study, antibody to the ameba surface was used as a label. This did not produce pinocytosis unless a 30-min exposure was used. It is possible that such a label resulted in fewer connections between surface and cytoplasm. If so, new surface could have been made more slowly because the existing membrane was free to move forwards over a new pseudopod. In fact, Jeon and Bell [lS], using a fluorescent-labelled basic protein derived from papain, reported clear areas at the surfaces of new pseudopods; and their results using amebae confined in capillaries (to prevent dye diffusion) were similar to the results reported here with Alcian Blue. A basic protein would be expected to be a good pinocytosis inducer at neutral pH [O, 211. Thegeneral prediction to be drawn from this speculative discussion is that when a strong pinocytosis inducer is used as label, rapid formation of new coat should occur in pre-chilled amebae; while a weak inducer should result in a slower formation. This prediction can be tested with a series of compounds. It is interesting to note that investigators using different conditions and techniques have concluded that surface in amebae was formed either rapidly [16, 17, 18, 221 or slowly [14, 20, 291. Some of the discrepancies in these findings may be explained if one assumes that the ameba surface may either move forwards over a new pseudopod, or may be attached to the underlying cortical gel in such a way that for a new pseudopod to form new surface must be formed, as in the present experiments. During normal movement, there might be a delicate balance between these two processes such that small changes in environmental conditions could favor one or the other. If this is so, it is hard to see how formation of cell surface could play a primary role in providing the force for cell movement [l], although it might be highly significant in determining its direction.
SUMMARY
The cell surface of the ameba Chaos chaos was “coated” with Alcian Blue by exposure of the cells to dilute dye solutions at 5°C. At this temperature the amebae are immobilized. Formation of new surface was studied after rinsing the specimens and removing them to a fresh drop of medium at room temperature. Direct observations of amebae indicated that complete turnover of surface occurred in about 35 min but that the resultant new surface was probExperimental
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600
Vivianne
T. Nachmias
ably not as convoluted as the original. Nate of formation of surface when amebae were recycled through the dye treatment was much slower. As previously reported, electron micrographs of fixed specimens showed that in dye-treated cells the surface filaments seen in untreated cells were replaced by dense clumps occurring at irregular intervals. New surface possessed all the layers of surface found in untreated cells. In sections including recently formed surface, the plasmalemma was smooth and few vesicles were seen in the underlying cytoplasm. In some sections clumps of old surface were found with surface filaments passing through them. More typically, a series of transitional profiles between typical filaments and typical clumps could be seen. Frequency counts of the clumps on photomicrographs before and after new surface formation provided no evidence for the hypothesis that there may be slippage between surface coat and underlying plasmalemma. In the cytoplasm of the uroid of amebae 15 min after return to room temperature, extensive areas of convoluted membranes bearing typical dense clumps were found. It is concluded that under the present conditions there is rapid and extensive formation of new surface which possesses all the morphological layers of the original surface. A mechanism involving interpolation of ground cytoplasm into the new surface appears to account better for the findings than one involving fusions of vesicles with the surface.
APPENDIX
1. Estimation of the change in frequency of surface clumps to be expected in Condition II if the surface coat were moved over the plasmalemma: assume that there is complete renewal of the surface in 35 min but that the new surface is only 4 as redundant as the original. After 25 min, 25135 x 4, or about l/3 of the original coat is replaced. Then the original coat must be moved so as to occupy only 2/3 of the original plasmalemma, since new coat occupies the other l/3. Because sections of equal thickness are used, the same number of clumps must occur on 2/3 of the perimeter or 3/2 times as many clumps/cm. Original number of clumps/cm = 1.01 (cold condition) 1.5 X 1.01 = 1.52 clumps/cm Found 1.05 clumps/cm 2. Similarly, if fusion of clumps had occurred, their volumes would have to be increased by the same factor, i.e., 1.5 x volume in order to fit 1.5 times the amount of clump material into the same perimeter. Assuming approxiExperimental
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Surface formation mate spheres, r is necessary?
in Chaos by electron microscopy
601
413 nr3 would become 1.5 x 413 zr3. How much r’, or the new r, would have to be 1.15 X r.
of a change in
Original r = 3.04 1.15 x 3.04 = 3.49 Found 3.14 REFERENCES 1. 2. 3. 4. 5. 6. 7.
BELL, L. G. E., J. Theoret. Riol. 1, 104 (1961). BHOWNICK, D. K. and WOHLFARTH-BOTTERMAN, K. E., Exptl Cell Res. 40, 252 (1965). BRANDT, P. W., Exptl Cell Res. 15, 300 (1958). BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cyiol. 8, 675 (1960). -J. Cell Biol. 15, 55 (1962). CHAPMAN-ANDRESEN, C., Compl. rend. lab. Carlsberg 33, 73 (1962). -in J. LUDVIK, J. LOM and J. VAVRA (eds.), Progress in Protozoology. Czech. Akad. Prague,
Profozool. 8. -9. CHAPMAN-ANDRESEN,
1963. Suppl.
Sci.
11, 11 (1964).
C. and HOLTZER, H., J. Biophys. Biochem. Cytol. 8, 288 (1960). R. G. and MARSHALL, J. M., J. Cell Biol. 25, 443 (1965). 10. CHRISTIANSEN, 11. CLAUDE, A., in SYDNEY S. BREESE, Jr. (ed.), Fifth International Congress for Electron Microscopy, vol. 2, L-14. Academic Press, New York, 1962. 12. GASIC, G. and GASIC, T., Nature 196, 170 (1962). 13. GOLDACRE, R. J., Expfl Cell Res. Suppl. 8, 1 (1961). 14. GRIFFIN, J. L. and ALLEN, R. D., Exptl Cell Res. 20, 619 (1960). 15. HAYWARD, A. I<‘., Compt. rend. lab. Carlsberg 33, 535 (1963). 16. JEON, K. W. and BELL, L. G. E., Expfl Cell Res. 33, 531 (1964). 17. LANDAU, J. V., .I. Cell Biol. 24, 332 (1965). 18. LANDAU, J. V., and THIBODEAU, L., Expfl Cell Res. 27, 591 (1962). J. M. and NACHMIAS, V. T., .J. Histochem. Cytochem. 13, 92 (1965). 19. MARSHALL, 20. MAST, S. O., .J. Morphol. 41, 347 (1926). V. T., J. Cell Biol. 23, 183 (1964). 21. NACHMIAS, 22. -Exptl Cell Res. 38, 128 (1965). 23. NACHMIAS, V. T. and MARSHALL, J. M., in T. W. GOODWIN and 0. LINDBER~ (eds), Biological Structure and Function, Vol. 2, p. 606. Academic Press, New York, 1961. 24. PALADE, G. E., in T. HAYASHI (ed.), Subcellular Particles. Ronald Press, New York, 1959. 25. PAPPAS, G. D., Ann. hr. Y. Acad. Sci. 78, article 2, 448 (1959). 26. REVEL, J. P. and HAY, E. D., Z. Zellforsch. Mikroskop. Anaf. 61, 110 (1963). 27. SCOTT, J. E., QUINTARELLI, G. and DELLOVO, M. C. Hisfochem. 4, 73 (1964). 28. STEEDMAN, H. F., Quart. .J. Microscop. Sci. 91, 477 (1950). 29. WOLPERT, L., Intern. Reu. Cyfol. 10, 163 (1960). 30. WOLPERT, L. and ONEILL, C. H., Sature 196, 1261 (1962).
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