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Patterns of cellular organisation in limax amoebae
Experimental
Cell Research 26, 497.-519 (1962)
PATTERNS
AN
497
OF CELLULAR ORGANISATION LIMAX AMOEBAE ELECTRON
MICROSCOPE
IN
STIJDY
K. VICKERMANl Department
of Zoology,
University
College London, England
Received July 28, 1961
THE majority
of the small amoebae vvhich are abundant in the soil have a single broad pseudopodium and a vesicular nucleus with a large central nucleolus. They are the “limax” amoebae of earlier writers, but this epithet has little taxonomic significance for their superficial similarity belies fundamental differences in cellular organization. These differences reveal themselves to the light microscopist only at certain stages in the life cycle, notably at nuclear division and when the amoebae undergo cellular transformation (metaplasia) in the face of certain environmental changes [38]. It is reasonable to suppose that differences in mitotic mechanisms and ability to transform have a more profound ultrastructural basis. It is the aim of this investigation to discern patterns of ultrastructure in the Zimax amoebae and, if possible, to correlate them with events in the cellular cycle. Strains
of Amoebae
Details of the strains investigated are given below together with brief notes on their cytological characteristics as seen with the light microscope. Singh’s classification [38] has been adopted. The main features of nuclear division for each strain were determined from phase-contrast observations on living material and from smears of amoebae stained by a modified Delamater technique [43].
Amoebae with “promitotic” division of the nucleus, i.e. the nucleolus persists throughout division and divides to form two polar masses; the nuclear membrane remains intact and amoeboid movement continues unimpaired. l Wellcome
Research
Fellow. Experimental
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K. Vickerman
498
*Vaegleria gruberi (Schardinger). This amoeba can temporarily transform into a flagellate under conditions of divalent cation deprivation [47]. It forms spherical cysts with pores in the cyst wall. The strain used vvas from the Cambridge Culture Collection, No. 151813. The flagellate stage of Naegleria will not be dealt with in this paper. Schizopyrenus sp. Similar to Naegleria in amoeboid and encysted stages but the cyst wall lacks pores. A flagellate stage is never produced. This clonal strain isolated from soil resembles S. russelli Singh, except for the fact that in the present species there is a single smooth cyst vv-all. S. russelli forms a double-walled cyst, the outer wall being slightly vv-rinkled. A preliminary study of Schizopyrenus russelli has shown that its ultrastructure is very similar to that of the species described here.
Amoebae in which nuclear division is very reminiscent of that found in most metazoan somatic cells. The nucleolus and nuclear membrane disappear and amoeboid movement ceases during mitosis. There is never a flagellate stage in the life-cycle. Acanthamoeba sp. This strain was isolated in clonal culture from soil and conforms closely to Singh’s Harmanella rhysodes, also to Volkonsky’s [44] account of Acanthamoeba castellanii. As the organism in culture has the slender mitotic spindle characterizing Volkonsky’s genus, this is the name adopted here without further specification. Hartmanella astronyxis Hay. Only a few observations have been made on this species, the ultrastructure of which has been briefly described from osmium-fixed material by Deutsch and Swarm [ll]. Cambridge Collection No. 1534/l. METHODS
All strains were grown in monoxenic culture with Aerobacfer aerogenes on agar plates (0.2 per cent peptone, 1.5 per cent agar) at 25°C. Two hr before fixation the plates were flooded with Lang’s colloidal gold suspension in distilled water. The organisms were washed off the plates and spun down to a loose pellet at 2500 rpm before dispersal in cold fixative. Fixation was in Palade’s 1 per cent osmium tetroxide buffered at pH 7.2 for 30 min to 2 hr, or in 0.6 per cent potassium permanganate, similarly buffered, for 2 hr and always at 4°C. Dehydration was in graded ethanolwater mixtures (30 min changes) with three l-hr changes of absolute ethanol and in some cases 1 hr staining in 1 per cent phosphotungstic acid in ethanol. After 3 hr immersion in Araldite resin M the pellets were imbedded in Araldite mixture in open troughs, then hardened at 50°C for at least two days. Grey, silver and gold Experimental
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Electron
microscopy
of Limax
amoebae
499
sections were cut on the Porter-Blum microtome and mounted on carbon films. Sections of unstained material were then stained overnight in either lead hydroxide or many1 acetate as prescribed by Watson [46]. Sections were examined in the Siemen’s Elmiskop I at an accelerating voltage of X0 kV.
OBSERVATIONS Bulfered permanganate was found to be superior to osmium tetroxide in t)reserving the natural form of the amoebae and the following descriptions apply principally to material fixed in this way. M’here striking differences have been observed between osmium and permanganate fixed material, these are noted.
Trophic
Amoebae
Ectoplasm and endopZasm.-With the light microscope the ectoplasm of amoebae is seen as the clear cytoplasm forming the pseudopodium or surrounding the rounded-up amoeba; the ectoplasm is packed with inclusions-mostly mitochondria and food vacuoles. The two zones are also readily distinguished with the electron microscope; here the ectoplasm is seen to be not entirely devoid of inclusions for the profiles of small membranous structures occur throughout its substance (Figs. 1, 2, em). Serial sections indicate that these membranes do not form a continuous canalicular system but are isolated vesicles and parts of tubules. Plasma membrane and membrane structure.-Surrounding the amoeba is a membrane which in some sections is seen to have the “unit membrane” structure described by Robertson [X3], i.e. it has two electron-dense layers enclosing an electron transparent layer, the thickness of the whole being approximately 80 A. The unit membrane structure is evident in other membrane systems of amoebae fixed in permanganate (Figs. 8, 9). Organisms fixed for short periods in 0~0, occasionally exhibit membranes with unit organisation (Fig. 4). Endoplasmic reticulum-Well-defined membrane systems are present in the endoplasm of all amoebae but their form in the hartmannellids is not identical with that observed in the schizopyrenid amoebae. In Acanthamoeba the elements of the reticulum are numerous flattened vesicles and tubuli, arranged in stacks or bundles and usually lying parallel to the surface of the amoeba (Fig. 1, er). The membranes are approximately 200 A apart. Golgilike stacks of membranes have been seen in Hartmannella astronyzis (Fig. 8). limax
Experimental
Cell Research 26
500
K. Vickerman
In Schizopyrenus and Nueglerin the reticulum is seen as branching tubules and compressed saccules, but here the latter are in very intimate association with the mitochondria and often partially envelope one or more of these structures (Fig. 2). In Schizopyrenus the membranes have a spacing of only 150 A in the trophic amoeba. Osmium-fixed specimens have the reticulum obscured to some extent 1~~ a coarse precipitate in the surrounding matrix (Figs. 6, 7). This presumably represents the ribosomal granules; it is difficult to distinguish between granular and agranular reticulum. There is a tendency for the reticular membranes to form rounded vesicles when fixed for long periods in GO,. Mitochondria.-Perhaps the most prominent endoplasmic inclusions are the mitochondria. These differ in form according to the species of amoeba. The mitochondria of Acanthamoeba have been briefly described elsewhere [43]. They are commonly oval in shape, 0.5-1.5 ,U in length and 0.5-l p across. However, they are frequently distorted and may appear branched or bent in sections. They are rarely cup-shaped, but an extensive invagination may occur towards one end giving the appearance of an empty vesicle in sections. The outer mitochondrial membrane is separated by a space of 100 L%from the inner bounding membrane which gives rise to tubular cristae. These project into the mitochondrial lumen in an irregular fashion and are occasionally seen to branch or traverse the lumen. Sometimes one or, more rarely, two small spherical inclusions can be seen lvithin a mitochondrion. These bodies are actually intra-cristal in location, up to 1000 Ai in diameter, and have an electron-dense cortex (Figs. 1 inset, 6). In Hartmnnnelln ctstronyri.s intra-crystal bodies lacking an electron-dense cortex have been observed (Fig. S), but as yet it is not possible to pronounce on their homology with the structures in Acanthcrmoebn. The mitochondrial matrix is very electron-dense follo\ving osmic fixation, and has a honeycomb structure (Fig. 6).
Aer, bacteria; cq, colloidal gold particles; cv, contractile vacuole; CW, cyst wall; em, ectoplasmic ert, reticulum tubule; fu, food vacuole; ich, intramembrane profiles; er, endoplasmic reticulum; vacuole; cristal body; mit, mitochondrion; mf, myelin figure; nl, nucleolus; n/v, nucleolar nm, nuclear envelope; nmp, pore in nuclear envelope; sm, surface membrane; sp, spongiome tubules: WU, water vacuoles. Pb(OH),, lead hydroxide tate stained.
stained;
PTA, phosphotungstic
acid stained;
Fig. I.-AcanthamoeDa sp. Trophic amoeba and part of cyst. Note in region of spongiome associated with the contractile vacuole, “Kernspalten” (ks). The difference in size of intra-cristal bodies cyst is readily seen; inset shows location of intra-cristal body x 15,000 (inset x 240,000). Pb(OH),. Experimental
Cell Research 26
and t.rAc,
uranyl
ace-
extensive reticulum and tubules also nucleus with nucleolus and in mitochondria of amoeba and in mitochondrion. KMnO, and
Electron microscopy of Limax amoebae
501
502
K. Vickerman
In Schizopyrenus the mitochondria are ovoid, cylindrical, discoid or cupshaped in form. Those belonging to this last category are particularly common; their three-dimensional shape has been determined from serial sections. The cristae are more flattened in cross-section than those of Accrnthamoeba and they lack internal bodies. The mitochondria of Naeglericr are usually ovoid, but otherwise similar to those of Schizopyrenus. Food vacuoles.-These are lined by unit membranes and can be identified by their contents, ingested Aerobacter and their remains, and colloidal gold particles. Most noticeable, particularly in permanganate-fixed preparations, are concentric or spirally-wound membranes surrounding food remains within the vacuole (Figs. 2, 10, 11) or lying outside the amoeba. These “myelin forms” are common in phagocytic cells (e.g. see 113, 14, 20, 251). They are probably artifacts of fixation of phospholipid [SO, 411 and appear to be produced in the digestion of bacteria (Fig. 11). Occasionally the membranes can be seen to have unit structure and a definite periodicity (Fig. 12). There is no evidence for the continuity of these membranes \vith the membrane lining the vacuole. Although bacteria are often seen sticking to the surface of the pseudopodium, the principal site of ingestion seems to be along the body of the amoeba, anterior to the contractile vacuole and most likely on its under side Invaginations of the surface memwhen it is crawling along a substratum. brane are seen in this area. The evidence that the food vacuoles are simply dilatations of a permanent intracellular canalicular system, continuous with the surface membrane, is equivocal. Certainly reticular tubules can be seen abutting on the vacuole membrane and occasionally these approach the surface of the organism colloidal gold particles have been observed in dilated (Fig. 10). Moreover, tubules, but in such numbers as to make distinction from a small food vacuole difficult in an isolated section. Single gold particles have not been seen in reticular tubules as might be expected if these formed part of a permanent alimentary system.
