190 J. PROTOZOOL. 7 ( 2 ) , 190-199 (1960).

The Fine Structure of the Heliozoan, Actinosphaerium nucZeofi!urn*’

EVERETT ANDERSON and H. W. BEAMS

Department of Zoology, State University of Iowa, Iowa City , f o w a

SYSOPCIS. Actinosphaeriirnt nucleofilum has been studied by light and electron microscopy. Thin sections of this organism reveal the relatively compact endoplasm to consist of numer- ous \-acuoles and mitochondria. Scattered in the cytoplasm are dense particles, presumably ribonucleoprotein particles, en- doplasmic reticulum of the rough variety and a tubular endo- plasmic reticulum of the smooth variety. The many nuclei do not appear to be randomly scattered in the cytoplasm but rather to be arranged in a circular pattern. Each is bounded by a double membrane envelope. A Golgi complex, consisting of isolated bodies with an ultrastructure similar to that de-

GCH of our knowledge of the cytology of Actino- R/’1 sphoerium comes from a long series of investiga- tions originally initiated by the description of the genus in 1 8 5 7 by Stein( 1 2 ) . The wealth of informa- tion accumulated since the refinement of electron mi- croscopical techniques on protozoan ultra-structure has been primarily concerned with ciliates (3 1,34,36, 37) . Ra~ellates(1.2,30) and to a lesser extent organ- isms of the class Sarcodina(3,29) and Sporozoa(8,21, 35). The aforementioned studies have helped to re- solve certain of the more controversial morphological problems. Despite the better resolution obtained by the electron microscope virtually no reports have been made on organisms of the order Heliozoa other than that of Ivohlfarth-Bottermann & Kruger (41 ) who studied whole mounted specimens of Actinophrys sol and Hfterophrys marina. There is need, therefore, for further analysis of the cytoarchitecture of organ- isms belonging to this group. Such structural data may proside new insight for studies aimed a t discern- ing cellular dynamics of such organisms. These ends are partially approached below by a description of the fine structure of Actinosphaerium nucleofi2um.

MATERIALS AND METHODS

The organisms used in this study, Actinosphaerium nucleofiIunz,l were obtained originally from Carolina Biological Supply and subsequently from Dr. James 11. Barrett of the Department of Biology, Marquette University. Milwaukee, Wisconsin. Culture medium

:&Supported by grants (RG 4706 and 5479) from the Na- tional Institutes of Health, United States Public Health Service.

1This organism was referred to in an abstract(1) as Actino- sphaerium eichhorni and is now known to be Actinosphaerium nucleofilziin.

scribed by others, is usually seen associated with each nucleus. The ectoplasm contains many vacuoles of varying diameter,

each of which is limited by a thin membrane; vacuoles con- taining dense granular material, and mitochondria.

The radially arranged axiopodia are extensions of the ecto- plasm. The periphery is largely composed of vacuoles, some of which contain dense granules, and mitochondria. The axial rod is birefringent and consists of many fine filaments oriented parallel to the longitudinal axis of the axiopodium. The con- stituent filaments penetrate deep into the endoplasm where they end in the vicinity of nuclei. The fibrillar component of the axiopodia may represent contractile units.

containing living organisms was either placed on a slide or in a rotocompressor and studied by phase contrast and polarized light microscopy. Whole mounts were made from organisms fixed in Champy’s and Schaudinn’s solutions and subsequently stained with Heidenhain’s hematoxylin or with a 0.1% aque- ous solution of thionin; other organisms, to be used for electron microscopic studies, were concentrated by slight centrifugation and fixed for 30 minutes in a 27% osmium tetroxide solution buffered a t pH 8.5 with acetate veronal buffer. The latter were dehydrated, infiltrated and embedded in methacrylate. Thin sec- tions were cut on a Porter-Blum microtome and ex- amined with a RCA EMU-3D electron microscope.

