Electron microscope observations on the nuclear pores of HeLa cells

620

H. W. Fisher and T. W. Cooper REFERENCES

1. 2. 3. 4. 5. 6.

HEREDIA, C. F., DE LA FUENTE, G. and SOLS, A., Biochim. JOHNSON, B. F., Exptl Cell Res. 39, 613 (1965). __ ibid. In press. __ J. Bacterial. 94, 192 (1967). MEGNET, R., J. Bacterial. 90, 1032 (1965). MITCHISON, J. M., Exptl Cell Res. 13, 244 (1957).

ELECTRON

MICROSCOPE

NUCLEAR

OBSERVATIONS

Acta 86, 216 (1964).

ON THE

PORES OF HELA CELLS

H. W. FISHER Biophysics

Biophys.

and T. W. COOPER

Laboratory, University of Rhode Island, Kingston, R. I. 02881, USA

Received June 16, 1967

THE

size and distribution of pores in the nuclear envelope may be important in considering the transfer to the cytoplasm of macromolecules or ultrastructures synthesized in the nucleus. Since, for technical reasons, many exquisite studies on the nuclear synthesis of RNA have been made with mammalian cells grown in vitro [8], it is useful to examine the nuclear cytoplasmic junction of these cells by electron microscopy. Unfortunately, most ultrathin section techniques do not permit a satisfactory evaluation of the distribution of pores in a thin membrane, and nucleus isolation techniques [2] may cause distortion or removal of the outermost nuclear membrane [5]. Recently, a vertical section technique was devised [l] which is extremely useful in the electron microscopic examination of cultured mammalian cells. With this technique it was possible to fix and embed the cells at different stages of monolayer culture and obtain sections without the distortion introduced by dispersal or centrifugation of the cells. An electron micrograph of such a vertical section of HeLa cells is shown in Fig. I. By careful alignment of such preparations, it was possible to obtain reasonably large areas of the nuclear envelope as a flat layer in a thin section. This was done by sectioning the preparation at right angles to the illustration in Fig. 1, until sections in the region (S) were obtained. Fig. 2 is an electron micrograph of such a section. Fig. 1.-A vertical section of a HeLa cell grown on a palladium film (Pd), showing nuclear pores (np) in the envelope between the nucleus (N) and cytoplasm (Cy). The region indicated (S) is the transverse section prepared for nuclear pore analysis. The glass side (Gs) of the palladium film is at the bottom. x 59,500. Fig. 2.-An electron micrograph of a transverse section from the region indicated in Fig. 1. The nuclear pores with corresponding holes in the dense chromatin are indicated (npl), also areas containing the membrane only (np.2). Microtubules (mt) are present in the cytoplasm. x 14,900. Figs 3 and 4.-The nuclear pores subjected to S-fold symmetry analysis by the procedure Markham et al. [7]. Fig. 3 with chromatin and Fig. 4 without chromatin. x 163,000. Experimental

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of

621

Nuclear pores of HeLa cells

Experimental

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622

L. Nicander

and Birgitta

Hellstrb’m

Although it is difficult to define the edge of the nuclear membrane in these sections, one can establish a lower limit on the density of the pores in the envelope. From a set of micrographs like Fig. 2, we made counts, measured the areas, and determined that there are about 13 nuclear pores/p2. We determined the diameter of the pores to be 650 A, which is close to published values for other preparations [3, 4, 61. From this we calculated that the pores give an open area to the nucleus of about 5 per cent. Nuclear pores found in oocytes, prepared by negative staining, have been described as octagonal [4] when subjected to analysis by the rotation technique described by Markham et al. [7]. Fig. 3 shows an g-fold symmetry analysis of a pore from the area in Fig. 2 with chromatin, and Fig. 4 from an area without chromatin. Although not directly comparable to negatively-stained preparations, the results of the rotation analysis of the sections was consistent with an octagonal shape. In the areas of sections which included chromatin, each pore had an opening in the chromatin of approximately twice the diameter of the pore. The pore count density in those areas was equal to that of areas which did not include chromatin. Also, no significant difference was found between the lower nuclear membrane and the upper one obtained by sectioning through the nucleus. This suggests that perhaps the nuclear pores and chromatin layer, with corresponding holes, are uniform over the entire nuclear-cytoplasmic junction. Preliminary examination of the cultured mammalian cell line 3T3 [9] revealed similar sizes and distribution of the nuclear pores. This work was supported by grant GB 2043 from the National Science Foundation and Public Health Service Research Grant CA-07787-03 from the National Cancer Institute. We thank F. Parlin and P. Wilson for technical assistance. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.

FISHER, H. W. and COOPER, T. W., J. Cell Biol. 34, 569 (1967). FISHER, H. W. and HARRIS, H., Proc. Roy. Sot. Ser. B 156, 521 (1962). FRANKE, W. W., J. Cell Biol. 31, 619 (1966). GALL, J. G., J. Cell Biol. 32, 391 (1967). HOLTZMAN, E., SMITH, I. and PENMAN, S., J. Mol. Biol. 17, 131 (1966). KESSEL, R. G., J. Ulfrasfrucf. Res. 1.5, 181 (1966). MARKHAM, R., FREY, S. and HILLS, G. J., Virology 20, 88 (1963). PENMAN, S., J. Mol. Biof. 17, 117 (1966). TODARO, G. J. and GREEN, H., J. Cell Biol. 17, 299 (1963).

INCREASED

THICKNESS

MEMBRANE

OF THE INNER

DURING

SPERM MATURATION

DOMESTIC L. NICANDER Department

of Anatomy

and Histology,

IN THE

ROOSTER

and BIRGITTA Received

Tm

MITOCHONDRIAL

Royal

HELLSTROM

Veterinary

College, Stockholm, Sweden

July 3, 1967

sperm mitochondria of some invertebrate groups are known to show remarkable structural specialization. Mammalian sperm mitochondria are less modified but Experimenfaf

Cell Research 48