J. Cell Sci. II , 403-414 (1972) 403

Printed in Great Britain

RIBOSOME CRYSTALS IN NECROTIZING CELLS

FROM THE POSTERIOR NECROTIC ZONE OF

THE DEVELOPING CHICK LIMB

N. K. MOTTET AND S. P. HAMMARDepartment of Pathology, University of Washington School of Medicine,Seattle, Washington 98195, U.S.A.

SUMMARY

Ribosome and ' protein crystals' were observed in electron micrographs of degenerating cellsfrom the posterior necrotic zone (PNZ) of developing chick limbs, stages 22 through 24.

Ribosome crystals were made up of basic units of 4 ribosomes arranged in a square tetramer.They were monomorphic with the surface lattice being of the p4 plane group. The i-ribosome-thick sheets of crystals were often seen stacked upon one another forming a 3-dimensional P422configuration. The percentage of crystallized ribosomes within degenerating PNZ cells isnearly directly proportional to the degree of degeneration of the cell. Crystals identical to thishave been observed in numerous types of embryonic chick cells subjected to hypothermia.

The 'protein crystals' were composed of straight filamentous structures separated by adistance of 20-30 nm. They were always closely associated with the ribosome crystals. Thesecrystals are similar to those seen in cells treated with high doses of vinblastine and vincristine.

The pathogenesis of the ribosome and protein crystals occurring in degenerating PNZ cellsis unknown. They are not associated in any way with hypothermia or vinblastine-vincristinetreatment.

INTRODUCTION

Cellular degeneration and necrosis is a common feature of embryogenesis in manyanimal species (Gliicksmann, 1951; Saunders, 1966). Death of cells has been describedas part of such diverse morphogenetic processes as fusion of Anlage, delamination,cell migration and differentiation. The morphologic features of the necrosis vary some-what depending on the cell type involved. For example, in the chick embryo, regions ofnecrosis appear at the anterior and posterior aspects of the developing limb buds atstage 24. The scattered necrotic cells appear interspersed with non-necrotic ones,later to be phagocytosed by the latter. As the cells are undergoing necrosis they shrinkand the nuclei become pyknotic. This cell death has aroused considerable interest asto whether it is genetically programmed or whether it occurs incidental to develop-mental events. Saunders & Fallon (1966) have shown that the posterior necrotic zone(PNZ) is clearly apparent at stage 24 in the chick embryo. However, the cells are' scheduled' for death at stage 17. Between stages 17 and 22 there are no distinguishingfeatures to identify the cells scheduled to die. At stage 22 the process is irreversibleand by stage 24 the features of necrosis are obvious by both light and electron micro-scopy.

During the course of our investigation correlating succinic dehydrogenase enzyme

404 N. K. Mottet and S. P. Hammar

activity as an index of energy turnover in differentiating cells (Hammar & Mottet,1971) with cell ultrastructure, we noted the presence of peculiar crystalline structuresin the cytoplasm of many dying cells in the PNZ. These crystals are identical to theribosome crystals described in embryonic chick cells subjected to hypothermia (Byers,1966, 1967; Maraldi & Barbieri, 1969; Carey & Read, 1971). This report describesand illustrates the ultrastructural features of the crystalline structures hitherto un-described in the PNZ.

MATERIALS AND METHODS

Limb buds of chick embryos stages 22-24 according to Hamburger & Hamilton (1951) wereused for this study. Fresh fertile eggs from White Leghorn hens were refrigerated for 1-2days prior to being incubated at 38 °C for 3-5-4 days (stages 22-24). Incubation was begunwithin a week of laying and temperatures were continuously monitored by recording thermo-meters. No periods of hypothermia occurred throughout embryogenesis. Posterior necroticzones were quickly excised from hind limb buds, fixed in a modification of Karnovsky'sfixative (Mottet & Jensen, 1968) for 4 h, postfixed in 1 % osmium tetroxide for 1 h and de-hydrated in ethanol and propylene oxide prior to embedding in Epon 812, using the procedureof Luft (1961). Thin sections were stained with uranyl acetate and lead citrate. Necrotic andnormal mesenchymal cells were photographed from 560 samples of 18 specimens.