Fig. 2.-S’chizopyrenus sp. Trophic amoeba with nucleus in metaphase. The nuclear envelope is still intact and amoeboid movement continuing, but the nucleolar material is distributed in two polar masses (pm) separated by the chromosome plate (cp). Note food vacuoles with enclosed myelin forms, collapsed contractile vacuoles near uroid (see Fig. 13), and association of reticulum with mitochondria. Insets 2 a and 2 b are two preceding serial sections through cup-shaped mitochondrion at top left. KMnO, and UrAc. x 10,000. Experimental
Cell Research 26
Electron microscopy of Limax amoebae
Experimental
503
Cell Reseurch 26
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K. Vickerman
Contractile vncuole.-Under phase contrast the contractile vacuole system of the living amoeba is seen to be composed of a principal vacuole and several subsidiaries apparently emptying into it. It discharges near the uroid or posterior end of the amoeba. With the electron microscope the vacuole and its subsidiaries in diastole are seen as membrane bounded vesicles. In the schizopyrenids the vesicles collapse at systole to give profiles which are reminiscent of the dictyosomes or Golgi sacs of other protozoa [18]-that is, flattened saccules expanded at either end (Fig. 4); Nassanov’s homology of the contractile vacuole and Golgi element is recalled. Many workers with larger amoebae (e.g. see [24, 281) have described the association of mitochondria and numerous small vesicles with the contractile vacuole. In the 1ima.x amoebae there is no marked accumulation of mitochondria in this region and, judging from their profiles in section, the associated membranous elements are tubules rather than vesicles in Acrrntharound the vacuole, comparable to rrmoebcz sp. These form a “spongiome” that observed in some ciliates [la, 361. Fig. 5 shows that in Acanthaznoebn sp. the tubules of the spongiome apparently empty into a common duct or into the main vacuole. A permanent pore for discharge of the contents of the contractile vacuole, so common in ciliates, has not been discerned in amoebae. Other cytoplczsznic inclusions.-Fat globules are common in the cytoplasm of dccznthnznoebrz and are readily distinguished in osmium-fixed material visible in the cytoplasm of the same amoeba (Fig. ‘i, fg). Al so occasionally are irregular masses of granules (Fig. 13, gm), each granule measuring about 200 A4 across. The masses do not have a crystalline structure and as they occur in permanganate-fixed material it seems likely that they are glycogen reserves [22]. Other inclusions in trophic amoebae of Acnnthnznoebn are ovoid structures ~111 to 2000 .i in length and 1000 -4 wide (Figs. 15, 16, up). They are surrounded by a unit membrane which is separated from an inner, concentric and similar membrane by a space of 200 ,$. These particles bear considerable resemblance to certain tumour viruses in their form (e.g. see [as]), but they are 10&20 times larger than such virus particles. The possibility of their being symbionts
Fig. 3.-Xaeyleriu gruheri. iVucleus of trophic amoeba showing nuclear arrows) and nucleolus with central vacuole. 0~0, and PTA. x 40,000. Fig. 4.-Xuegleria “unit membrane” Experimentul
envelope
with
gruheri. Connexion between outer nuclear membrane and reticulum structure at points arrowed. 0~0, and PTA. x 78,000.
Cell Research 26
pores (at showing
Electron microscopy of Limax amoebae
Experimentul
505
Cell Research 26
K. Vickerman is an interesting one as symbiotic micro-organisms are knolvn to he associated with large amoebae [34]. Another possibility is that they represent precursors of mitochondria, the cristae arising as outgrowths from the inner membrane. In view of the large space between the hounding membranes this seems unlikely, holvever. At the moment these inclusions cannot be accounted for with any confidence. TIze nzzcZeus.-Notwithstanding the remarkable differences in their mitotic processes, the amoebae have interphase nuclei of very similar structure. The nucleolus is electron-dense in osmium preparations (Fig. 3) and motierately so in permanganate-fixed material (Fig. 1). In the latter, electrondense granules approximately 200 a across are found. There is an electronpale space (Figs. 3, 17, nlv) to the nucleolus; it can be seen as a vesicle with the light microscope. The nuclear envelope consists of two apposed membranes separated 1)~ a space of about 250 ,% in 4cunthamoehn and Srreglerirr only 150 A in Schi:opyrenus. Pores are present in the envelope and these hare a diameter of approximately 700 A. In Acnnthumoehn sp. the nuclear membrane often shows numerous invaginations enclosing cytoplasm (Fig. 1, I;s) anti these described by Altman [3] in certain cancer are similar to the “Kernspalten” ~11s; they have not been observed in schizopyenid amoebae. On the other hand, although the outer membrane of the nuclear envelope in these amoebae often shows connexions \vith the reticular tubules (Fig. 4), as described in metazoan cells, such connexions have not yet been seen in hartmannellicls. Light microscope studies sho\v “Feulgen positive” material between the nucleolus and nuclear membrane [31, 381, but a fine granular prwipitatc is all that is usually found here with the electron microscope in osmiumfixed material; in permanganate-fixed specimens this region lacks inclusions altogether. Dividing forms.-Division stages are seldom encountered in fixed smears examined with the light microscope, so it is not surprising to find that the! Fig. 5.-Hartmurmellu astronyxis. Part of mitochondrion bodies. KMnO, and PTA. Y 50,000.
showing what appear lo be intra-cristal
Fig. (i.-,iccmlhamoeba sp. Mitochondria of trophic forms showing dense honeycomb of matrix after osmium fixation. Note intra-cristal bodies. 0~0, and PTA. x 27,000.
structure
1.‘ig. 7.--Acanthamoebu
*: 30,000.
sp. Fat globules
(fq) and reticulum
elements.
Fig. X.-Hurfmanne/la astrompis. Golgi-like stacks of membranes unit membrane structure. KMnO, and PTA. x 54,000. I;ig. Cl-Acanthamoeba and PTA. Experimental
sp. Endoplasmic
Cell Research
26
reticulum
showing
unit
0~0,
and 1’TA.
from trophic membrane
amoeba, showing structure.
KRfnO,
Electron microscopy of Limax amoebae
Experimental
507
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508
K. Vickerman
are also rare in sections examined with the electron microscope. Obvious have never been seen, but a series of sections mitotic figures of Acanthnmoebo was obtained through Schizopyrenrzs which matches well the appearance of the amoeba stained in metaphase (Fig. 2). The nucleolus has divided to form two polar masses, each of which has a spongy structure as seen in section. A space of low electron density separates the two masses and this is interpreted as the metaphase plate. The nuclear envelope is still intact and there are no traces of centrioles either inside or outside the nuclear membrane [al, X3!, nor have these been seen in sections of non-dividing amoebae. Encysted Forms soil amoebae encyst. In culture the strains described here do so 1-4 days after subculture. The changes at encystation as observed under the phase contrast microscope may he summarized briefly as follows. The individual amoebae stop moving and feeding and become spherical. Compacted food remains are cast out and numerous small lvater vacuoles appear in the c;vtoplasm; these can be seen bursting randomly over the surface of the organism. This results in shrinkage in size of the Tvhole cell and this active “dehydration” process continues during and after the laying down of the cyst \\-all. The nucleolus becomes much smaller, and rcfractile granules are often conspicuous on the nuclear membrane in the fully formed cyst. The cyst zutrll.-In all amoebae the cyst wall appears to be secreted at the surface membrane. This is the “multiple-layered membrane” seen 1)~ Deutsch and Swami ill] in Hartmrtnelln nstronyxis. In dcnnthcrmoebrr it is composed of many sheets or fihres, 100 LX thick, lying parallel to the membrane. I)uring incipient encystation the wall is of uniform thickness and the cyst rounded. As encystation proceeds, shrinkage of the organism results in buckling of the wall, an effect w-hich is exaggerated by the continual discharge of \vatcr vacuoles and food remains into the spaces bet\\-een the sheets formChanges
Fig. i&Fig.
at encystation-All
13.--All
Schizopyrenus
sp. KRlnO,
and ITrAc.
Fig. lo.-Showing food vacuoles with myelin forms and ingested colloidal gold. The \acuole Lo the right has a diverticulum (diu) from which a tubule proceeds to the surface of the amoeba. Y 40,000. Fig. Il.-Aerobacfer Fig. ia.-Part Fig. 13.-Uroid Eqwimenfal
in process of being digested inside food vacuole.
of food vacuole
showing
periodicity
region of amoeba showing (Ml Kesearch 26
collapsed
of myelin contractile
forms.
x 31,000. x 250,000.
vacuoles.
x 16.000.
