J. Cell Sa. 72, 75-87 (1984) 75 Printed in Great Britain © The Company of Biologists Limited 1984 NUCLEAR PORES AND INTERPHASE CHROMATIN: HIGH-RESOLUTION IMAGE ANALYSIS AND FREEZE ETCHING C. NICOLINI Temple University, Philadelphia, U.SA. and Eminent Chair of Biophysics, Faculty of Medicine, University of Genova, Italy G. VERNAZZA, A. CHIABRERA Istituto di Elettrotecnica, Sezione di Ingegneria Biofisica ed Elettronica, Universita di Genova, Italy I. N. MARALDI AND S. CAPITANI Istituto di Anatotnia, Universita di Bologna, Italy SUMMARY Computer-enhanced analysis of electron micrographs of thin-seCtioned rat liver nuclei, combined with three-dimensional reconstruction of the same Feulgen-stained nuclei, points to a unique clustering of chromatin DNA fibres near the nuclear border. Computer-enhanced image analysis has been applied to electron micrographs of the envelopes of the same rat liver nuclei prepared by freeze etching and a few essential geometrical parameters characterizing the pores and their distribution have been determined. During interphase, clusters of nuclear pores, closely paralleling the clustering of membrane-attached chromatin fibres, have been identified on the envelope, the number of these being similar to the number of homologus pairs of metaphase chromosomes. Furthermore, rapid changes induced in chromatin distribution appear to be associated with rapid changes in pore number, but not in the number of pore clusters. INTRODUCTION The double membrane surrounding the nucleus contains as its most conspicuous features the nuclear pore complexes, which have been assumed to be the sites of molecular and ionic exchange between nucleus and cytoplasm (Maul, 1977). Curiously, these channels in the nuclear envelope are referred to as pores, even if they are usually filled with a dense plug (Krohne, Franke & Schree, 1978; Unwin & Milligan, 1982). Biochemical characterization suggests that the nuclear pore complex is formed only of a few majdr polypeptides and some RNA (Krohne et al. 1978), but no direct identification has yet been possible within the boundaries of this structure and all speculations about its function are based on scanty, circumstantial and at times contradictory data obtained by limited, ultrastructural and autoradiographic methods (Franke & Scheer, 1970; Maul, 1977). While the complex has long been known to consist of a cylindrical assembly that spans the inner and outer nuclear membranes and is arranged with octagonal symmetry about a central axis, new detailed informa- tion has recently been obtained from electron micrographs of an intact oocyte nuclear Key words: nuclear pores, chromatin, image analysis. 76 C. Nicolini and others envelope by modern image-processing methods (Unwin & Milligan, 1982) at a resolution of 90 A. By these high-resolution methods the pore structure appears as a three-layered sandwich, consisting of a disk between two thin rings co-planar with one half of the double membrane. The disk, consisting of a central plug and eight broad spokes, is suspended between the two rings by extension of the spokes, at the peri- phery of which, at a diameter of about 100 nm, the two membranes fuse. These findings, which also provide evidence for the attachment of eight ribosomal particles to the cytoplasmic face of this structure (Unwin & Milligan, 1982), raise numerous further questions about the structure and function of the nuclear pores. Pore complexes are no longer considered to be randomly distributed but present at specific sites (Maul, 1977). Furthermore, rapid changes in pore pattern and number have been found to occur in nuclear membranes of various cell types in different functional states (Markowicz, Glass & Maul, 1974). A biphasic increase in pore frequency, associated with sharp transitions from a clumped to a uniform pore distribution, has been found in synchronized HeLa cell and phytohaemagglutinin- stimulated human lymphocytes in the very early part of G\ and before DNA replica- tion (Markovicz et al. 1974). Curiously, this biphasic change is exactly correlated with abrupt changes in the higher-order chromatin structure from a clumped to a uniform distribution, occurring at the same time in these cells (Kendall et al. 1980). Evidence suggesting the attachment of chromatin fibres to the nuclear envelope at the pores has recently been provided by freeze-etching of rat liver nuclei (Nicolini et al. 1983) and by fluorescence microscopy (Agard & Sedat, 1983), but conclusive proof of the presence of thin chromatin threads in the pore complexes is difficult by any present technique of electron microscopy. In a mammalian cell, the nuclear pores are rapidly formed in early telophase, when membrane pieces are seen attached to the chromosomes (Maul, 1977), and they are