RESULTS

A low-power electron micrograph of the PNZ from a stage 22 embryo is shown inFig. 1. Several degenerating cells are seen among the normal viable cells; the degen-erating cell in the upper right portion of the micrograph has been phagocytosed. Thesecells appear shrunken compared to the viable cells and are characterized by electron-dense cytoplasmic matrix, dense pyknotic nuclei, partial loss of nuclear and cytoplas-mic membranes, vacuolization, and the formation of distinct crystalline structureswithin the cytoplasm. The fraction of degenerating cells and their stage of degenera-tion varies with the stage of development. At stage 21-22 (35—3"75 days incubation)approximately one quarter to one third of the cells of the PNZ show evidence ofdegeneration; at stage 24 (4 days incubation) approximately two thirds are eitherdegenerating or frankly necrotic. In the earlier stages of limb development (stage 21-22)few degenerating cells have been phagocytosed and the majority show the distinctribosome crystals to be described. By stage 24 (4 days incubation) however, the majo-rity of cells in the PNZ have undergone degeneration and have been phagocytosed;their remains are seen morphologically as residual bodies within the phagocytic cells.

Fig. 2 shows 4 representative degenerating cells from the PNZ of stages 22-24embryos (3-5—4 days incubation); in each, distinct crystalline structures are seen.In Fig. 2 A only a few small crystals are seen, while in 2B-D there is extensive crystalformation. As can be observed, there is a wide variation in size and shape of thecrystals; in some cells they exceed 1-5 /wn in each dimension; also, especially inFig. 2 A, B, there appear to be several different patterns of the crystalline lattice.

Fig. 3 shows higher magnifications of the ribosome crystals. While they havevariable surface dimensions, each crystal is a sheet 1 ribosome thick which appearsto be quite flexible. The crystals are made up of basic units of 4 ribosomes in a square

Ribosome crystals in necrotizing cells 405

tetramer; the square tetramers are bonded together with a resultant p4 surface lattice.The ribosomes vary slightly in shape but are generally round and average 20 nm indiameter. Their centres lie approximately 24 nm apart. Although separate large andsmall subunits of the ribosomes could not be identified, a few ribosomes were eitherkidney-shaped or clefted.

The 'unit cell' of the ribosome crystals, as defined by Byers (1966, 1967) consistsof a square with its vertices at the centre of 4 square tetramers. The sides of the unitcell of ribosome crystals from degenerating PNZ cells measured between 53 and 55 nm.This is in agreement with side lengths of unit cells from crystals of hypothermic chickembryo cells.

The ribosome crystals are commonly arranged in parallel sheets stacked upon oneanother. There is a bonding between these sheets resulting in a 3-dimensional crystalof the P422 configuration. When in this arrangement and when viewed on edge,parallel rows of ribosomes displaying 3 typical configurations are seen: (1) a dashedline, as seen in Fig. 3 A, B, and D; (2) a dotted line, 3 c and D; and (3) a solid line, Fig.3 D. These 3 edge views are dependent upon which axis of the lattice is collinear withthe electron beam. We are in agreement with Barbieri, Simonelli, Simoni & Maraldi(1970) that definite measurable spaces exist between the aggregates of ribosomes pro-ducing the dashed-line configuration seen on edge view. These spaces have measuredbetween 3 and 5 nm.

We have also observed that the percent of crystallized ribosomes seems to be nearlydirectly proportional to the degree of degeneration of the PNZ cells. By the time thenucleus has become completely pyknotic and has become eccentrically located withinthe cell, 50-75 % of the ribosomes have crystallized.

