Nuclear Pores and Interphase Chromatin: High-Resolution Image Analysis and Freeze Etching

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 periphery 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 replication (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 (pores) through their closest distance, the longest edges are progressively obtained. For the MST method the basic feature is to select the maximum length for'cutting', i.e. the length above which the corresponding interconnected pores belong to different clusters; normally a reference is made to the average value (d) of all tree connections and Kd is taken as the maximum length, where 1

RESULTS The pore distribution on the nuclear membrane, as apparent in freeze-etched micrographs, is shown in Fig. 1 for rat liver nuclei in different functional states; namely, with low and high metabolic activity. The abrupt change in metabolic activity brought about by phospholipid treatment (Manzoli et al. 1982) is accompanied by abrupt changes in both pore number (Fig. 2 and Table 1) and chromatin distribution (Fig- 2). However, an independent statistical analysis of each nuclear envelope, extrapolated to the whole nucleus, yields a similar number of 19—21 total clusters of pores for both unstimulated and phospholipid-stimulated nuclei, using either the ISODATA (see Table 1) or the MST (see Fig. 3) algorithms. From the ISODATA algorithm, for every cluster the distribution of the pores from the centre of the cluster has also been computed; a toroidal distribution consistently results, with a mean value typically of about l-Ofim (the nuclear diameter is about 7^tm) and with a quasi-Gaussian form. Conversely, by means of the MST algorithm the influence of the cutting parameter (see Materials and Methods) on the resulting number of clusters has been investigated (see Fig. 3). As is apparent from Fig. 3, a step-like variation in the number of clusters (NC) versus the parameter K (see Materials and Methods) is achieved at A— 1-75, whereby for A" values greater than this wide variations in A." introduce relatively small changes in NC, and for K values less than this value wide variations in NC occur, even for small variations in K. The average value of d, taking into account all the closest distances among adjacent pores (Fig. 4), i,s223nm. Therefore we obtain about 20 clusters whenKd is about 390 nm, which would represent the maximum distance between pores belonging to the same

Table 1. Average number of clusters (NC), as integer value, for each nuclear image (1M) as computed from ISODATA (see Fig. 1)

IM NP m S.D. iV NC Controls 1 580 18-6 3-5 30 19 5 788 19-5 5-0 39 20 6 514 20-8 5-6 25 21 Stimulated 1 390 18-6 2-6 20 19

NP, number of pores for the whole nuclear surface after the specular extrapolation; m, mean value of clusters obtained with a wide range of values for the parameters at the beginning of the iteration process; S.D., standard deviation of the clusters obtained with different initial values; N, avarage number of pores for each cluster.

Fig. 1. Freeze-etch micrographs of nuclear membrane from rat liver cells. Nuclei arc shown before (A) and after (B) stimulation with phospholipid vesicles. Nuclear pores and interphase chromatin 79

* •• * "/sv

Vv,

B Fig. 1 80 C. Nicolini and others

Stimulated ;h y

t I J2 • t S ! "5. • II 5 I :

Black Grey level White

Fig, 2. Number of pixels with given transmittance as a function of transmittance for 500 A thick nuclear sections, stained with uranyl acetate, from rat liver nuclei before (A) and 1 min after (B) phospholipid vesicle stimulation. A redistribution of chromatin from peripheral regions towards the inner part of the nucleus is also apparent (see insert). The bars indicate the positions of states I and II of chromatin condensation, in both A and B. Nuclear pores and interphase chromatin 81

90-

70-

60-

50-

40-

30-

20-

10-

1-5

120- 90- 60- 30-

1 1-5 4 K Fig. 3. A. Number of clusters versus the value Kof the cutting parameter, as determined with the Minimal Spanning Tree algorithm (see the text for the cluster analysis), B. Derivative of NC per unit K, as a function of K. cluster. The number of pores per cluster is shown in Table 2 for four different nuclear envelopes. Another reproducible finding from the frequency distribution of the closest distance between adjacent pores (Fig. 4) is that most pores are separated by about 160 nm, from centre to centre. Finally, about 18 stained areas are present near the nuclear periphery in similar cells Feulgen-stained and three-dimensionally reconstructed (Table 3 and Fig. 5). There is a wide variation in the fraction of total DNA present in each chromatin body (similar to the wide variation in pore number per cluster). 82 C. Nicolini and others

Table 2. Typical number of pores per cluster on the nuclear membrane for four different nuclear envelope preparations

Number of pores > Cluster no. 1 2 3 4

Total 280 282 251 198 1 37 55 31 23 2 47 43 18 20 3 55 18 12 14 4 29 24 53 22 5 15 10 15 10 6 7 11 39 19 7 8 32 15 8 8 14 13 10 12 9 16 16 13 5 10 19 11 — — Nuclear envelope major diameter (^m) 7-2 7-0 6-7 6-2

As justified in the text and shown in Fig. 4, the combination of pores interconnected by a distance equal to or less than 320 nm forms a cluster. Only a few pores (10%) cannot be grouped in any cluster using this criterion and they are either isolated or interconnected to form doublets or triplets; furthermore, they are concentrated near the periphery, where some deformation of the envelope is evident and tails connected to clusters of the opposite hemisphere are possible.