Electron microscopy of Limax amoebae
Er~erimental
509
Cell Resectrch 26
510
K. Vickerman
ing the wall (Figs. 17, 19). The result is a cyst of irregular shape, often with two distinct cyst walls, an inner and an outer, the two meeting only at “pores” in the wall (Fig. 20). This appearance is more pronounced in H. astronysis in which the inner cvst wall is star-shaped and the outer more or less circular [32]. Electron micrographs show that it is doubtful that true pores exist: the so-called pores are simply those parts of the cyst wall that hare not been inflated by matter discharged from the encysting amoeba. The ejected matter in the cyst wall appears to he composed of disrupted membranes and in eludes the virus-like particles mentioned above (see p. 504). The cyst wall of Schixpyren~s (Fig. 1X) is not layered, nor does it contain ejected food material. It is of fairly uniform thickness (0.5-l ,u) in any one cyst; its outermost parl is probabl\- gelatinous, as colloidal gold particles from the surrounding medium may be found imbedded in it. Srze~ylerirr {Irdwri has a cyst wall of similar structure, but the pores visible \vith the light microscope have not been identified in electron micrographs. Cyst contents.-These are difficult to fix satisfactorily with osmium and the following account is taken from permanganate-fixed material. The most obvious change in the cytoplasm at encystation is the appearance of man! small water vacuoles. In the schizopyrenids it appears that water is expressed into the lumina of some of the reticulum tubules as these are distended but empty in micrographs. They occasionally show openings at the surface menmanes. In Acrrnthnmoehrr more delinite vacuoles are found scattered throughout the cytoplasm and in some there is a sign of continuity with an cndoplasmic tubule (Fig. 21). The contractile vacuole svstem is seen only as the spongiome in encysted amoebae. The most striking ultrastructural change occurring at encystation lies in these are nearly all spherical and diminthe mitochondria. In Schizopyrenus ished in size, 0.5-l ,U in diameter, and more completely enveloped by the associated reticulum (Fig. 18). In dcnnthnmoeha the mitochondria also assume a spherical shape and decrease in over-all diameter. The intra-cristal granules, however, increase in size to become vesicles 3000-3000 a across. Their cortical electron-dense material is distributed as particles of irregular size and shape (5OG300 *%) at the boundary of the vesicles (Figs. 17, 20).
Fig. 14.--9negleria
gruheri. Contractile
vacuole
region in systole.
KMnO,
and PT,4.
x 25,000.
Fig. 15.-Acanthamoeba sp. Contractile vacuole in partial diastole showing tubules of spongiome draining into common duct leading to vacuole. Note virus-like particles (up) and glycogen masses (gm) in surrounding cytoplasm. KMnO, and PTA. x 25,000. Fig. 16.-Single Experimental
virus-like
inclusion.
Cell Research 26
KhlnO,
and PTA.
x 60,000.
Electron microscopy of Limax amoebae
Experimental
511
Cell Research 26
512
K. Vickerman DISCUSSION
Compcxratiue cytology of schizopyrenid crnd hartm<~nnellid nmoebne.Amoebae are popular experimental organisms, and their ability to gronunder strictly defined cultural conditions [l ] enhances their value to the cell biologist. As they have few characters on which to make a tasonomic diagnosis, however, experimental \vork is frequently carried out on unidentified or misidentified strains. Attempts to classify amoebae on such characters as amoeboid movement and type of pseudopod formation [8, ‘31 are scnsibl? practical for the ecologist who Avishes to identify organisms at sight, but are often regarded as suspect by experimentalists who find some of these characters labile under laboratory conditions [3‘2]. The form of the dividing nucleus may be a reliable criterion, but it is often revealed only after intensive perusal of thousands of stained specimens. It is interesting to note that in the two families of limcrr amoebae studied here, differences in cell division are associated xvith differences in cgtoplasmic organization of the non-dividing cell. From this preliminary \\-ork it n-oultl appear possible to distinguish the schizopyrenids and hartmannellids on the ultrastructural form of such structures as the endoplasmic reticulum, contractile vacuole, mitochondria and cyst \\-all. It is hoped that an extension of this comparative study will eventually enable determination of the taxohe nomic status of an amoeba from its ultrastructure. Although this would a laborious task it v-oulti perhaps be less arduous than identification on the dividing nucleus! In view of these discovered cytoplasmic tiifl‘erences and the known tlifferences in mitotic mechanisms between the Schizopyrenidae and Hartmannellidae, it is surprising to find that the ultrastructure of the interphase nucleus is so uniform in the limnx amoebae. ‘I’he only possible familial tlifference lies in the relationship of the nuclear envelope to the endoplasmic reticulum. In the schizopyrenids the outer nuclear membrane is frequentl! seen to be in continuity with a tubule of the reticulum, n-hereas in the hartmannellitls no such continuity has been observed. Conncsions between the two membrane systems have often been noted in metazoan cells. Barer et rrl. [5] contend that such links rcprescnt the transformation of one system to Fig. 17.-Acanlhamoekt sp. Part of cyst showing and Pb(OH),. x 20,000.
stratified
wall and various
Fig. 18.-Schizopyrenus sp. Part of cyst showing lack of organized structure reticulum around each mitochondrion. KMnO, and PTA. x 25,000. Experimental
Cell Research 26
inclusions.
KBlnO,
in wall and fold of
Electron microscopy of Limax amoebae
513
514
K. Vickerman
another, a process which takes place on a grand scale \\-hen the nuclear envelope disappears at metaphase and, in the reverse direction, \vhen it is reformed around the chromosomes at telophase. The fact that Saeglerirr and Schizopyrenus show continuity bctxveen the outer nuclear membrane and reticulum indicates that this continuity reflects more than just a stage in lvithdrawal or reformation of the nuclear membrane. The links probably have a necessary function in the interphase cell, such as feeding membrane into the grooving nucleus, or conversely, by membrane flow [C;] in the opposite direction, in the transfer of nuclear material to the cytoplasm [43]. Compari.son of limax amoebae with other forms.-The large fresh\\-ater amoebae have been extensively studied under the electron microscope by many workers (e.g. see [24, 27, 28, 31, 371. The so-called limrrx amoeba, Hycrlodisczrs simplex, studied by M’ohlfarth-Bottermann [48, 491 is quite dinerent from the forms described here and belongs to another family, the Hyalodiscidae. Its fine structure is more akin to that of the large freshjvater amoebae. It seems probable that variations in the life-cycle can accourit for the main differences in ultrastructure hetlveen the large amoebae and soil limrrx forms. The evidence for a life-cycle involring alternation of trophic and encysted forms in the large amoebae is unconvincing. In an\- case their occurrence in large permanent bodies of water almost precludes the necessity for encystation. Birheck and Mercer [7], in an interesting article on the dif’ferences between secreting and non-secreting cells, hare cited the slime-mold amoebulae as examples of the former and the large amoebae as examples of the latter. The Zirnn.r amoebae are in many \vays comparable to the amoebulae of mycetozoans in their fine structure [13, 14, 15, 2.3, 261, and the distinction applies just as \vell to the organisms under discussion here. At encystation, in addition to secreting the cyst wall, the 1inm.r forms are actively pumping out fluid from their cytoplasm; it has been assumed that this fluid is water but there is nothing to indicate that it does not contain osmotically active
Fig. 19-Fig.
21.-Acnnthamoeba
sp. All KMnO,
Fig. lg.-Part of cyst wall showing and outer cyst walls. s 20,000.
and PTA.
debris in outer cyst wall (oao) and space between inner (icw)
Fig. 20.-Part of cyst showing so-called pore at lower right. Cytoplasm and mitochondria with intra-cristal bodies. x 12,000.
has numerous fat globules
Fig. 21.-Early stage in cyst wall formation. empties into the water vacuole. x 50,000.
thickness.
