Hamamoto, Susan T.

Hohl, Hans R.

Pacific Science

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Lamellate Structures in the Nucleolus of the CellularSlime mold Acrasis rosearHANS R. HOHL and SUSAN T . HAMAMOT02DURING A STUDY on the morph ogenesis ofA crnsis rosea, a cellular slime mold, we haveencountered lamellar structures appearing aspart of the nucleolus of spores and stalk cellsin the fruiting body. This report describes indetail these structures, their occurrence, andtheir possible nature.MATERIALS AND METHODSA crasis rosea originally obtained from Dr.A. Kahn, Syracuse University, was grown onDifco cornmeal-dextrose agar supplementedwith 0.1% yeast extract (Weitzman, 1962)with Rh odotomla mucilagin osa as food orga-nism. Myxamoebae and fruiting bodies weremost successfully prepared for electron micros-copy by one of the following two methods :(1 ) Fixation for 1 hour in osmium vapor,followed by 1 hour in 2.5% phosphate-bufferedglutaraldehyde at pH 7.3, and post-fixation for1 hour in 1% phosphate-buffered osmiumtetroxide at pH 7.3. (2) Fixation for Y2 hourin Karnovsky's (1965) cacodylate-bufferedglutaraldehyde-paraformaldehyde at pH 7.0,dilu ted to 1;4 (pers onal communication fromDr. S. Ito, H arvard University) , followed byfixation in 1% Palade's osmium tetroxide atpH 7.3 for Y2 hour. For both methods dehydra-tion was carried out in ethyl alcohol followed bypropylene oxide, and the specimen was thenembedded in epon. In some cases the tissue wasincubated in 0.5% uranyl acetate in 70% ethylalcohol for Yz hour dur ing dehydration. Sec-tions were cut on an LKB ultramicrotome, post-stained in lead citrate and viewed in a H itachiHU- l l A electron microscope.1 Th is research was supported by a research gran t(GM 11758-03) from the National Institutes ofHealth of the U. S. Public H ealth Service.2 Pacific Biomedical Research Center and D epart-ment of Microbiology, Uni versity of Hawaii, Hono-lulu , Hawaii 96822 . Manuscrip t received May 23,1967.For demonstration of RN A, glutaraldehyde-paraformaldehyde fixed (Yz hour, 4°C, pH 6.0)samples were treated with 0.7 mg/ rnl RNAase(heated for 10 minutes at 90°C prior to use) .After incubation, the cells were washed with5% trichloracetic acid for 30 minutes at O°C,postfixed in osmium tetroxide, and embedded asdescribed above.For histochemical purposes at the light -microscope level the cells were fixed for 10minutes in acetone at 4° C. The cells werestained for (1) RN A using pyronin Y accord-ing to Kurnick ( 1955), for ( 2) DN A andRNA with acridine orange followed by fluores-cence microscopy, and for (3) protein withmercury bromphenol blue following the proce-dure outlined by Pearse (1960) . Controls weretreated with DNAase or RN Aase ( Loh andSoergel, 1965) before staining.RESULTSThe nucleolus of the myxamoebae of Acrasisrosea appears to be built from three morpho-logically distinct components . The most striking,and the one of particular interest for our dis-cussion, consists of a series of roundish massesof granular material (Figs . 1 and 2) . Thesemasses are either dispersed as separate unitswithin the nucleus (F ig. 2) or condensed intclarge aggregates (Fig. 3) . Each mass is madeup of a large number of electron dense granuleswhich stain heavily with lead citrate and arecomparable in size to cytoplasmic ribosomes.The granules are more densely packed at theperiphery of each clump, the central part ofwhich then appea rs as a light core revealing asomewhat fibrous matrix.The second component has not been observedfrequently. It appears as an intensely stained,homogeneous body containing electron trans-parent cavities and is always situated within thefirst component (Figs . 