Interestingly (Fig. 5 and Table 3), similar total numbers of chromatin DNA bodies are present in both stimulated and unstimulated nuclei, which differ only in the state of condensation of the chromatin. The quantal nature of the transition between the two states, upon metabolic activation, is more conclusively indicated by the uranyl acetate data obtained at the electron-microscope level (Fig. 2), which show the absorbance ratio between the two states of condensation to be 2-75 (very close to the 2-5 apparent from the Feulgen-stained large chromatin bodies at the light-microscope level).

DISCUSSION Rigorous statistical analysis carried out on computer-enhanced nuclear membranes from various types of rat liver interphase cells demonstrates that the number of pore clusters is independent of the total number of pores and the chromatin distribution, but close to the number of chromatin bodies near the nuclear border. The fraction of total DNA present in most chromatin clusters is about 0-9-7% (Table 3), which is in striking agreement with the fraction of total DNA present in each metaphase chromosome from the same cells (0-8-5%). Curiously, the proportion of the total number of pores in each pore cluster of the envelope also ranges between 1-2 and 9-9%.

C Nicolini and others

e u o

Si E

i i i

4 12 20 28 Average optical density (arbitrary units)

Fig. 5. Number of chromatin bodies with given average absorbance as identified in three- dimensional reconstructed nuclei from Feulgen-stained 2^m serial sections (see insert), as a function of average absorbance from Go ( ) and G\ (—) nuclei.

1979; Kendall et al. 1980; Nicolini et al. 1983). More direct evidence for a close association of interphase chromatin with the nuclear membrane has been recently provided in unfixed nuclei (Agard & Sedat, 1983), resulting in a predominantly chromosome-free central cavity, which confirms earlier findings in fixed nuclei (Kendall et al. 1980; Nicolini, 1979, 1980; Olins & Olins, 1979). By optical fluorescence microscopy and three-dimensional chromosome Nuclear pores and interphase chromatin 85

Table 3. Typical amount of DNA (integrated optical density) per identifiable chromatin body around the periphery of Feulgen-stained mammalian diploid nuclei (in Gq), reconstructed three-dimensionally for 2 \on thick sections as previously described (Kendall et al. 1980)

Chromatin body I.O.D. (arbitrary units) 1* 10629 2* 14911 3 1472 4 1268 5 817 6 611 7 311 8 1598 9 520 10 2073 11 241 12 524 13 822 14 929 15 901 16 1250 17 2180 18 3170

The total I.O.D. for the reconstructed nucleus is 44-228 arbitrary units. • Very large regions possibly resulting from the overlappiing of two or more discrete regions ('chromatin bodies' or islands of absorbance higher than the background) displaying an unusually large condensation (Fig. 5). topography of intact unfixed nuclei (Agard & Sedat, 1983) a complex mixture of intertwined coils and parallel chromosome segments have been shown to be closely packed against the inner surface of the nuclear membrane. The presence along the giant 60 cm long (~10"Mr) chromatin fibres of laminar fragments - having dimensions centred around 120, 240 and 360 nm — and of an upper limit in the fibre length between pieces of nuclear membrane (Nicolini et al. 1983) provide an indirect justi- fication for the clustering of pores and for the upper limit to the distance between pores within each cluster. Furthermore, the existence of a repeating unit of about 160 nm in the interpore distance is compatible with the observation of a repeating unit of 480 nm in the chromatin segments delimited by fragments of the nuclear lamina (as shown in figs 2 and 5 of Nicolini et al. 1983), whereby a 480 nm contour length of the same fibre, when depending from the nuclear envelope, corresponds to a pore distance typically of 160 nm. However, it does not escape our notice that our findings raise more questions than they answer, concerning the actual role of nuclear pores and the higher-order organization of chromatin during interphase. There is an enormous amount of literature on this subject (Maul, 1977; Nicolini, 1983) and a complete discussion of 86 C. Nicolini and others the difficulties in fitting our findings with all other relevant work is beyond the scope of this paper.

This work was partially supported by a grant from C. N. R. Finalized Project 'Oncology', Consiglio Nazionale delle Ricerche.

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(Received 12 December 1983—Accepted, in revised form, 25 April 1984)