Experimental
Cell Research 26
The wall is of uneven
A small tubule
Electron microscopy of Limax amoebae
Experimenful
Cell Research
26
516
K. Vickerman
substances. From the current studies it appears that the endoplasmic reticulum plays an important role in this process in that fluid is expressed into its channels for conduction to the surface. ,4n abundance of cytoplasmic membranes is characteristic of secreting cells on Birbeck and Mercer’s classification. The large amoebae have fe\v cptoplasmic membranes and these are in the form of Golgi stacks and vesicles. In view of the fact that descriptions of these organisms are taken from osmium-fixed material, however, and that lengthy osmium fixation leads to resiculation of tubular elements, it would be interesting to see what membrane systems are revealed in large amoebae following permanganate fixation. The fringed surface membrane found in Amoebrr, Pelomyxa and Hynlodiscus does not occur in soil amoebae. It has been suggested that its function is to provide an increased surface for absorption [‘~7] and there is some evidence that the hair-like projections do act as binding sites for material taken in by pinocytosis. The presence of membrane projections in small amoebae may be rendered unnecessary by the relatively larger surface area. It is worth noting, however, that in hartmannellids, micropseudopodia (Fig. 1) of the determinate type [8] are a common feature and these may serve to increase binding areas. That pinocytosis is common in the hartmannellids is witnessed by the readiness with which they gro\\- \\-ithout solid food in a liquid medium. Relntionships of membrune systems.-The problem of the continuity or discontinuity of cell membrane systems has been reviewed by Robertson [33] and Sjiistrand [39]. It has been argued that amoeboid movement is incompatible with permanent association between cptoplasmic elements [24], but in the present study continuity of some of these elements with one another has been noted. The theories of amoeboid movement advanced by Jlast and Doyle [‘23!, Goldacre and Larch [17] and Goldacre [16] involve synthesis of new membrane at the tip of the advancing pseudopod and resorption of membrane accompanying solation of the cortical gel at the uroid. Is this resorption and resynthesis in actual fact an inward-tucking of the surface membrane to form a continuous tubular system \vhich later re-emerges as surface membrane‘? From the present work this seems unlikely. The membrane profiles in the pseudopod of Schizopyrenzzs represent discrete vesicles or tubules as shown by serial sectioning. However, the formation of new membrane by a coalescence of these vesicles cannot be ruled out, and this effect would parallel membrane formation as envisaged by Afzelius [2] in the cleaving sea urchin egg, by Stewart and Stewart [M] in the sclerotizing slime-mold Experimental
Cell Resenrch 26
Electron
microscopy
of Limax
amoebae
517
plasmodium, and in other cases reviewed by these authors. The site of membrane resorption is most likely not the uroid itself but just anterior to it. In-tuckings of the surface membrane have been seen in this region. The uroid is most likely an excretory organelle [19, 501. In 1ima.r: amoebae it can be shed with no ill effect. The fragmentary evidence obtained so far points to interconversion of membrane systems in amoebae rather than permanent fusion to form a continuous system. Changes in mitochondrin rzt encystation.--The vast literature on changes in mitochondrial morphology induced bp certain physiological and pathological conditions has been reviewed by Rouiller [%I. The changes observed in the mitochondria of Schixopyrenzzs and Acanthamoebn at encystation are among the most striking of mitochondrial transformations. Cup-shaped mitochondria of the type found in Schiropyrenrzs have been observed previously in rat testicular cells by Christensen and Chapman [10] and their interpretation and occurrence elsewhere are discussed by these authors. The relatively greater surface area presented by this shape is believed to facilitate diffusion of respiratory substrates into the mitochondrion and, as this is the rate-limiting factor in aerobic respiration 7211, the result is increased metabolism. In the cyst the mitchondria are spherical, thus presenting minimum surface area and presumably minimum metabolic activity. In Acrznthnmoebrz the change in shape of the mitochondria at encystation is not so marked as in Schkopyrenzzs, but they arc reduced in volume in the cyst and once again tend to be spherical. The internal bodies of the mitochondria of Acnnthnnzoebn are unique in their location in the cristae: other electron-dense bodies in mitochondria lie in the matris. The increase in size of these bodies at encystation is of special interest in view of the work ol’ Klein and Sell’ [21] on the isolated mitochondria of ilcrzntizrznzoebrr. They found that the respiratory rate was proportional to mitochondrial volume and that both \\-ere affected by the tonicity and ionic concentration in the surrounding medium, increasing tonicity having a depressing effect on respiratory activity. At encystation it \vas found here that the mitchondria decrease in volume and that the volume of the mitochondrial lumen is decreased further by the swelling of the intra-cristal body. In the cyst respiration is virtually negligible, yet it has been shown [A] that cncystation demands an ample supply of oxygen. It is probable that the high oxygen requirement is associated with the osmotic work involved in the expression of water from the cytoplasm. The increasing tonicity results in shrinkage of the mitochondria anal accompanying depression of respiratory activity. Perhaps the swelling Experimental
Cell Research
26
518
K. Vickerman
of the intracristal bodies provides the final clamp on respiration, but at the moment the constitution of these bodies is unknown and their function purely speculative. SUMMARY The fine structure of some of the linmx amoebae of the soil is described. These amoebae are similar in appearance when viewed xvith the light microscope but can be broadly classified into two families on their mode of nuclear division. The dif’ference in mitotic mechanisms is correlated with dill’erent patterns of ultrastructure in the non-dividing cells. Amoebae of the family Hartmannellidae have mitosis similar to that found in metazoan cells. The endoplasmic reticulum is characteristically in the form of flattened saccules and shows neither close association with the mitochondria nor a continuity \vith the nuclear envelope. There is a “spongiome” of tubules associated with the contractile vacuole. The cyst \\-a11 is laminated. The mitochondria in dccznthnmoebn have intra-cristal granules lvhich increase in size at encystation. The Schizopgrenidae have “promitotic” division of the nucleus. The endoplasmic reticulum consists of tubules and isolated flattened saccules which are closely applied to the mitochondria. Its elements are in continuity with the outer nuclear membrane. There is no well-developed “spongiomc” and the cyst \\-a11 is unstratified. The mitochondria lack intra-cristal bodies and in Schi:opyrenm are often cup-shaped in the trophic amoeba. amoebae and the The difTerences in fine-structure between the limnr large fresh-water forms are associated with differences in the life cycle. ,1n alternation of trophic and encysted phases is characteristic of the life cycle of soil amoebae. It is suggested that the abundant endoplasmlc reticulum in these forms is active in “dehydration” of the amoeba at encystation, and the changes in mitochondrial morphology at this time are associated \\ith depressed respiratory activity. It is a pleasure to thank professor J. Z. Young for the facilities of his electron microscope laboratory, Dr. E. G. Gray, Dr. J. D. Robertson and Professor J. 0. Corliss for their advice, and Mrs. R. Tilly for help with the photography.
REFERENCES 1. ADAM, K. h1. G., J. Gen. Microbial. 21, 519 (1959). 2. AFZELIUS, B. A., Ezpt[. Cell Kesearch 10, 257 (1956). 3. ALTMANN, H. \V., 2. Krebsforsch. 58, 632 (1952).
Experimentul
Cell Research 26
519
Electron microscopy of Limas amoebae
4. Bask, R. N., J. Protozoof. 5 (Suppl.), 13 (1958). 5. BARER, S., JOSEPH, J. and MEEK, G. A., Proc. Roy. Sot., Lond. B. 152, 353 (1960). 6. BESNETT, H. S., J. Biophys. Biochem. Cylol. 2, Suppl. 99 (1956). 7. RIRBECK, M. S. C. and MERGER, E. H., Nature, Load. 189, 558 (1961). 8. BOVEE, E. C., Proc. Iowa Acad. Sci. 60, 599 (1953). 9. Box& E. C. and JAHN, T. L., J. Profozoof. ? (Suppl.), 8 (1960). IO. CHRISTENSEN, A. I<. and CHAPMAN, G. B., Ezpff. Cell Research 18, 576 (1959). 11. DEUTSCH, K. and SWANN, M. X, Quarf. J. Microscop. Sci. 100, 13 (1959). 12. FAUR&FREMIET, E. and ROUILLER, C., J. Profozoof. 6, 29 (19.59). 13. GEZELIUS, K., Ezpff. Cell Research 18, 425 (1959). 14. __ ibid. 23, 300 (1961). 15. GEZELIC'S, K. and RKNBY, B. G., ibid. 12, 265 (1957). 16. GOLDACRE, R. J., Ezpff. Cell Research Suppl. 8, 1 (1961). 17. GOLU~CRE, R. J. and LORCH, I. .J., Xafure, Lond. 166, 497 (1950). 1X. GRASSI?, P. P., Proceedings of the Stockholm Conference on Electron Microscopy, p. 143, Stockholm, 1956. 19. HOPKINS, D. L. and WARSER, K. L., J. Parasifof. 32, 175 (1946). 20. KARRER, H. K., .I. Biophys. Biochem. Cytof. 7, 357 (1960). 21. KLEIN, R. L. and NEFF, R. J., Expff. CefZ Research 19, 133 (1960). 22. LUFT, J. H., J. Hiophys. Biochem. Cyfof. 2, 799 (1957). 23. MAST, S. 0. and DOYLE, W. L., Arch. Profisfenk. 86, 155 (1935). 24. MERGER, E. H., Proc. Roy. Sot., Lond. B 150, 216 (1959). 25. MERCER, E. H. and SCHAPFER, B. M., .I. Biophys. Wiochem. Cyfol. 7, 353 (1960). 26. ~IUHLETI~ALER, I(.; Am. J. Botany 43, 673 (1956). 27. PAPPAS, G. D., Ann. S. 1.. Acad. Sci. 78, 488 (1959). 28. PAPI~AS, G. D. and BRAX~T, P. \V., .I. Biophys. Biochem. C!yfof. 4, 485 (1958). 29. P.4RSOSS, D. IJ., DAKUEN, K. B., LINDSEY, D. L. and PRATT, G. T., ibid. 4, 485 (1958). 30. POLICARI>, A., COLLRT, A. and PREOERMAIN, S., Buff. Microscop. Appf. 7, 49 (1957). 31. RAFALKO, J. S., .7. Morphof. 81, 1 (1947). 32. RAY, I). L. and HAYES, R. E., ibid. 95, 159 (1954). 33. RORERTSON, .I. D., Niochem. Sot. Symp. 16, 3 (1959). 34. ROTH, I,. E. and I~ANIELS, E. \1’., J. Biophys. Biochem. (,‘yfof. 9, 317 (1961). 35. ROUILLER, (:., Intern. Rro. Cyfol. 9, 227 (1960).
36. SCHNEIUF.H, I,.. .I. Profozoof. 7, 75 (1960). 3i. SCHNEIl)ER, L. Xlti ~~OHLFARTH-BoTTEK1M.\SS, li. E:., Protoplasma 3X. SINGH, B. N., Phil. Trans. Roy. Sot., Load. H. 236, 405 (1952). 39.
40. 41.
42. 43.
44.
51, 377 (1959).
SJ~STR~ND, F., in Riophysical Scienrc--A Study Program, p. 301. Ed. Sew York, 1959. STEWART, P. A. and STEWART, B. T., Expff. Cell Research 23, 471 (1961). STOECKENIUS, W., ibid. 13, 410 (1957). VICKERRIAN, K., Xafure, Lond. 188, 248 (1960). --Parasitology 50, 351 (1960). VOLKONSKY, hf., Arch. Zoof. Expf/. (kn. 72, 317 (1931). N'ATSON, RI. L., J. Riophys. Biochem. C{yfol. 1, 257 (1955).