1 and 5) . The thirdcomponent of the nucleolus consists of a large402Acrasis rosea Nucleolus-c-Hom, AND HAMAMOTO 403FIG. 1. Part of a cell of Ac rasis rosea showing nucleus (NU) with two components of the nucleolus(1 ,2 ) as described in the text . X 25,000. Th e black bar in all pictur es repr esents 1~.FIG. 2. N ucleus of Acrasis rosea with component s 1 and 3 of nucleolus . Note light er and darker areaswithin component 1. X 20,800.FIG. 3. Example of component 1 aggregated into a large mass. Cytoplasmic invagination (I) and nuclearevagination (E) are seen at upper left . X 20,000.404roundish mass of finely granular material some-what more dense than the rest of the nucleusbut considerably less dense than the first com-ponent (Fig. 2).In the resting stages, that is, in the stalk cellsand the spores, the first granular componentoften contains lamellar elements which arestacked in parallel fashion (Fi g. 4) . The num-ber of lamellae per stack has been observed tovary from 1 to 12. The lamellae can be flat orcurved, may cross thr ough the middle of thegranular component (Figs. 4 and 5), or mayfollow its contour (Figs. 6 and 7). Yet theynever extend beyond the granular mass. Thelamellae are separated by a constant distanceof approximately 150 A. The constant thicknessof the lamellae together with the constantspacing conveys to the whole structure a regu-larity of appearance (Fig. 4).Each lamella consists of a single layer ofgranules ( Figs. 1, 6, and 7) apparently iden-tical to the granules making up the first com-ponent (Figs. 1, 2, and 8) . In fact, the layersare often continuous with the granular com-ponent (Figs. 8 and 9) and are always closelyadjacent to it. Oblique sections through thestacks pr ovide additional evidence that thelayers are built from granules and not, forexample, from a filamentous component (Fig.8) . Furthermore, we see that the granules arenot oriented in any particular way but arepacked rather tightly , though randomly, withinthe planes.By applying the histochemical tests described,ribonucleoprotein-containing areas can bedemonstrated within the nucleus comparable insize and location with the components describedat the electron microscope level. This suggeststhat the granular masses contain ribonucleo-protein and are probably aggregates of ribo-somes or ribosomal precu rsors. The nucleolushas been shown to be a source of ribosomes(B irnstiel , Chipchase, and Hyde , 1963). Ourbest evidence supporting this idea is obtainedfrom the electron microscope study of RNAasetreated cells in which the granular componenthas largely faded (Fig. 10).DISCU SSIONThe lamellae described in this report bear acertain resemblance to the "core" structures ofPACIFIC SCIENCE, Vol. XXII, July 1968spermatocytes (M oses, 1956) as well as to thechromatoid bodies in parasitic protozoa and ingerm cells of plants and animals (B arker,1963) . In the case of the "cores" a close simi-larity can be noted between the outer layer ofthe complex cores described by Schin (1965)and our lamellae. In both cases the layers aregranular, show continuity with the nucleolus,and are often attached to or partly surroundedby the latter (Schin, 1965; Guen in, 1965) .In the case of Acrasis, however, all the othercomponents of the "core" structure are lacking ;also, the cells are at a resting stage and nodivision of cells or nuclei occurs.In the chromatoid bodies of Entamoeba in-uadens, granules identified as ribonucleopro-tein particles also are arranged in layers. Acloser examination of those lamellae reveals,however, that the granules within the layersform helices (Siddiqui and Rudzinska, 1963)and thus differ from the arrangement describedhere for A crasis.All of these examples demonstrate the capac-ity of ribonucleoprotein granules to assembleinto orderly structures. In our case particularly,we attribute this