In many degenerating cells from the PNZ a different type of crystalloid structurewas observed. This was always closely associated with the ribosome crystals and con-sisted of parallel rows of filamentous structures measuring up to o-8 fim in lengthand 0-3 /im in width. The individual filaments were difficult to measure, averagingless than 1-5 nm in diameter; the distance between them varied between 20 and 30 nm.These crystals are essentially identical in morphological structure to the proteincrystals described by Krishan & Hsu (1969) in vincristine-treated Earle's L-cell fibro-blasts and in vinblastine-treated hypothermic cell cultures of chick embryo hepatictissue described by Maraldi, Simonelli, Pettazzoni & Barbieri (1970). Both investiga-tors observed these crystals in association with helical polyribosomes; Maraldi et al.(1970) observed them also to be associated with ribosome crystals. We are not ableto prove that these crystalline structures are derived from protein but will refer tothem as protein crystals.

DISCUSSION

The morphology of cellular degeneration and necrosis in most experimentallyinduced conditions is characterized by generalized cellular oedema, morphologicallyexpressed as dilated, swollen mitochondria and endoplasmic reticulum cisternae, witha decrease in density of the matrix of both cytoplasm and nucleus (Ashworth, Luibel,

406 N. K. Mottet and S. P. Hammar

Sanders & Arnold, 1963; Trump, Goldblatt & Stowell, 1965; Smuckler & Trump,1968; Gritzka & Trump, 1968; Churg & Richter, 1971). Unlike that in experimentalconditions, cellular degeneration and necrosis occurring in the PNZ during chick limbbud morphogenesis is characterized by dehydration, most vividly displayed morpho-logically as coalescence and precipitation of the nuclear chromatin with resultantpyknotic nuclei. The most unique morphological feature of cellular necrosis occurringin limb development, as described in this paper, is the formation of ribosome crystals.

Bellairs (1961) described crystals of this type in degenerating cells from chickblastoderms; she referred to them as 'beaded granules' and suggested they repre-sented altered ribonucleoprotein particles. Her electron micrographs showed pre-dominantly edge views of the ribosome crystals.

Porte & Zahnd (1961) observed crystals in ovarian cells of the lizard Lacerta stir-pium. Ghiara & Taddei (1966) have re-examined these crystals and have found themto be morphologically identical to ribosome crystals.

Byers (1966) has described ribosome crystals morphologically identical to thoseseen in degeneration cells from the PNZ in numerous types of chick embryo cellssubjected to hypothermia. He observed the crystals only in tissue which had beensubjected to a temperature of 5 °C for 3 h or more or 10 °C for 8 h or more. Nocrystals were found in cells treated with higher temperatures (15-20 °C) nor after 5 ° or10 °C for shorter periods than those indicated above. Byers (1967) further demonstratedthat prolonged hypothermia was necessary for ribosome crystallization in interphasiccells, whereas rapid cooling resulted in crystallization in mitotic cells. He attributedthis difference to the fact that free ribosomes were already present in mitotic cells,whereas in interphasic cells the ribosomes had to be released from polyribosomes.

Maraldi & Barbieri (1969) studied hypothermically induced ribosome crystallizationin several types of chick embryo cells and found that the number and size of thecrystals were unique for a given tissue and depended upon its degree of differentiation.They demonstrated that the percentage of crystallizing ribosomes decreased in allembryonic organs during differentiation.

Barbieri et al. (1970) described nuclear as well as cytoplasmic ribosome crystals inhepatic cell cultures subjected to hypothermia; the nuclear crystals occurred only inmitotic cells. Their occurrence in nuclei was explained on the basis that RNA syn-thesis and formation of ribonucleoprotein particles occurs in the nucleus during themitotic cycle.