Oncley,
\Viley,
45. 46. -ibid. 4, 475 (1958). 47. WILLMER, IX. N., Expff. Cell Research Suppl. 8, 32 (1961). 4X. \\'OIILI~~RTH-BOTTERMANS, I<. E., Protoplasma 52, 58 (1960). 49. --ibid. 53, 259 (1961). 50. Z.\#.IN, v., Trans. Roy. Sot. Trap. Med. Hqg. 55, 263 (1961).
Experimental
Cell Research 26
Cell Research 26, 497.-519 (1962)
PATTERNS
AN
497
OF CELLULAR ORGANISATION LIMAX AMOEBAE ELECTRON
MICROSCOPE
IN
STIJDY
K. VICKERMANl Department
of Zoology,
University
College London, England
Received July 28, 1961
THE majority
of the small amoebae vvhich are abundant in the soil have a single broad pseudopodium and a vesicular nucleus with a large central nucleolus. They are the “limax” amoebae of earlier writers, but this epithet has little taxonomic significance for their superficial similarity belies fundamental differences in cellular organization. These differences reveal themselves to the light microscopist only at certain stages in the life cycle, notably at nuclear division and when the amoebae undergo cellular transformation (metaplasia) in the face of certain environmental changes [38]. It is reasonable to suppose that differences in mitotic mechanisms and ability to transform have a more profound ultrastructural basis. It is the aim of this investigation to discern patterns of ultrastructure in the Zimax amoebae and, if possible, to correlate them with events in the cellular cycle. Strains
of Amoebae
Details of the strains investigated are given below together with brief notes on their cytological characteristics as seen with the light microscope. Singh’s classification [38] has been adopted. The main features of nuclear division for each strain were determined from phase-contrast observations on living material and from smears of amoebae stained by a modified Delamater technique [43].
Amoebae with “promitotic” division of the nucleus, i.e. the nucleolus persists throughout division and divides to form two polar masses; the nuclear membrane remains intact and amoeboid movement continues unimpaired. l Wellcome
Research
Fellow. Experimental
Cell Research 26
K. Vickerman
498
*Vaegleria gruberi (Schardinger). This amoeba can temporarily transform into a flagellate under conditions of divalent cation deprivation [47]. It forms spherical cysts with pores in the cyst wall. The strain used vvas from the Cambridge Culture Collection, No. 151813. The flagellate stage of Naegleria will not be dealt with in this paper. Schizopyrenus sp. Similar to Naegleria in amoeboid and encysted stages but the cyst wall lacks pores. A flagellate stage is never produced. This clonal strain isolated from soil resembles S. russelli Singh, except for the fact that in the present species there is a single smooth cyst vv-all. S. russelli forms a double-walled cyst, the outer wall being slightly vv-rinkled. A preliminary study of Schizopyrenus russelli has shown that its ultrastructure is very similar to that of the species described here.
Amoebae in which nuclear division is very reminiscent of that found in most metazoan somatic cells. The nucleolus and nuclear membrane disappear and amoeboid movement ceases during mitosis. There is never a flagellate stage in the life-cycle. Acanthamoeba sp. This strain was isolated in clonal culture from soil and conforms closely to Singh’s Harmanella rhysodes, also to Volkonsky’s [44] account of Acanthamoeba castellanii. As the organism in culture has the slender mitotic spindle characterizing Volkonsky’s genus, this is the name adopted here without further specification. Hartmanella astronyxis Hay. Only a few observations have been made on this species, the ultrastructure of which has been briefly described from osmium-fixed material by Deutsch and Swarm [ll]. Cambridge Collection No. 1534/l. METHODS
All strains were grown in monoxenic culture with Aerobacfer aerogenes on agar plates (0.2 per cent peptone, 1.5 per cent agar) at 25°C. Two hr before fixation the plates were flooded with Lang’s colloidal gold suspension in distilled water. The organisms were washed off the plates and spun down to a loose pellet at 2500 rpm before dispersal in cold fixative. Fixation was in Palade’s 1 per cent osmium tetroxide buffered at pH 7.2 for 30 min to 2 hr, or in 0.6 per cent potassium permanganate, similarly buffered, for 2 hr and always at 4°C. Dehydration was in graded ethanolwater mixtures (30 min changes) with three l-hr changes of absolute ethanol and in some cases 1 hr staining in 1 per cent phosphotungstic acid in ethanol. After 3 hr immersion in Araldite resin M the pellets were imbedded in Araldite mixture in open troughs, then hardened at 50°C for at least two days. Grey, silver and gold Experimental
Cell Research 26
Electron
microscopy
of Limax
amoebae
499
sections were cut on the Porter-Blum microtome and mounted on carbon films. Sections of unstained material were then stained overnight in either lead hydroxide or many1 acetate as prescribed by Watson [46]. Sections were examined in the Siemen’s Elmiskop I at an accelerating voltage of X0 kV.
OBSERVATIONS Bulfered permanganate was found to be superior to osmium tetroxide in t)reserving the natural form of the amoebae and the following descriptions apply principally to material fixed in this way. M’here striking differences have been observed between osmium and permanganate fixed material, these are noted.
Trophic
Amoebae
Ectoplasm and endopZasm.-With the light microscope the ectoplasm of amoebae is seen as the clear cytoplasm forming the pseudopodium or surrounding the rounded-up amoeba; the ectoplasm is packed with inclusions-mostly mitochondria and food vacuoles. The two zones are also readily distinguished with the electron microscope; here the ectoplasm is seen to be not entirely devoid of inclusions for the profiles of small membranous structures occur throughout its substance (Figs. 1, 2, em). Serial sections indicate that these membranes do not form a continuous canalicular system but are isolated vesicles and parts of tubules. Plasma membrane and membrane structure.-Surrounding the amoeba is a membrane which in some sections is seen to have the “unit membrane” structure described by Robertson [X3], i.e. it has two electron-dense layers enclosing an electron transparent layer, the thickness of the whole being approximately 80 A. The unit membrane structure is evident in other membrane systems of amoebae fixed in permanganate (Figs. 8, 9). Organisms fixed for short periods in 0~0, occasionally exhibit membranes with unit organisation (Fig. 4). Endoplasmic reticulum-Well-defined membrane systems are present in the endoplasm of all amoebae but their form in the hartmannellids is not identical with that observed in the schizopyrenid amoebae. In Acanthamoeba the elements of the reticulum are numerous flattened vesicles and tubuli, arranged in stacks or bundles and usually lying parallel to the surface of the amoeba (Fig. 1, er). The membranes are approximately 200 A apart. Golgilike stacks of membranes have been seen in Hartmannella astronyzis (Fig. 8). limax
Experimental
Cell Research 26
500
K. Vickerman
In Schizopyrenus and Nueglerin the reticulum is seen as branching tubules and compressed saccules, but here the latter are in very intimate association with the mitochondria and often partially envelope one or more of these structures (Fig. 2). In Schizopyrenus the membranes have a spacing of only 150 A in the trophic amoeba. Osmium-fixed specimens have the reticulum obscured to some extent 1~~ a coarse precipitate in the surrounding matrix (Figs. 6, 7). This presumably represents the ribosomal granules; it is difficult to distinguish between granular and agranular reticulum. There is a tendency for the reticular membranes to form rounded vesicles when fixed for long periods in GO,. Mitochondria.-Perhaps the most prominent endoplasmic inclusions are the mitochondria. These differ in form according to the species of amoeba. The mitochondria of Acanthamoeba have been briefly described elsewhere [43]. They are commonly oval in shape, 0.5-1.5 ,U in length and 0.5-l p across. However, they are frequently distorted and may appear branched or bent in sections. They are rarely cup-shaped, but an extensive invagination may occur towards one end giving the appearance of an empty vesicle in sections. The outer mitochondrial membrane is separated by a space of 100 L%from the inner bounding membrane which gives rise to tubular cristae. These project into the mitochondrial lumen in an irregular fashion and are occasionally seen to branch or traverse the lumen. Sometimes one or, more rarely, two small spherical inclusions can be seen lvithin a mitochondrion. These bodies are actually intra-cristal in location, up to 1000 Ai in diameter, and have an electron-dense cortex (Figs. 1 inset, 6). In Hartmnnnelln ctstronyri.s intra-crystal bodies lacking an electron-dense cortex have been observed (Fig. S), but as yet it is not possible to pronounce on their homology with the structures in Acanthcrmoebn. The mitochondrial matrix is very electron-dense follo\ving osmic fixation, and has a honeycomb structure (Fig. 6).