present with varying numbers and distribution throughout interphase (G\-S-Gz). A question that arises is whether, when pore clusters appear, there is any correlation between the number of these clusters and the number of chromosomes (even for changing chromatin and pore distributions). It may also be asked whether there is a similar number of clumps of chromatin near the envelope during interphase. These questions may be answered by further exploring the exact distribution of nuclear pores, using a statistical method, in relation to the parallel changes in pore number and chromatin distribution taking place in cells in different functional states but of constant chromosome number. To increase the resolution, computer-enhanced image analysis has been utilized in conjunction with light and electron microscopy (EM), for the studies reported here. MATERIALS AND METHODS Isolated rat liver nuclei (Widnell & Tata, 1964) were fixed in 2-5 % (v/v) glutaraldehyde in 0-1 M-phosphate buffer (pH 7-2) for 1 h and then rinsed in 0-15 M-phosphate buffer. In such isolated nuclei, before fixation, RNA synthesis is readily induced (within 1 min) by exposure to the appropriate concentration of phospholipid vesicles (namely, 1-5 min of phosphotidylserine). This method has been used to obtain nuclei in a state of high metabolic activity. The nuclear pellet was then processed in four ways. Nuclear pores and interphase chromatin 11 (1) Directly stained with acridine-orange for differential monitoring of chromatin DNA, using fluorescence microscopy (Nicolini, 1979). (2) Stained with uranyl acetate following thin sectioning (500 A thick), for subsequent electron microscopy (Manzoli et al. 1982). (3) Sectioned at 2//m and Feulgen-stained for differential chromatin DNA absorption, by means of quantitative light microscopy (Kendall et al. 1980). (4) Resuspended in 30 % glycerol in distilled water for 30 min, frozen in Freon and processed for cleaving and replication in a Balzers 360 M freeze-etch device. Analytical image acquisition and processing High-resolution image analysis was conducted as described in detail recently (Nicolini et al. 1983; Kendall et al. 1980; Belmont, Kendall & Nicolini, 1984), either on Feulgen-stained nuclear sections or on electron micrographs of freeze-etched nuclear envelopes and of sections stained with uranyl acetate. In the latter case, the EM pictures were imaged through a macroepidiascope (final optical magnification = 24). Individual EM pictures were acquired in an array of several thousand picture points. Images were acquired on a European standard TV scanner target, equipped with a Plum- bicon tube (which ensures a highly linear transfer function between light intensity and electrical signal) and analysed by means of the ACTA system built and installed at the Biophysical and Electronic Engineering Section, Institute of Electrotechnics, University of Genova (Italy) (Beltrame et al. 1980). The final linear dimensions of each approximately square picture point, characteristic of the Plumbicon-equipped image analyser, were determined to be 0-6 or 0-9 nm under our conditions of illumination and magnification. Individual transmittance values for each picture point (termed a 'pixel') were acquired in a calibrated linear scale of 256 grey levels, where 0 and 256 correspond to 0 % ('black') and 100 % ('white') transmittance, respectively. The analogue video signal was typically fed through a fast A/D conversion group (8 bit, 30 MHz, a monolitic integrated circuit) and each video frame could be stored in real time on a memory according to the format 512x512pixels, 8bit resolution per pixel. Images were transferred on a mass-memory device, such as magnetic tape or disc, interfaced to a HP 21MX minicomputer (which controls the ACTA system). High-resolution densitometric and geometric image analysis of the Feulgen-stained sections were performed on a Quantimet 720-D, as previously described at length (Kendall et al. 1980; Belmont etal. 1984). Cluster analysis Many methods can be used for cluster analysis. We have selected two different methods in order to verify independently pore cluster assignments on the nuclear envelope. The first one is called ISODATA (Ton & Gonzales, 1974) and it is characterized by an iterative procedure, which determines the number of clusters and the co-ordinates of each cluster centre by grouping sample means. Widely different values are given initially for these parameters to verify optimal convergence on the 'true' final number of clusters (NC) (the term 'ISODATA' stands for Iterative Self- Organizing Data Analysis Technique A). The second graph-theoretical method is called MST (Minimal Spanning Tree) (Dude & Hart, 1973). After the construction of a tree graphically connecting all points