JOURNAL OF ULTRASTRUCTURE RESEARCH 56, 304-311 (1976)

A Possible Mode of Format ion of Tubuloret icu lar Structures 1

SATISH CHANDRA AND STEFANO S. STEFANI

Service of Therapeutic Radiology, VA Hospital Hines, Illinois 60141

Received January 26, 1976, and in revised form, April 19, 1976

Serial electron micrographs of a cell containing tubuloreticular structures or undulating tubules associated with endoplasmic reticulum (ER), as they were previously designated, suggested that these structures are derived from ER. The endoplasmic reticulum forms paired or compound reticulum when two or more elements of this organelle come close together. In such a juxtaposition, the inner paired lamellae lose the attached ribonucleoprotein (RNP) granules and the lamellae and material between them become more electron opaque. The outermost lamellae of the compound reticulum retain RNP granules. Although the mechanism of tubule formation is not known, the paired lamellae form undulating tubules in the dilated cisterna of ER. The amount of undulation and the packing of tubules which produce various profiles seen in the electron microscope probably depend on the physiopathologic state of the cell. A three-dimensional reconstruction of one of the possible modes of formation of tubuloreti- cular structure is provided.

Undulating tubules associated with granular endoplasmic reticulum were first described in normal, pathologic and malig- nant human tissues both in vivo and in vitro (4). Similar structures have since been described in a variety of tissues in different species and the reader is referred to an excellent compilation on the subject by Schaffet al. (19) and Uzman et al. (23). That these structures are neither virus- like (2, 3, 15) nor phagocytosed matter (11) has been well documented (4, 6, 13, 19, 23). They appear to be morphologic manifestations of some pathologic process (4). The name tubuloreticular structure (TRS) suggested by Schaff et al. (19) for these structures appears more appropriate and will be used henceforth. Because of the close association of these structures with granular endoplasmic reticulum (ER) they were believed to be derived from ER. It was suggested that a system of undulat- ing tubules emanating from the endoplas- mic reticulum could produce various pro-

Part of this study was made as an independent investigation under the Special Virus Leukemia Program (Contract No. NIH 70-2080) of the National Cancer Institute, Bethesda, at the John L. Smith Memorial for Cancer Research, Maywood, N.J. 07607.

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

files seen in the electron microscope (4). In one plane they formed a crystalline pat- tern made up of small membrane bound bodies, approximately 24 nm in diameter. The uniformity in their size suggested tu- bules of uniform diameter. The present study suggests, on the basis of serial elec- tron micrographs of a portion of a cell, a possible mode of formation of tubuloreticu- lar structure from ER.

MATERIALS AND METHODS

Tissue Culture Cells

A culture of Burkitt lymphomal designated as P3- J was received from the National Cancer Inst i tute . Another culture, designated M-l, was derived from the peripheral blood leukocytes of a pat ient with acute lymphocytic leukemia (6). Cells grew in sus- pension as lymphoblasts. Their growth and culture characteristics are similar to other leukocyte cul- tures described by many investigators (5, 8, 14, 19).

Electron Microscopy For electron microscopic examination cells were

processed as described earlier (7). Sections stained with uranyl acetate and lead citrate were examined in a Siemens Elmiskop I electron microscope.

RESULTS

The micromorphology of lymphoblast- like cells in cultures derived from different

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hemopoiet ic t issues from hea l thy individ- uals and from pa t ien ts with var ious malig- nancies has been described in detai l by many invest igators (5, 8, 14, 19). Cul- tured cells contain small amounts of rough endoplasmic r e t i cu lum (ER). Somet imes e lements of two ER come close together and run paral le l to each other with a sepa- ra t ion of approximate ly 24 nm (Fig. 1). In such a juxtaposi t ion the segment of the lamel la of one re t i cu lum which is in close proximity to the lamel la of the other loses the a t tached r ibonucleoprote in (RNP) granules. Concomi tan t with this loss of granules, there appears to be an increase in electron opacity of apposing lamel lae and of the mate r i a l be tween them. This increase in densi ty cannot be considered an ar t i fact since two adjacent membranes of e i ther Golgi appara tus or mi tochondr ia in the same cell do not exhibi t this in- crease in electron opacity. Such a configu-

ra t ion by two apposed e lements of ER was refer red to as pa i r ing of ER (6). When three or more e lements of ER come close together only the outermost lamel lae ex- hibi t the presence of RNP granules on them and as described above the inner two or more paired lamel lae exhibi t increased electron opacity (Figs. 2-4). These com- pound endoplasmic re t iculum, CER, some- t imes extend over large portions of the cytoplasm (Figs. 2-4).