Maraldi et al. (1970) have subsequently demonstrated polyribosome helices, 'tubu-lar crystalline bodies', 'cytoplasmic tubular-like structures' and ribosome crystals inhypothermic cultures of hepatic cells treated with vinblastine sulphate. They hypo-thesized that tubular crystalline bodies and cytoplasmic tubular structures werederived from proteins normally forming microtubules and demonstrated that the sizeand number of these crystals increased as the dose of vinblastine increased. Theydemonstrated a very close association of the polyribosome helices and ribosome crystalswith the other protein crystals and postulated that the 2 crystalline formations of ribo-somes (helices and crystals) suggested at least 2 distinct functional classes of ribosomes.

The exact morphology of the ribosomes producing the crystalline lattice is not

Ribosome crystals in necrotizing cells 407

entirely clear. Byers (1967) considered the crystallized ribosomes as isodiametric par-ticles approximately 20 nm in diameter; he occasionally observed a cleft in the outeredge of the ribosome and suggested this represented the site of association betweenthe large and small subunit of the ribosome. Barbieri et al. (1970) observed the ribo-somes to have a major axis 20—21 nm high and a width of 15 nm. He demonstratedthat ribosomes of these dimensions could produce the observed edge views, and explainthe 5-nm spaces seen between the aggregates of ribosomes when in the dashed-lineconfiguration. Carey & Read (1971), using centrifugation and biochemical methods,have concluded that the small subunit is located above the large subunit in such away as to enhance the elongated shape of the ribosome, and produce the observedkidney-shaped or clefted ribosome. The crystallized ribosomes from degeneratingPNZ cells have generally been isodiametric, ranging between 20 and 25 nm in dia-meter ; a few ribosomes did have a definite kidney shape.

Recent biochemical studies have further elucidated the structure and potentialfunction of the ribosome crystals. Humphreys & Bell (1967) demonstrated that ribo-nuclease-resistant square aggregates of 4 ribosomes are formed from monosomes invivo in response to low temperatures. Carey & Read (1971) and Byers (1971) haveshown that the ribosomes of the square tetramers are joined by bonds involving thelarge, 60-S subunits. Morimoto, Blobel & Sabatini (1972) have documented the factthat the tetramers are composed of mature, 80-s ribosomes and have shown them tocontain all species of ribosomal RNA and a complete set of ribosomal proteins. Byers(1971) and Morimoto et al. (1972) have demonstrated that the tetramers are capableof polypeptide synthesis; however, they do not exhibit endogenous messenger-RNAactivity.

The pathogenesis and significance of the ribosome crystals and protein crystalsoccurring in degenerating PNZ cells remains unknown. The ribosome crystals areclearly not the result of hypothermia. While fertilized eggs are refrigerated at approxi-mately 5-10 °C prior to being incubated, essentially no development takes place untilincubation at 38 °C is begun. Furthermore, the limb bud does not begin to developuntil approximately stage 17 (2-5 days incubation) and degenerating cells are not seenin the PNZ until stage 22 (3-5 days incubation). Also, necrosis in chick embryogenesisoccurs only in certain areas and is not a widespread event.

Likewise, the protein crystals observed in degenerating PNZ cells are not the resultof vinblastine or vincristine treatment. To our knowledge, crystals of this type haveonly been observed in cells treated with high doses of these drugs. Krishan & Hsu(1969) have demonstrated that formation of these crystals is a reversible phenomenon;the crystals are apparently capable of transforming into filaments. Their data supportthe hypothesis that the materials forming filaments seen in c-mitotic cells, the proteincrystals in vinblastine-vincristine treated cells, microtubules, neurofibrils and neuro-tubules all originate from a pool of similar, interchangeable cellular proteins. Whilefilaments and microtubules are not obvious in non-mitotic or degenerating PNZ cellsthe proteins forming these structures are undoubtedly present and probably serve asa source for the observed protein crystals. As with the ribosome crystals the exactpathogenetic mechanism resulting in protein crystal formation remains a mystery.