OBSERVATIONS AND DISCUSSION

Since the general cytology of Actinosphaerium nu- cleofilum is known from the light microscopic study of Barrett(5) it is not necessary for us to enter into a detailed description of the organism a t this level of observation. I t will suffice to point out, for purposes of orientation of the electron microscope observations to follow, some general cytological features of the or- ganism. A ctinosphaerium nucleofilum (see insert, Fig. 1) is multinucleated, spherical in shape and ranges from 200-400 p in diameter. I t is divided into a highly vacuolated ectoplasm (VE) and a less vacuo- lated endoplasm (EN) . Radiating out from the body are long, slender, specialized pseudopodia referred to as axiopodia (AX). Each axiopodium consists of a central, relatively stiff, axial rod which is surrounded by a thin cortical layer of ectoplasm. The axial rods penetrate deep into the endoplasm where they termi- nate freely or in close association with nuclei.

THE FIXE STRUCTURE OF THE HELIOZOAN, .Ictinosphaerizim nucleofilum 19J

Endo plasm relatively low power electron micrograph illustrated in Fig. 1. Here the nuclei ( N ) , sectioned a t different levels, appear to be arranged in circular pattern. Each nucleus is limited by a double membrane en-

The mdtinuckated condition of the compact and somewhat vacuolated endoplasm can be seen in the

Fig. 1. .it the top of the plate is a photomicrograph ( X 110) of a living organism showing the vacuolated ectoplasm (VE). ir less vacuolated endoplasm ( E S ) and axiopodia (AX). The rkctron micrograph illustrates a section made through (D.4) on the lateral aspect of the filament bundle. X 7,000.

the surface of the animal showing large vacuoles (Vl), nuclei (N) , nucleoli (SCL), mitochondria (M) and filaments of the deeper portion of .the axial rod (Ft l ) . Piote the dense area

192 THE FINE STRUCTURE OF THE HELIOZOAN, ilctinosphaerium nucleofilum

velope which encloses a granular nucleoplasm (Figs. 10 and 14, N) . The multinucleoli are composed of masses of dense granules which occupy a position subjacent to and in close contact with the inner nuclear envelope (Figs. 1, 9 and 11, NCL). In preparations stained with Heidenhain’s hematoxylin and thionin, the nucleolar masses are basophilic and Barrett ( 5 ) states that such masses are Feulgen-negative. In the interphasic nuclei of Amoeba( 29) the nucleoli are distributed in a pattern similar to that described here for A . nucleofilum, as is also the case in many other interphasic nuclei of protozoan and metazoan cells.

Many dense granules, - 100-200 A in diameter. can be observed scattered throughout the cytoplasm (Fig. 12, SG). These correspond to the fine particulate component originally described by Palade (2 7 ) which has subsequently been shown to be rich in ribonucleo- protein (28). In some preparations the previously mentioned granules are confined to the surfaces of flattened cisternae of the endoplasmic reticulum (Figs. 11 and 14, ER) . In addition to the aforementioned elements of the endoplasmic reticulum another system is observed that is typically in the form of slender, linear or twisted tubules and is characterized by the absence of ribonucleoprotein particles on its surfaces (Figs. 8, 10, 11 and 14, T E R ) . The circular profiles (Figs. 10 and 11, V,) scattered throughout the endo- plasm may be sections of the tubular endoplasmic reticulum.

A Golgi complex is usually found in the vicinity of nuclei. This substance, which is in the form of dictyo- somes, must be distributed widely within the organism since it is not uncommon to find as many as six dis- crete bodies within a given section. This material typically appears to be composed of many broad flat- tened membranous sacs which appear in sections as a system of smooth double membraned lamellae in paral- lel array (Figs. 10 and 14, GC). In close association with the ends of the lamellae is a vesicular component which may or may not be budded off from the edges of the lamellae. Ritter (32), in an abstract on A . eich- horni, referred to certain rod-shaped or ovoid granules found predominately in the ectoplasm as Golgi gran- ules. After certain cytochemical tests he also stated that these granules contain lipid and protein and that they were neutral red-stainable and osmiophilic. The problem of neutral red staining with respect to Golgi material has been described in detai1(6,40) and need not be taken up here. The question might be raised, however, since the ectoplasm harbors many vacuoles, whether the bodies described by Ritter (32) as Golgi granules are in reality Golgi material. In the present study Golgi bodies were never seen in the ectoplasm. They were always found deep in the endo-

plasm among vacuoles and other cytoplasmic organ- elles.