Tubulore t icular s t ruc tures occur in 2- 5% of cu l tu red lymphoblast- l ike cells in one section of specimen embedded for elec- t ron microscopy (personal observation). In the experience of this invest igator all cul- tures of lymphoblas t - l ike cells (4, 5, 7 and others not listed in these references) con- ta in tubulore t icu lar s t ruc tures (TRS) asso- ciated with endoplasmic ret iculum. In one cell from the cul ture P3-J, undula t ions of paired lamel lae (Fig. 2, unlabeled arrow)

FIG. 1. Pairing of endoplasmic reticulum, ER, in a cell from the culture M-1 exhibiting the loss of RNP granules from the surface of inner apposing lamellae (arrows). Note also that these lamellae and the material in between them have increased electron opacity. × 72 000.

FIo. 2. A portion of a cell from the culture P3-J exhibiting extensive compound endoplasmic reticulum, CER, formed by the juxtaposition of three or more elements of ER. RNP granules are present only on the outer lamellae of such reticulum. Towards the center and the lower left corner of the electron micrograph, the inner paired lamellae appear to form tubuloreticular structures, TRS. The unlabeled arrow points to an undulation within TRS which is continuous with the paired lamellae of CER. Note that at the arrow head the outer lamella of compound reticulum forms a boundary of TRS. A cytoplasmic body CB appears continuous with elements of compound reticulum (double arrows). Nucleus (N), mitochondria (M), lipid (L). x 23 000.

Fio. 3. An electron micrograph of the adjoining serial section of the portion of cell in Fig. 2 exhibiting a slight increase in the size of the tubuloreticular structure, TRS. x 23 000. -

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FIG. 4. The seventh serial section of the portion of cell in Fig. 2. In this plane, the tubuloreticular structure, TRS, has become extensive, yet the compound reticulum. CER. has the same lateral dimension as in Figs. 2 and 3. The unlabeled arrow points to a continuity between paired lamellae and a tubule within TRS. Also, the cytoplasmic body, CB, which is enveloped with granular ER (small arrow heads) appears continuous with elements of compound reticulum (double arrows). At the large arrow head, images of paired lamellae from where the tubules may be emanat ing are represented (see Discussion and Chart 1). Mitochon- dria (M), lipid (L). x 23 000.

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308 CHANDRA AND STEFANI

are observed in a region of cytoplasm where three or four compound reticulum converge to form tubuloreticular struc- tures, TRS. In the adjoining serial section (Fig. 3), the same cell exhibits a greater amount of TRS which is found to be exten- sive in the seventh serial section (Fig. 4). The compound reticulum on the other hand, do not exhibit any increase in their lateral dimension in the thickness of cell contained within seven sections, which suggests that they have a nearly straight lamellar ~structure. It will be noticed that whereas the inner paired lamallae of the compound reticulum have uniform spac- ing, the outer lamella which has RNP granules on its surface is unevenly sepa- rated from the adjoining inner lamellae (Figs. 2-5). In proper plane of sectioning, this outer lamella can be seen forming the boundary of the tubuloreticular structures (small arrow heads, Figs. 2 and 5). At a higher magnification (Fig. 5) various im- ages of a system of undulating tubules within the tubuloreticular structure, namely "granules" (short arrows) and cir- cular profile (long, thin arrows), described earlier (4), are clearly seen. The electron micrographs also show similar electron opacity of various sections of TRS and of the inner paired lamellae of the compound reticulum. Continuity between the tubules of TRS and the paired lamellae of the com- pound reticulum is seen at the unlabeled arrows in Figs. 2 and 4.

At the bottom of the electron micro- graphs, Figs. 2-4, is seen an unusual cyto- plasmic body, CB, which is enveloped by a lamella of granular endoplasmic reticulum (small arrow heads, Fig. 4). This body ap- pears to have a continuity with elements of compound reticulum (double arrows, Figs. 2-4).

DISCUSSION

Ultrastructural modification of endo- plasmic reticulum (ER) occur in a variety of pathologic processes. Whorl-like forma- tions of ER are induced by certain chemi-

cals (9, 10, 12, 16, 22). In cells infected with group B togavirus "complex tangle or crystalline array of membranes and tu- bules" was observed (21). "Labyrinthine tubular aggregates" were seen in liver pa- renchymal cells following exposure to car- bon tetrachloride (18). These latter forma- tions are different from those described earlier (4) and in the present study.

Pairing or stacking of endoplasmic retic- ulum has been described in cultured pe- ripheral blood leuk0cytes of a patient with acute lymphocytic leukemia (6). The char- acteristic feature of such a pairing is the loss of RNP granules from the surface of the paired lamellae of ER and an increase in the electron opacity of these lamellae. The same phenomena are also observed if three or more elements of endoplasmic re- ticulum stack together and from a com- pound reticulum. Even in this case, the paired lamellae maintain a uniform sepa- ration of approximately 24 nm throughout their three-dimensional configuration, or at least over a portion of a P3-J cell ap- proximately 0.5 t~m thick, assuming an average thickness of 70 nm for each sec- tion.