Aer, bacteria; cq, colloidal gold particles; cv, contractile vacuole; CW, cyst wall; em, ectoplasmic ert, reticulum tubule; fu, food vacuole; ich, intramembrane profiles; er, endoplasmic reticulum; vacuole; cristal body; mit, mitochondrion; mf, myelin figure; nl, nucleolus; n/v, nucleolar nm, nuclear envelope; nmp, pore in nuclear envelope; sm, surface membrane; sp, spongiome tubules: WU, water vacuoles. Pb(OH),, lead hydroxide tate stained.
stained;
PTA, phosphotungstic
acid stained;
Fig. I.-AcanthamoeDa sp. Trophic amoeba and part of cyst. Note in region of spongiome associated with the contractile vacuole, “Kernspalten” (ks). The difference in size of intra-cristal bodies cyst is readily seen; inset shows location of intra-cristal body x 15,000 (inset x 240,000). Pb(OH),. Experimental
Cell Research 26
and t.rAc,
uranyl
ace-
extensive reticulum and tubules also nucleus with nucleolus and in mitochondria of amoeba and in mitochondrion. KMnO, and
Electron microscopy of Limax amoebae
501
502
K. Vickerman
In Schizopyrenus the mitochondria are ovoid, cylindrical, discoid or cupshaped in form. Those belonging to this last category are particularly common; their three-dimensional shape has been determined from serial sections. The cristae are more flattened in cross-section than those of Accrnthamoeba and they lack internal bodies. The mitochondria of Naeglericr are usually ovoid, but otherwise similar to those of Schizopyrenus. Food vacuoles.-These are lined by unit membranes and can be identified by their contents, ingested Aerobacter and their remains, and colloidal gold particles. Most noticeable, particularly in permanganate-fixed preparations, are concentric or spirally-wound membranes surrounding food remains within the vacuole (Figs. 2, 10, 11) or lying outside the amoeba. These “myelin forms” are common in phagocytic cells (e.g. see 113, 14, 20, 251). They are probably artifacts of fixation of phospholipid [SO, 411 and appear to be produced in the digestion of bacteria (Fig. 11). Occasionally the membranes can be seen to have unit structure and a definite periodicity (Fig. 12). There is no evidence for the continuity of these membranes \vith the membrane lining the vacuole. Although bacteria are often seen sticking to the surface of the pseudopodium, the principal site of ingestion seems to be along the body of the amoeba, anterior to the contractile vacuole and most likely on its under side Invaginations of the surface memwhen it is crawling along a substratum. brane are seen in this area. The evidence that the food vacuoles are simply dilatations of a permanent intracellular canalicular system, continuous with the surface membrane, is equivocal. Certainly reticular tubules can be seen abutting on the vacuole membrane and occasionally these approach the surface of the organism colloidal gold particles have been observed in dilated (Fig. 10). Moreover, tubules, but in such numbers as to make distinction from a small food vacuole difficult in an isolated section. Single gold particles have not been seen in reticular tubules as might be expected if these formed part of a permanent alimentary system.
Fig. 2.-S’chizopyrenus sp. Trophic amoeba with nucleus in metaphase. The nuclear envelope is still intact and amoeboid movement continuing, but the nucleolar material is distributed in two polar masses (pm) separated by the chromosome plate (cp). Note food vacuoles with enclosed myelin forms, collapsed contractile vacuoles near uroid (see Fig. 13), and association of reticulum with mitochondria. Insets 2 a and 2 b are two preceding serial sections through cup-shaped mitochondrion at top left. KMnO, and UrAc. x 10,000. Experimental
Cell Research 26
Electron microscopy of Limax amoebae
Experimental
503
Cell Reseurch 26
504
K. Vickerman
Contractile vncuole.-Under phase contrast the contractile vacuole system of the living amoeba is seen to be composed of a principal vacuole and several subsidiaries apparently emptying into it. It discharges near the uroid or posterior end of the amoeba. With the electron microscope the vacuole and its subsidiaries in diastole are seen as membrane bounded vesicles. In the schizopyrenids the vesicles collapse at systole to give profiles which are reminiscent of the dictyosomes or Golgi sacs of other protozoa [18]-that is, flattened saccules expanded at either end (Fig. 4); Nassanov’s homology of the contractile vacuole and Golgi element is recalled. Many workers with larger amoebae (e.g. see [24, 281) have described the association of mitochondria and numerous small vesicles with the contractile vacuole. In the 1ima.x amoebae there is no marked accumulation of mitochondria in this region and, judging from their profiles in section, the associated membranous elements are tubules rather than vesicles in Acrrntharound the vacuole, comparable to rrmoebcz sp. These form a “spongiome” that observed in some ciliates [la, 361. Fig. 5 shows that in Acanthaznoebn sp. the tubules of the spongiome apparently empty into a common duct or into the main vacuole. A permanent pore for discharge of the contents of the contractile vacuole, so common in ciliates, has not been discerned in amoebae. Other cytoplczsznic inclusions.-Fat globules are common in the cytoplasm of dccznthnznoebrz and are readily distinguished in osmium-fixed material visible in the cytoplasm of the same amoeba (Fig. ‘i, fg). Al so occasionally are irregular masses of granules (Fig. 13, gm), each granule measuring about 200 A4 across. The masses do not have a crystalline structure and as they occur in permanganate-fixed material it seems likely that they are glycogen reserves [22]. Other inclusions in trophic amoebae of Acnnthnznoebn are ovoid structures ~111 to 2000 .i in length and 1000 -4 wide (Figs. 15, 16, up). They are surrounded by a unit membrane which is separated from an inner, concentric and similar membrane by a space of 200 ,$. These particles bear considerable resemblance to certain tumour viruses in their form (e.g. see [as]), but they are 10&20 times larger than such virus particles. The possibility of their being symbionts
Fig. 3.-Xaeyleriu gruheri. iVucleus of trophic amoeba showing nuclear arrows) and nucleolus with central vacuole. 0~0, and PTA. x 40,000. Fig. 4.-Xuegleria “unit membrane” Experimentul
envelope
with
gruheri. Connexion between outer nuclear membrane and reticulum structure at points arrowed. 0~0, and PTA. x 78,000.
Cell Research 26
pores (at showing
Electron microscopy of Limax amoebae
Experimentul
505
Cell Research 26
K. Vickerman is an interesting one as symbiotic micro-organisms are knolvn to he associated with large amoebae [34]. Another possibility is that they represent precursors of mitochondria, the cristae arising as outgrowths from the inner membrane. In view of the large space between the hounding membranes this seems unlikely, holvever. At the moment these inclusions cannot be accounted for with any confidence. TIze nzzcZeus.-Notwithstanding the remarkable differences in their mitotic processes, the amoebae have interphase nuclei of very similar structure. The nucleolus is electron-dense in osmium preparations (Fig. 3) and motierately so in permanganate-fixed material (Fig. 1). In the latter, electrondense granules approximately 200 a across are found. There is an electronpale space (Figs. 3, 17, nlv) to the nucleolus; it can be seen as a vesicle with the light microscope. The nuclear envelope consists of two apposed membranes separated 1)~ a space of about 250 ,% in 4cunthamoehn and Srreglerirr only 150 A in Schi:opyrenus. Pores are present in the envelope and these hare a diameter of approximately 700 A. In Acnnthumoehn sp. the nuclear membrane often shows numerous invaginations enclosing cytoplasm (Fig. 1, I;s) anti these described by Altman [3] in certain cancer are similar to the “Kernspalten” ~11s; they have not been observed in schizopyenid amoebae. On the other hand, although the outer membrane of the nuclear envelope in these amoebae often shows connexions \vith the reticular tubules (Fig. 4), as described in metazoan cells, such connexions have not yet been seen in hartmannellicls. Light microscope studies sho\v “Feulgen positive” material between the nucleolus and nuclear membrane [31, 381, but a fine granular prwipitatc is all that is usually found here with the electron microscope in osmiumfixed material; in permanganate-fixed specimens this region lacks inclusions altogether. Dividing forms.-Division stages are seldom encountered in fixed smears examined with the light microscope, so it is not surprising to find that the! Fig. 5.-Hartmurmellu astronyxis. Part of mitochondrion bodies. KMnO, and PTA. Y 50,000.
showing what appear lo be intra-cristal
Fig. (i.-,iccmlhamoeba sp. Mitochondria of trophic forms showing dense honeycomb of matrix after osmium fixation. Note intra-cristal bodies. 0~0, and PTA. x 27,000.
structure
1.‘ig. 7.--Acanthamoebu
*: 30,000.
sp. Fat globules
(fq) and reticulum
elements.
Fig. X.-Hurfmanne/la astrompis. Golgi-like stacks of membranes unit membrane structure. KMnO, and PTA. x 54,000. I;ig. Cl-Acanthamoeba and PTA. Experimental
sp. Endoplasmic
Cell Research
26
reticulum
showing
unit
0~0,
and 1’TA.
from trophic membrane
amoeba, showing structure.
KRfnO,
Electron microscopy of Limax amoebae
Experimental
507
Cell Research 26
508
K. Vickerman
are also rare in sections examined with the electron microscope. Obvious have never been seen, but a series of sections mitotic figures of Acanthnmoebo was obtained through Schizopyrenrzs which matches well the appearance of the amoeba stained in metaphase (Fig. 2). The nucleolus has divided to form two polar masses, each of which has a spongy structure as seen in section. A space of low electron density separates the two masses and this is interpreted as the metaphase plate. The nuclear envelope is still intact and there are no traces of centrioles either inside or outside the nuclear membrane [al, X3!, nor have these been seen in sections of non-dividing amoebae. Encysted Forms soil amoebae encyst. In culture the strains described here do so 1-4 days after subculture. The changes at encystation as observed under the phase contrast microscope may he summarized briefly as follows. The individual amoebae stop moving and feeding and become spherical. Compacted food remains are cast out and numerous small lvater vacuoles appear in the c;vtoplasm; these can be seen bursting randomly over the surface of the organism. This results in shrinkage in size of the Tvhole cell and this active “dehydration” process continues during and after the laying down of the cyst \\-all. The nucleolus becomes much smaller, and rcfractile granules are often conspicuous on the nuclear membrane in the fully formed cyst. The cyst zutrll.-In all amoebae the cyst wall appears to be secreted at the surface membrane. This is the “multiple-layered membrane” seen 1)~ Deutsch and Swami ill] in Hartmrtnelln nstronyxis. In dcnnthcrmoebrr it is composed of many sheets or fihres, 100 LX thick, lying parallel to the membrane. I)uring incipient encystation the wall is of uniform thickness and the cyst rounded. As encystation proceeds, shrinkage of the organism results in buckling of the wall, an effect w-hich is exaggerated by the continual discharge of \vatcr vacuoles and food remains into the spaces bet\\-een the sheets formChanges
Fig. i&Fig.
at encystation-All
13.--All
Schizopyrenus
sp. KRlnO,
and ITrAc.