Many mitochondria are found scattered in the en- doplasm. They are oval to elongate in shape and possess a double limiting membrane. In a few prepa- rations the internal structure of these organelles ap- pears tubular, but typically the interior is filled with small vesicles (Figs. 8, 10, 11 and 14, AI). In some electron micrographs the double limiting membrane appears broken suggesting a release of its internal contents (Fig. 10, M ) (cf. 42). I t is of interest to note that mitochondria herein described bear some internal structural resemblance to multivesicular bod- ies, however, the latter are usually limited by a single membrane(38).

The ingested nutritive particulates form conspicuous food vacuoles which are bounded by a single layered membrane (Figs. 12, 13 and 14, FV). The most in- teresting feature of the food vacuoles is the many smaller vacuoles of varying diameter in the area im- mediately surrounding it (Figs. 12 , 13 and 14, PV). These vacuoles may have formed as a result of pino- cytosis and if so it is possible that they contain dis- solved nutrient materials( 11,13,23,29).

Ectoplasm

The ectoplasm consists of many vacuoles of a non- contractile nature. They are so closely spaced and are often so large that only thin cytoplasmic areas are found between them (Figs. 2, 8, 12 and 14, IT1). These vacuoles are bounded by a single-layered mem- brane. In some electron micrographs there appears to be a slight indication of some amorphous internal substance ; however, they typically appear empty. Such non-contractile vacuoles have been implicated as hydrostatic devices which aid in flotation ( 7 ) . In ra- diolarians, it has been suggested that similar ectoplas- mic vacuoles maintain the organisms at particular depths in the ocean(l6). A phenomenon like that which apparently exists in the heliozoans and radio- larians occurs in an unrelated fresh water protozoon ArceZZa. Bles(9) in his detailed investigation of the gas-filled vacuoles of Arcella suggested that they influ- ence the specific gravity of the organism according to environmental-physiological conditions.

The axiopodia, which a t times may be branched, consist of a cortical layer of cytoplasm and a central axial rod (Fig. 3, AP). At the base of each axio- podium, the cortical ectoplasm is continuous with that of the body. Within the cortical ectoplasm are found mitochondria and tubular elements of the endoplasmic reticulum similar to those which exist in the body of the organism. The most conspicuous element of this surface are vacuoles, some of which consist of a mass

THE FINE STRUCTURE OF THE HELIOZOAN, .4 ctinosphaerium nucleofiluin 193

of dense particles (Figs. 3 , 4 and 14, V,). These cor- respond in position to the highly refringent bodies observed by light microscopy and may be related to the excretory granules of Borowsky( 10) and crystal- line inclusions of iVIcArdle(24,25). Recent studies on the crystalline inclusions of different amebas have in-

dicated that these structures are the end products of nitrogen metabolism( 14,lS). Whether the apparent crystalline granules found in A . nucleofilum are waste products similar to those of amebas is not known.

The axial rod is composed of filaments 60-125 ‘4 in diameter and of varying lengths, oriented parallel to

Figs. 2 and 3. Sections showing some aspects of the ecto- plasm. The branched axiopodium (.4P, Fig. 3 ) shows mito- chondria ( M ) , tubular endoplasmic reticulum (TER), fila- ments oi the axial rod (FA) and vacuoles, many of which plasmic kacuoles V1 (Fig. 2 ) . X 12,800.

appear empty (V,) and others filled with dense granules (Vz). Similar vacuoles are seen in the ectoplasm (V2 and V3, Fig. 2 ) . A mass of dense granular material is labeled G and large ecto-

194 THE FINE STRUCTURE OF THE HELIOZOAN, .4 ctinosphaeriunz mcleofi lum

the long axis of the axiopodium (Figs. 3. 4-7. and 14, FA). The constituent filaments penetrate deep into

the endoplasm where many terminate in close associa- tion with the nucleus (Figs. 1, 9. and 14, FA). a con-

Figs. 4-7. Sections through portions of axiopodia demon- strating filaments of the axial rod (FA), tubular endopiasmic reticulum (TER), mitochondrion ( M ) , vacuoles containing

dense granules (V,) and masses of a dense granular material (G) . Figs. 4 and 5 , X 40,000; Figs. 6 and 7 , X 20.000.

