The presence of intranuclear annulate lamellae shortly after mitosis in human melanoma cells in vitro

© 1970 by Academic Press, Inc.

J. ULTRASTRUCTURERESEARCH 31, 375-380 (1970)

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The Presence of Intranuclear Annulate Lamellae Shortly after Mitosis in Human Melanoma Cells in vitro GERD G. MAUL

Department of Pathology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received November 3, 1969 Intranuclear annulate lamellae were found in human melanoma cells in vitro shortly after mitosis. Their origin is thought to be due to membranes being "trapped" during nuclear envelope formation at the end of mitosis. Intranuclear annulate lamellae are not seen during interphase. Membrane systems which resemble the nuclear membrane have been observed only rarely within the nucleus. Most of these membranes, termed intranuclear annulate lamellae (IAL), were found in germ cells (2, 3, 7, 9-12, 17, 19) and in undifferentiated embryonic cells (5, 8), but also in fully differentiated gland cells (4, 6). The morphogenesis of I A L was described (9, 10) as a sequence that started with a blebbing of the inner layer of the nuclear envelope. The resulting vesicles were said to touch along a small portion of their surface without completely fusing. A small dense channel, which later forms the annulus, developed in this region of partial fusion of two vesicles. This morphogenesis is dependent on vesicles and the blebbing process which were never observed in human melanoma cells in vitro (9, 10, 12). A different mode of I A L formation was therefore assumed.

MATERIALS AND METHODS Human melanoma cells were grown as previously described (18). They were fixed first in 2 % glutaraldehyde and then in 1% OsO 4 in phosphate buffer (pH 7.4), and flat embedded (1) in Epon (13). Cells were preselected with a light microscope and then prepared for electron microscopy as described earlier (14-16). A Siemens 101 electron microscope was used at 80 kV. RESULTS AND DISCUSSION Intranuclear annulate lamellae were found in extremely rare cases in human mela n o m a ceils in vitro. It was recognized, however, that if these types of membranes were found in one cell they could be seen in only one neighboring cell. The possible

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FIG. 1. Survey micrograph of telophase. Nuclear membrane reformation is nearly completed, x 3000. Fro. 2. Detail of Fig. 1. There is a double membrane in close association with two chromosomes (arrows at the upper right), x 25,000.

reason for this became apparent during a study of different mitotic stages (16). It was observed that during telophase double membrane sheets would attach to the chromosomes, eventually leading to nuclear envelope reformation (Fig. 1). This process begins in early telophase after cytokinesis has begun. Three possible ways of I A L

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FIG. 3. Survey micrograph of a late telophase, x 3000. FIG. 4. Detail of Fig. 3. Part of the trapped membrane can be seen within the nucleus. The other part pointing in the same direction was apparently separated by the closing nuclear membrane. x 72,000.

formation seem probable: (a) In telophase some chromosomes seem to continue to move closer to the centriole; these may have membranes attached to them (Fig. 2, arrow), pulling them between chromosomes. (b) Adjacent chromosomes might by chance attach to the same sheet of double membranes (Fig. 2, double arrow). (c) Small sheets of double membranes might become trapped in a fashion suggested in the top left corner of Fig. 2, where a bigger sheet of double membranes makes contact with two chromosomes enclosing a pocket of cytoplasm. Membranes covering spaces as such were observed to have no nuclear pores in late telophase despite being part of the nuclear membrane. In Fig. 3 a late prophase is shown. The chromatin is not yet decondensed, but midbody formation is completed. Part of this micrograph is magnified in Fig. 4. It shows a small piece of what must be called IAL, as it already contains pores. (This cannot be seen in this section.) It also shows a piece of double membrane outside the nuclear envelope, which in its direction seems to be a continuation of the IAL, but interrupted by the completed nuclear envelope. When such areas are analyzed by serial sections, a direct continuity of IAL with the nuclear envelope could never be established unequivocally (Fig. 5). Five areas

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Fro. 5. Serial sections through the intranuclear annulate lamellae. This short type was most frequently found in late telophase and in cells in early G1. x 70,000.

have been checked critically. However, the sample is not big enough to prove these negative findings. In Fig. 5b and c, one can see the closest spacing routinely observed. In Fig. 5e, fine tubular membranes (arrow pointing to the left) seem to approach closer. There were never any vesicles found that would support a mechanism of I A L formation as has been proposed previously (9-12). The apparent vesicle in Fig. 5b

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FI~. 6. The longest intranuclear annulate lamellae was found in a cell of unknown phase during the cell cycle. The nuclear membrane is sectioned obliquely, x 95,000. FIG. 7. The only intranuclear lamellae found in the "middle" of the nucleus was in close proximity to an indentation of the nuclear membrane, x 175,000.

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(arrow) can be found to be part of an underlying membrane tubule seen in Fig. 5c and d (arrows). Arrows are also pointing to pores in Fig. 5 d and e. A vast number of cells have been scanned with the electron microscope for other projects, but only two IAL were found in cells which did not resemble those shortly after mitosis (Figs. 6 and 7). Both IAL were in close proximity to the nuclear membrane. This observation was made in all previous reports on IAL. Continuity with the innermost nuclear membrane has been claimed in the crayfish Orconectes virilis (12), but the evidence is suggestive only. In tunicate oocytes, no continuity between the nuclear envelope and IAL has ever been observed (12). The extremely rare observation of IAL in melanoma cells in vitro could be due either to sampling problems, or to the presence of IAL during a very short period of the cell cycle. The observation that if one cell has IAL one other cell in its immediate vicinity also has them, indicates together with the evidence presented on nuclear membrane reformation in mitosis that IAL are present shortly after mitosis only. Cytoplasmic annulate lamellae also appear shortly after mitosis (15), but they persist. The careful investigation of other cells shortly after mitosis may possibly show that IAL occur more commonly. The process which eliminates IAL during interphase is not known. At this time, speculations on a probable function of IAL (9, 10, 12) are purely hypothetical. This investigation was supported by Grant No. CA 11654-01 from the National Institutes of Health. REFERENCES

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