to a process of spontaneousself assembly within or at the periphery of ahighly concentrated mass of granules in a stateof low metabolic activity.The fact that the lamellae have so far beenobserved only in resting cells, and then not withregul arity, indicates that they do not representan organelle involved in an essential funct ion.SUMMARYLamellate structures have been demonstratedwithin the nucleoli of spores and stalk cells ofthe slime mold A crasis rosea. The lamellae,numbering up to about 12 , are separated fromeach other by about 150 A and are arrangedin a parallel fashion to form stacks which eithercross the main nucleolar mass or line it periph-erally. Each lamella is composed of a singlelayer of granules apparently identi cal to the onesmaking up the bulk of the nucleolus . Thegranules are fairly tightly but randomly packedwithin the layer. The lamellate structures areinterpreted to represent an arrangement of ribo-somal type particles formed from a concentratedpool of particles by a process of self assembly.Acrasis rosea Nudeolus-c-Ho m. AND H AMAMOTO 405FIG. 4. Nucleolus of Ac rasis rosea with a series of parallel lamell ae trans versing it. X 16,000. The blackbar in all pictures represents 1IL.FIG. 5. Massed component 1 of nucleo lus with lamellar materi al in between port ions of mass. X 22,000 .FIG. 6. Nucleolus with lamellae along contours . Lamellae are sectioned tangenti ally in parts. X 22,000.FIG. 7. Nucleolus with per ipheral lamell ae. X 22,000.406 PACIFIC SCIENCE, Vol. XXII, July 1968FIG. 8. Higher magnification of lamellar material cut obliquely. The lamellae appear to be made upentirely of granules and seem to be continuous with dark mass of component 1 (arrow) . X 56,000. Theblack bar in all pictures represents tu,FIG. 9 . Continuity between lamellar material and component 1 of nucleolus (arrows). X 53,000.FIG. 10. Nucleus after treatment with RNAase . The nucleolar masses have largely faded. X 20.000Acrasis rosea Nucleolus-i-Hou r, AND HAMAMOTO 407We would like to thank Dr. H ans Ris foradvice and Dr. Philip Loh for the use of thefluorescence microscope.REFERENCESBARKER, O. C. 1963. A ribonucleoprot ein In-clusion body in Entamoeba inoadens. Z.Zellforsch. 58 :641- 659.BIRNSTIEL, M. 1., M. I. H . CHIPCHASE, andB. B. H YDE. 1963. The nucleolus, a sourceof ribosomes. Biochim. Biophys. Acta 76 :454-462.GUENIN, H . A. 1965. Observations sur la struc-ture submicroscopique du complexe axialdans les chromosomes meiotiques chez Gl'yl-las campestris 1. et G. bimaculatus de Geer(Orthopt. GryII.) . J. Microscopie 4 :749-758.KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality foruse in electron microscopy. J. Cell BioI. 27 :137A-138A.KURNICK, N . B. 1955. Pyronin Y in the methyl-green-pyronin stain. Stain Technol, 30 :213-230.LOH, P. c., and M. SOERGEL. 1965. Grow thcharacteristics of Reovirus type 2: actinomycinD and the synthesis of viral RNA. Proc.Nat!. Acad. Sci. 54 :857- 863 .MOSES, M. J. 1956. Chromosomal structure incrayfish spermatocytes. J. Biophys. Biochem.Cyto!. 2:215- 218.PEARSE, A. G. E. 1960. Histochemistry. 2nd ed.little, Brown and Company , Boston. 998 pp .SCHIN, K. S. 1965. Core-Strukturen in denmeiotischen und post-meiotischen Kernen derSpermatogenese von Gryllns domesticus.Chromosoma 16:436-452.SIDDIQUI, W . A., and M. A. RUDZINSKA. 1963.A helical structure in ribonucleoproteinbodies of Entamoeba inuadens. N ature 200:74-75.WEITZMAN, I. 1962. Studies on the nutrition ofAcrasis rosea. Mycologia 54:11 3-115.

Lamellate Structures in the Nucleolus of the Cellular Slime mold Acrasis rosea

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Lamellate Structures in the Nucleolus of the Cellular Slime mold Acrasis rosea

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