The continuity between the undulating tubules and the dense paired lamellae of endoplasmic reticulum (Figs. 2 and 4) and the similar electron opacity of these two structures suggest that the tubules are de- rived from the paired lamellae. A possible mode of formation of tubular structures from paired laminated sheets is diagra- matically illustrated in Chart 1 where a compound reticulum contains three paired lamellae. Just as the granular endoplas- mic reticulum is closed at its ends, it is postulated that the paired lamellae are also joined together at their extremities. Tubules cannot form from open ends of two parallef~lamellae; but how they emanate from the closed ends of these lamellae is not known. Section A of Chart i shows straight or convoluted tubules emanating from the three paired lamel lae of com- pound reticulum. Sections B and C repre-

FIo. 5. Tl~e large body of tubuloret icular structure, TRS, in Fig. 4 at a higher magnificat ion exhibits various images of a system of undula t ing tubules, namely, '~granules" (short arrows) and circular profiles (long, th in arrows). Small arrow heads point to a lamella of ER with at tached granules forming the boundary of TRS. The region pointed by the large arrow heads and enlarged in the inset shows the end view of the paired lamellae from where the tubules may be emana t ing (see Discussion and Char t 1C). x 60 000; inset, x 110 000.

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310 CHANDRA AND STEFANI

::.:.[:.:...

A. B. C.

CHART 1.(A) A three-dimensional representation of three paired lamellae constituting a compound reticu- lure. The two outermost lamellae have RNP granules which are absent from the surface of inner paired lamellae. The latter are closed at the ends from where tubules of the same diameter as the paired lamellae emanate. Some tubules are undulating while others are straight. (B) A paired lamellae as seen from the top of the page. (C) A paired lamellae from which three straight tubules are emanating as seen from the right to the left.

sent views f rom the top and f rom r igh t to left, respect ively. L a r g e a r row heads in Figs. 4 and 5 point to s t ruc tu res which p robab ly r ep resen t the l a t t e r view. As re- por ted ear l ier (4) o ther profiles of TRS seen in the electron microscope resu l t f rom var ious p lanes of sect ioning t h rough a sys- tern consis t ing of a l a rge n u m b e r of undu- la t ing tubules localized in a smal l volume.

The n u m b e r of undu la t ions of a tubu le would p robab ly depend on the di lat ion of the endoplasmic r e t i cu lum and the length of tubules . Both of these, in tu rn , p robab ly depend on the physiopathologica l s ta te of the cell, as the tubu lo re t i cu la r s t ruc tu re has, except for a few cases (4, 19), been most ly observed in disease conditions. Shor t tubules m a y have l i t t le or no undu- la t ions and if they are packed in a smal le r volume, images r ep re sen t ing t r a n s v e r s e and longi tudina l sections of the s t r a igh t tubules will be obtained. A rev iew of the publ i shed electron mic rographs of tubulo- re t icu lar s t ruc tu res and of those t a k e n in our l abora to ry r a r e l y revea ls a long s t r a igh t profile of the tubules , sugges t ing t h a t they are e i ther very shor t or have a lways some undula t ion , however smal l it m a y be.

The proposed mode of deve lopment of tubu lore t icu la r s t ruc tu res necess i ta tes the fo rmat ion of compound re t i cu lum as a pre- requisi te. This appea r s to be a r a re phe- nomenon since in mos t of the repor ted studies no pa i r ing of ER or fo rmat ion of compound re t i cu lum has been observed. In these cases, the model sugges ted by Bar in- ger and Swoveland (1) would appea r to be the correct th ree -d imens iona l r epresen ta - t ion of tubu lo re t i cu la r s t ructure . Whe the r these s t ruc tu res or ig ina te f rom compound re t i cu lum or direct ly f rom the l amel lae of ER (1) would seem to depend on the host response of cells to var ious physiopatho- logic s t imuli . •

The tubu lo re t i cu la r s t ruc tu re has been repor ted in a va r i e ty of t i ssues f rom m a n y species (19, 23), yet its funct ional signifi- cance r e m a i n s obscure. Poth ier et al. (17) sugges ted t h a t the t u b u l a r a r r a y m a y be re la ted to the immunog lobu l in synthesiz- ing ac t iv i ty of lymphoid cells. These inves- t iga tors also showed t h a t the a r r a y s a re ~more closely associated to the g a m m a heavy-cha in synthes is t h a n wi th the m u heavy-cha in synthes is . " The b iochemical na tu r e of TRS was s tudied by Schaff et al. (19, 20) who found t h a t i t contains pro-

MODE OF FORMATION OF TRS 311

teinaceous matter. The loss of RNP gran- ules during the pairing of ER and the in- crease in the electron opacity of the paired lamellae and possibly of the contents be- tween them would suggest a chemical combination of ribonucleoprotein granules with the lamellae. However, the studies of Schaff et al. (19, 20) revealed no ribonu- cleic acid within these structures.

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