Fig. lo.-Showing food vacuoles with myelin forms and ingested colloidal gold. The \acuole Lo the right has a diverticulum (diu) from which a tubule proceeds to the surface of the amoeba. Y 40,000. Fig. Il.-Aerobacfer Fig. ia.-Part Fig. 13.-Uroid Eqwimenfal
in process of being digested inside food vacuole.
of food vacuole
showing
periodicity
region of amoeba showing (Ml Kesearch 26
collapsed
of myelin contractile
forms.
x 31,000. x 250,000.
vacuoles.
x 16.000.
Electron microscopy of Limax amoebae
Er~erimental
509
Cell Resectrch 26
510
K. Vickerman
ing the wall (Figs. 17, 19). The result is a cyst of irregular shape, often with two distinct cyst walls, an inner and an outer, the two meeting only at “pores” in the wall (Fig. 20). This appearance is more pronounced in H. astronysis in which the inner cvst wall is star-shaped and the outer more or less circular [32]. Electron micrographs show that it is doubtful that true pores exist: the so-called pores are simply those parts of the cyst wall that hare not been inflated by matter discharged from the encysting amoeba. The ejected matter in the cyst wall appears to he composed of disrupted membranes and in eludes the virus-like particles mentioned above (see p. 504). The cyst wall of Schixpyren~s (Fig. 1X) is not layered, nor does it contain ejected food material. It is of fairly uniform thickness (0.5-l ,u) in any one cyst; its outermost parl is probabl\- gelatinous, as colloidal gold particles from the surrounding medium may be found imbedded in it. Srze~ylerirr {Irdwri has a cyst wall of similar structure, but the pores visible \vith the light microscope have not been identified in electron micrographs. Cyst contents.-These are difficult to fix satisfactorily with osmium and the following account is taken from permanganate-fixed material. The most obvious change in the cytoplasm at encystation is the appearance of man! small water vacuoles. In the schizopyrenids it appears that water is expressed into the lumina of some of the reticulum tubules as these are distended but empty in micrographs. They occasionally show openings at the surface menmanes. In Acrrnthnmoehrr more delinite vacuoles are found scattered throughout the cytoplasm and in some there is a sign of continuity with an cndoplasmic tubule (Fig. 21). The contractile vacuole svstem is seen only as the spongiome in encysted amoebae. The most striking ultrastructural change occurring at encystation lies in these are nearly all spherical and diminthe mitochondria. In Schizopyrenus ished in size, 0.5-l ,U in diameter, and more completely enveloped by the associated reticulum (Fig. 18). In dcnnthnmoeha the mitochondria also assume a spherical shape and decrease in over-all diameter. The intra-cristal granules, however, increase in size to become vesicles 3000-3000 a across. Their cortical electron-dense material is distributed as particles of irregular size and shape (5OG300 *%) at the boundary of the vesicles (Figs. 17, 20).
Fig. 14.--9negleria
gruheri. Contractile
vacuole
region in systole.
KMnO,
and PT,4.
x 25,000.
Fig. 15.-Acanthamoeba sp. Contractile vacuole in partial diastole showing tubules of spongiome draining into common duct leading to vacuole. Note virus-like particles (up) and glycogen masses (gm) in surrounding cytoplasm. KMnO, and PTA. x 25,000. Fig. 16.-Single Experimental
virus-like
inclusion.
Cell Research 26
KhlnO,
and PTA.
x 60,000.
Electron microscopy of Limax amoebae
Experimental
511
Cell Research 26
512
K. Vickerman DISCUSSION
Compcxratiue cytology of schizopyrenid crnd hartm<~nnellid nmoebne.Amoebae are popular experimental organisms, and their ability to gronunder strictly defined cultural conditions [l ] enhances their value to the cell biologist. As they have few characters on which to make a tasonomic diagnosis, however, experimental \vork is frequently carried out on unidentified or misidentified strains. Attempts to classify amoebae on such characters as amoeboid movement and type of pseudopod formation [8, ‘31 are scnsibl? practical for the ecologist who Avishes to identify organisms at sight, but are often regarded as suspect by experimentalists who find some of these characters labile under laboratory conditions [3‘2]. The form of the dividing nucleus may be a reliable criterion, but it is often revealed only after intensive perusal of thousands of stained specimens. It is interesting to note that in the two families of limcrr amoebae studied here, differences in cell division are associated xvith differences in cgtoplasmic organization of the non-dividing cell. From this preliminary \\-ork it n-oultl appear possible to distinguish the schizopyrenids and hartmannellids on the ultrastructural form of such structures as the endoplasmic reticulum, contractile vacuole, mitochondria and cyst \\-all. It is hoped that an extension of this comparative study will eventually enable determination of the taxohe nomic status of an amoeba from its ultrastructure. Although this would a laborious task it v-oulti perhaps be less arduous than identification on the dividing nucleus! In view of these discovered cytoplasmic tiifl‘erences and the known tlifferences in mitotic mechanisms between the Schizopyrenidae and Hartmannellidae, it is surprising to find that the ultrastructure of the interphase nucleus is so uniform in the limnx amoebae. ‘I’he only possible familial tlifference lies in the relationship of the nuclear envelope to the endoplasmic reticulum. In the schizopyrenids the outer nuclear membrane is frequentl! seen to be in continuity with a tubule of the reticulum, n-hereas in the hartmannellitls no such continuity has been observed. Conncsions between the two membrane systems have often been noted in metazoan cells. Barer et rrl. [5] contend that such links rcprescnt the transformation of one system to Fig. 17.-Acanlhamoekt sp. Part of cyst showing and Pb(OH),. x 20,000.
stratified
wall and various
Fig. 18.-Schizopyrenus sp. Part of cyst showing lack of organized structure reticulum around each mitochondrion. KMnO, and PTA. x 25,000. Experimental
Cell Research 26
inclusions.
KBlnO,
in wall and fold of
Electron microscopy of Limax amoebae
513
514
K. Vickerman
another, a process which takes place on a grand scale \\-hen the nuclear envelope disappears at metaphase and, in the reverse direction, \vhen it is reformed around the chromosomes at telophase. The fact that Saeglerirr and Schizopyrenus show continuity bctxveen the outer nuclear membrane and reticulum indicates that this continuity reflects more than just a stage in lvithdrawal or reformation of the nuclear membrane. The links probably have a necessary function in the interphase cell, such as feeding membrane into the grooving nucleus, or conversely, by membrane flow [C;] in the opposite direction, in the transfer of nuclear material to the cytoplasm [43]. Compari.son of limax amoebae with other forms.-The large fresh\\-ater amoebae have been extensively studied under the electron microscope by many workers (e.g. see [24, 27, 28, 31, 371. The so-called limrrx amoeba, Hycrlodisczrs simplex, studied by M’ohlfarth-Bottermann [48, 491 is quite dinerent from the forms described here and belongs to another family, the Hyalodiscidae. Its fine structure is more akin to that of the large freshjvater amoebae. It seems probable that variations in the life-cycle can accourit for the main differences in ultrastructure hetlveen the large amoebae and soil limrrx forms. The evidence for a life-cycle involring alternation of trophic and encysted forms in the large amoebae is unconvincing. In an\- case their occurrence in large permanent bodies of water almost precludes the necessity for encystation. Birheck and Mercer [7], in an interesting article on the dif’ferences between secreting and non-secreting cells, hare cited the slime-mold amoebulae as examples of the former and the large amoebae as examples of the latter. The Zirnn.r amoebae are in many \vays comparable to the amoebulae of mycetozoans in their fine structure [13, 14, 15, 2.3, 261, and the distinction applies just as \vell to the organisms under discussion here. At encystation, in addition to secreting the cyst wall, the 1inm.r forms are actively pumping out fluid from their cytoplasm; it has been assumed that this fluid is water but there is nothing to indicate that it does not contain osmotically active
Fig. 19-Fig.
21.-Acnnthamoeba
sp. All KMnO,
Fig. lg.-Part of cyst wall showing and outer cyst walls. s 20,000.
and PTA.
debris in outer cyst wall (oao) and space between inner (icw)
Fig. 20.-Part of cyst showing so-called pore at lower right. Cytoplasm and mitochondria with intra-cristal bodies. x 12,000.
has numerous fat globules
Fig. 21.-Early stage in cyst wall formation. empties into the water vacuole. x 50,000.
thickness.