THE FINE STRUCTURE OF THE HELIOZOAN, Actinosphaerium nucleofilum 195

Fig. 8. A section showing filaments of an axial rod (FA) as showing vacuoles (V) , nucleus (N) and nucleolar material it penetrates deep into the endoplasm. Note also large vacu- (NCL) . Kote the close anatomical association of filaments of oles (V,), tubular endoplasmic reticulum (TER) and mito- the axial rod (FA) with that of the external nuclear envelope. chondrion (11). X 16.000. Fig. 9. Portion of the endoplasm X lj,80C.

196 THE FINE STRUCTURE OF THE HELIOZOAN, ilctinosphaerium nucleofilunz

dition similar to that demonstrated by Noirot- species of Zsot~icha. In some electron micrographs a Timothee(26) for filaments of the karyophore of two dense area was sometimes seen on the lateral sides of

Figs. 10 and 11. Portions of the endoplasm showing nuclei Golgi complex (GC) and vesicles (Vq). Fig. 10, X 40,000; (N) bounded by a double membrane envelope, nucleoli (NCL), mitochondria (M), endoplasmic reticulum (ER and TER) ,

Fig. 11, X 30,000.

THE FINE STRUCTURE OF THE HELIOZOAN, Actinosphaeriunz nucleofiluilz 197

the filament bundle located in the vicinity of nuclei (Fig. 1, D.1); however, no differentiated structure

similar to a “kinetosome” was ever observed. I t is well known that the axiopodia of heliozoans

Figs. 12 and 13. Sections showing food vacuoles (FV). Fig 12 shows a nucleus (N), small dense granules (SG) and Surroundicg the food vacuoles are smaller vacuoles (P1’). large vacuoles (1‘1). Fig. 12, X 7,600; Fig. 13, X 20,000.

198 THE FINE STRUCTURE OF THE HELIOZOAN, d ctinosphaerium nucleofilum

are primarily concerned with the capturing of food. Some investigators have reported that they are rela- tively rigid structures( 18) ; others state that they may contract and also exhibit some lateral bending(4,20, 22.33). Polarization optics demonstrate that the axial rod of A . nucleofilum is birefringent, a phenomenon

shown for the axial rod of A . eichhorizi by Mackinnon ( 2 2 ) , Roskin(33) and McArdle( 24). The birefring- ence of the axial rod indicates that this structure is composed of a highly oriented fibrillar component, as suggested by Roskin(33) ; this view has been con- firmed herein by means of the electron microscope.

Fig. 14. A schematic representation of Actinosphaeriunz nu- cleofilunz. AT, nucleus; GC, Golgi complex; TER, tubular en- doplasmic reticulum ; M, mitochondria ; .4X, axiopodium ; plasm : Y,3 vesicle containing dense granules. FA, filaments of axial rod ; ER, endoplasmic reticulum (rough

variety) ; FV, food vacuole ; PV. pinocytotic vesicles originat- ing from food vacuoles; Vl, non-contractile vacuoles of ecto-

THE FINE STRUCTURE OF THE HELIOZOAN, A ctinosphaerium nucleofilum 199

A similarly oriented fibrillar component was demon- strated for the axial rod of axiopodia of Actinophrys sol by Wohlfarth-Bottermann & Kriiger(41).

In the stalk of Vorticella and in the muscle fiber a submicroscopic fibrillar unit has been demonstrated which has been postulated to play a prominent role in the phenomenon of contraction in such systems(39, 17 ) . I t is not within the scope of this paper to dis- cuss mechanisms that may be involved in the move- ment of axiopodia ; however, the morphological fine structure of these appendages invites one to speculate that the constituent filaments are somehow related to and involved in contraction. Any theory advanced on contraction of axiopodia must take into account this filamentous component. For example, the theory sug- gested by Jahn and Rinaldi(l9) on protoplasmic streaming in reticulopodia of foraminiferea is also applicable to the streaming phenomenon in the axio- podia of heliozoans, for it takes into account the pres- ence of the axial rod.

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