408 N. K. Mottet and S. P. Hammar

Numerous investigators have demonstrated helical crystalline arrangements ofribosomes in several different cell types (Behnke, 1963; Siddiqui & Rudzinska, 1963;Waddington & Perry, 1963; Weiss & Grover, 1968). None of these reportsdemonstrated any association of crystalline arrangements of ribosomes and cellulardegeneration.

Nauman, Silverman & Volez (1971) demonstrated ribosomal helices in Escherichiacoli cells gradually subjected to acidic conditions. Interestingly, their data suggestedthat so-formed ribosomal helices were inactive or dormant. We have not observedribosomal helices in degenerating PNZ cells and pH measurements of whole crushedembryos have always been neutral or slightly alkaline (pH 7-0-7-4). This does notrule out the possibility that the PNZ itself may be of acidic pH.

While it seems very unlikely that ribosome and protein crystals occur naturally onlyin degenerating PNZ cells, we have not observed intracellular crystals of any typein several types of ectodermal, mesodermal and entodermal derived embryonic chicktissues. Similarly, numerous investigators (Jurand, 1964, 1966; Maruyama & D'Ago-stino, 1967; Farbman, 1968; Saunders & Fallon, 1966; Manasek, 1969; Shapiro &Sweney, 1969; Sweney & Shapiro, 1970; Hammar & Mottet, 1971) have studiedcellular degeneration and necrosis electron microscopically in several different speciesof embryos and have not described ribosome or other types of intracellular crystals.The reason for this lack of ribosome and protein crystalline formation in degeneratingembryonic cells, especially those occurring naturally in other areas of the chick embryo,is not clear; we are certain that their presence in degenerating PNZ cells was notfortuitous, since our sampling of this area was so extensive.

The exact mechanism by which ribosomes and proteins crystallize in degeneratingPNZ cells remains uncertain. From the observations presented in this paper, it is nota specific hypothermic or vinblastine-vincristine induced response. Studies are cur-rently under way to determine the pathogenesis of the crystalline formation in cellsfrom the PNZ.

This investigation was supported by the U.S. Public Health Service Grants AM 08-727 andGM 13543. We are indebted to Dr D. Lagunoff for his critical reading of the manuscript. Weare also indebted to Mr Ralph Body and Mr Johsel Namkung for technical assistance and toMrs Miriam A. Kappes for typing the manuscript.

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(Received 7 January 1972)

Fig. 1. Representative area from the PNZ of stage 22 embryo. Several degeneratingcells (dc) are obvious; the one in the right upper portion of the micrograph has beenphagocytosed. The cell in the lower portion of the micrograph has a distinct ribosomalcrystal (re) in its cytoplasm, pn, pyknotic nucleus, x 5500.

Ribosome crystals in necrotizing cells 411

N. K. Mottet and S. P. Hammar

2B

Fig. 2. Typical examples of degenerating cells from the PNZ of stage 22-24 embryos.The cells are characterized by shrunken pyknotic nuclei (pn), condensed cytoplasmcontaining distinct ribosome crystals (re) and numerous cytoplasmic vacuoles (v).The ribosome crystals are seen in at least 2 different configurations, x 12000.

Ribosome crystals in necrotizing cells

Fig. 3. Enlargement of the ribosome crystals (re) reveals the tetramer pattern as wellas what appears to be a parallel-row arrangement. The parallel-row arrangementrepresents stacks of the crystals viewed on edge. In 3 B and 3 c transition of the riboso-mal crystals from a dotted and dashed line appearance into a tetramer configuration(arrows) is seen. In Fig. 3 D and 3 E stacks of ribosomal crystals are viewed on edge asparallel rows of ribosomes in dotted, dashed, and solid line configurations, x 20000.

N. K. Mottet and S. P. Eammar

Fig. 4. Degenerating cells from the PNZ showing protein crystals (pc). Note theirassociation with the ribosomal crystals and their somewhat beaded nature, x 32000.