Experimental
Cell Research 26
The wall is of uneven
A small tubule
Electron microscopy of Limax amoebae
Experimenful
Cell Research
26
516
K. Vickerman
substances. From the current studies it appears that the endoplasmic reticulum plays an important role in this process in that fluid is expressed into its channels for conduction to the surface. ,4n abundance of cytoplasmic membranes is characteristic of secreting cells on Birbeck and Mercer’s classification. The large amoebae have fe\v cptoplasmic membranes and these are in the form of Golgi stacks and vesicles. In view of the fact that descriptions of these organisms are taken from osmium-fixed material, however, and that lengthy osmium fixation leads to resiculation of tubular elements, it would be interesting to see what membrane systems are revealed in large amoebae following permanganate fixation. The fringed surface membrane found in Amoebrr, Pelomyxa and Hynlodiscus does not occur in soil amoebae. It has been suggested that its function is to provide an increased surface for absorption [‘~7] and there is some evidence that the hair-like projections do act as binding sites for material taken in by pinocytosis. The presence of membrane projections in small amoebae may be rendered unnecessary by the relatively larger surface area. It is worth noting, however, that in hartmannellids, micropseudopodia (Fig. 1) of the determinate type [8] are a common feature and these may serve to increase binding areas. That pinocytosis is common in the hartmannellids is witnessed by the readiness with which they gro\\- \\-ithout solid food in a liquid medium. Relntionships of membrune systems.-The problem of the continuity or discontinuity of cell membrane systems has been reviewed by Robertson [33] and Sjiistrand [39]. It has been argued that amoeboid movement is incompatible with permanent association between cptoplasmic elements [24], but in the present study continuity of some of these elements with one another has been noted. The theories of amoeboid movement advanced by Jlast and Doyle [‘23!, Goldacre and Larch [17] and Goldacre [16] involve synthesis of new membrane at the tip of the advancing pseudopod and resorption of membrane accompanying solation of the cortical gel at the uroid. Is this resorption and resynthesis in actual fact an inward-tucking of the surface membrane to form a continuous tubular system \vhich later re-emerges as surface membrane‘? From the present work this seems unlikely. The membrane profiles in the pseudopod of Schizopyrenzzs represent discrete vesicles or tubules as shown by serial sectioning. However, the formation of new membrane by a coalescence of these vesicles cannot be ruled out, and this effect would parallel membrane formation as envisaged by Afzelius [2] in the cleaving sea urchin egg, by Stewart and Stewart [M] in the sclerotizing slime-mold Experimental
Cell Resenrch 26
Electron
microscopy
of Limax
amoebae
517
plasmodium, and in other cases reviewed by these authors. The site of membrane resorption is most likely not the uroid itself but just anterior to it. In-tuckings of the surface membrane have been seen in this region. The uroid is most likely an excretory organelle [19, 501. In 1ima.r: amoebae it can be shed with no ill effect. The fragmentary evidence obtained so far points to interconversion of membrane systems in amoebae rather than permanent fusion to form a continuous system. Changes in mitochondrin rzt encystation.--The vast literature on changes in mitochondrial morphology induced bp certain physiological and pathological conditions has been reviewed by Rouiller [%I. The changes observed in the mitochondria of Schixopyrenzzs and Acanthamoebn at encystation are among the most striking of mitochondrial transformations. Cup-shaped mitochondria of the type found in Schiropyrenrzs have been observed previously in rat testicular cells by Christensen and Chapman [10] and their interpretation and occurrence elsewhere are discussed by these authors. The relatively greater surface area presented by this shape is believed to facilitate diffusion of respiratory substrates into the mitochondrion and, as this is the rate-limiting factor in aerobic respiration 7211, the result is increased metabolism. In the cyst the mitchondria are spherical, thus presenting minimum surface area and presumably minimum metabolic activity. In Acrznthnmoebrz the change in shape of the mitochondria at encystation is not so marked as in Schkopyrenzzs, but they arc reduced in volume in the cyst and once again tend to be spherical. The internal bodies of the mitochondria of Acnnthnnzoebn are unique in their location in the cristae: other electron-dense bodies in mitochondria lie in the matris. The increase in size of these bodies at encystation is of special interest in view of the work ol’ Klein and Sell’ [21] on the isolated mitochondria of ilcrzntizrznzoebrr. They found that the respiratory rate was proportional to mitochondrial volume and that both \\-ere affected by the tonicity and ionic concentration in the surrounding medium, increasing tonicity having a depressing effect on respiratory activity. At encystation it \vas found here that the mitchondria decrease in volume and that the volume of the mitochondrial lumen is decreased further by the swelling of the intra-cristal body. In the cyst respiration is virtually negligible, yet it has been shown [A] that cncystation demands an ample supply of oxygen. It is probable that the high oxygen requirement is associated with the osmotic work involved in the expression of water from the cytoplasm. The increasing tonicity results in shrinkage of the mitochondria anal accompanying depression of respiratory activity. Perhaps the swelling Experimental
Cell Research
26
518
K. Vickerman
of the intracristal bodies provides the final clamp on respiration, but at the moment the constitution of these bodies is unknown and their function purely speculative. SUMMARY The fine structure of some of the linmx amoebae of the soil is described. These amoebae are similar in appearance when viewed xvith the light microscope but can be broadly classified into two families on their mode of nuclear division. The dif’ference in mitotic mechanisms is correlated with dill’erent patterns of ultrastructure in the non-dividing cells. Amoebae of the family Hartmannellidae have mitosis similar to that found in metazoan cells. The endoplasmic reticulum is characteristically in the form of flattened saccules and shows neither close association with the mitochondria nor a continuity \vith the nuclear envelope. There is a “spongiome” of tubules associated with the contractile vacuole. The cyst \\-a11 is laminated. The mitochondria in dccznthnmoebn have intra-cristal granules lvhich increase in size at encystation. The Schizopgrenidae have “promitotic” division of the nucleus. The endoplasmic reticulum consists of tubules and isolated flattened saccules which are closely applied to the mitochondria. Its elements are in continuity with the outer nuclear membrane. There is no well-developed “spongiomc” and the cyst \\-a11 is unstratified. The mitochondria lack intra-cristal bodies and in Schi:opyrenm are often cup-shaped in the trophic amoeba. amoebae and the The difTerences in fine-structure between the limnr large fresh-water forms are associated with differences in the life cycle. ,1n alternation of trophic and encysted phases is characteristic of the life cycle of soil amoebae. It is suggested that the abundant endoplasmlc reticulum in these forms is active in “dehydration” of the amoeba at encystation, and the changes in mitochondrial morphology at this time are associated \\ith depressed respiratory activity. It is a pleasure to thank professor J. Z. Young for the facilities of his electron microscope laboratory, Dr. E. G. Gray, Dr. J. D. Robertson and Professor J. 0. Corliss for their advice, and Mrs. R. Tilly for help with the photography.
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Experimentul
Cell Research 26
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Electron microscopy of Limas amoebae
4. Bask, R. N., J. Protozoof. 5 (Suppl.), 13 (1958). 5. BARER, S., JOSEPH, J. and MEEK, G. A., Proc. Roy. Sot., Lond. B. 152, 353 (1960). 6. BESNETT, H. S., J. Biophys. Biochem. Cylol. 2, Suppl. 99 (1956). 7. RIRBECK, M. S. C. and MERGER, E. H., Nature, Load. 189, 558 (1961). 8. BOVEE, E. C., Proc. Iowa Acad. Sci. 60, 599 (1953). 9. Box& E. C. and JAHN, T. L., J. Profozoof. ? (Suppl.), 8 (1960). IO. CHRISTENSEN, A. I<. and CHAPMAN, G. B., Ezpff. Cell Research 18, 576 (1959). 11. DEUTSCH, K. and SWANN, M. X, Quarf. J. Microscop. Sci. 100, 13 (1959). 12. FAUR&FREMIET, E. and ROUILLER, C., J. Profozoof. 6, 29 (19.59). 13. GEZELIUS, K., Ezpff. Cell Research 18, 425 (1959). 14. __ ibid. 23, 300 (1961). 15. GEZELIC'S, K. and RKNBY, B. G., ibid. 12, 265 (1957). 16. GOLDACRE, R. J., Ezpff. Cell Research Suppl. 8, 1 (1961). 17. GOLU~CRE, R. J. and LORCH, I. .J., Xafure, Lond. 166, 497 (1950). 1X. GRASSI?, P. P., Proceedings of the Stockholm Conference on Electron Microscopy, p. 143, Stockholm, 1956. 19. HOPKINS, D. L. and WARSER, K. L., J. Parasifof. 32, 175 (1946). 20. KARRER, H. K., .I. Biophys. Biochem. Cytof. 7, 357 (1960). 21. KLEIN, R. L. and NEFF, R. J., Expff. CefZ Research 19, 133 (1960). 22. LUFT, J. H., J. Hiophys. Biochem. Cyfof. 2, 799 (1957). 23. MAST, S. 0. and DOYLE, W. L., Arch. Profisfenk. 86, 155 (1935). 24. MERGER, E. H., Proc. Roy. Sot., Lond. B 150, 216 (1959). 25. MERCER, E. H. and SCHAPFER, B. M., .I. Biophys. Wiochem. Cyfol. 7, 353 (1960). 26. ~IUHLETI~ALER, I(.; Am. J. Botany 43, 673 (1956). 27. PAPPAS, G. D., Ann. S. 1.. Acad. Sci. 78, 488 (1959). 28. PAPI~AS, G. D. and BRAX~T, P. \V., .I. Biophys. Biochem. C!yfof. 4, 485 (1958). 29. P.4RSOSS, D. IJ., DAKUEN, K. B., LINDSEY, D. L. and PRATT, G. T., ibid. 4, 485 (1958). 30. POLICARI>, A., COLLRT, A. and PREOERMAIN, S., Buff. Microscop. Appf. 7, 49 (1957). 31. RAFALKO, J. S., .7. Morphof. 81, 1 (1947). 32. RAY, I). L. and HAYES, R. E., ibid. 95, 159 (1954). 33. RORERTSON, .I. D., Niochem. Sot. Symp. 16, 3 (1959). 34. ROTH, I,. E. and I~ANIELS, E. \1’., J. Biophys. Biochem. (,‘yfof. 9, 317 (1961). 35. ROUILLER, (:., Intern. Rro. Cyfol. 9, 227 (1960).
36. SCHNEIUF.H, I,.. .I. Profozoof. 7, 75 (1960). 3i. SCHNEIl)ER, L. Xlti ~~OHLFARTH-BoTTEK1M.\SS, li. E:., Protoplasma 3X. SINGH, B. N., Phil. Trans. Roy. Sot., Load. H. 236, 405 (1952). 39.
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Experimental
Cell Research 26