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 information 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 phytohaemagglutininstimulated 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 Plumbicon 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 SelfOrganizing 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 <K< 4.
While ISODATA seeks classical cluster configurations according to statistical properties (mean
and variance), MST can also find laminar clusters, through a very lengthy computer process. In
order to reduce border effects the entire nuclear envelope is assumed to consist of two identical
hemispheres, where the measured hemisphere is the mirror image of the unknown hemisphere; this
allows determination of the total number of pores for each nuclear sphere.
Numerous tests have been performed with positive results on the ISODATA and MST
programs, to establish their ability to recognize automatically clusters of points grouped in different
configurations on the surface of a sphere (Bozzo & Risola, 1982). For these studies we have selected
78
C. Nicolini and others
nuclear envelopes only of a diameter identical to the diameter (about 7/un) of nuclei isolated from
Go rat liver tissue.
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)
Controls
Stimulated
IM
NP
m
S.D.
iV
NC
1
5
6
1
580
788
514
390
18-6
19-5
20-8
18-6
3-5
5-0
5-6
2-6
30
39
25
20
19
20
21
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.
79
Nuclear pores and interphase chromatin
*
•• *
"/sv
i
V
P
Vv,
B
Fig. 1
80
C. Nicolini and others
;h
Stimulated
y
t
t
I
J2
•
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-
80
70-
605040302010-
1-5
1209060301
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
>
2
3
4
Cluster no.
1
Total
1
280
37
47
55
29
15
7
8
14
16
19
282
55
43
18
24
10
11
32
13
16
11
251
31
18
12
39
15
10
13
—
198
23
20
14
22
10
19
8
12
5
—
7-2
7-0
6-7
6-2
2
3
4
5
6
7
8
9
10
Nuclear envelope
major diameter (^m)
53
15
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
O
e
u
o
Si
E
i
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 threedimensional 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
1*
2*
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
I.O.D. (arbitrary units)
10629
14911
1472
1268
817
611
311
1598
520
2073
241
524
822
929
901
1250
2180
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"M r ) 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 justification 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.
REFERENCES
AGARD, A. D. & SEDAT, J. (1983). Three-dimensional architecture of a polytene nucleus. Nature,
Land. 302, 676-681.
Bozzo, W. & RISOLA, E. (1982). Analisi dei raggruppamenti dei pori dei pori sulla superficie
nucleare delle cellule. Doctoral thesis, University of Genova.
BELMONT, A., KENDALL, F. M. & NICOLINI, C. (1984). Three-dimensional intranuclear DNA
organization in situ: three states of condensation and their redistribution as a function of nuclear
size near the Gi-S border in HeLa S-3 cells. J. Cell Sci. 65, 123-138.
BELTRAME, F., CHIABRERA, A., GRATTAROLA, M., GUERRIN, P., PARODI, G., PONTA, D.,
VERNAZZA, G. & VIVIANI, R. (1980). The ACTA system. 2nd Ann. Conf. of the IEEE Engineering in Med. and Biol. Soc, Washington, D.C.
COMINGS, D. E. (1968). The rationale for an ordered arrangement of chromatin in interphase
nucleus. Am. J . hum. Genet. 20, 440-460.
DUDE, R. O. & HART, P. E. (1973). Pattern Classification and Scene Analysis. New York: John
Wiley.
FRANKE, W. & SCHEER, U. (1970). The ultrastructure of the nuclear envelope of amphibian
oocytes: a reinvestigation. J. Ultrastruct. Res. 30, 288-316.
KENDALL, F., BELTRAME, F., ZIETZ, S., BELMONT, A. & NICOLINI, C. (1980). The quinternary
chromatin-DNA structure. Three-dimensional reconstruction and functional significance. Cell
Biophys. 2, 373-404.
KROHNE, T., FRANKE, W. & SCHREE, U. (1978). The major polypeptides of the nuclear pore
complex. Expl Cell Res. 116, 85-102.
MANZOLI, A., CAPITANI, S. & MARALDI, N. (1982). In Cell Growth (ed. C. Nicolini), pp.
466-486. New York, London: Plenum.
MARKOVICZ, J., GLASS, L. & MAUL, G. (1974). Pore patterns on nuclear membranes. Expl Cell
Res. 85, 443-451.
MAUL, G. (1977). The nuclear and cytoplasmic pore complex: structure, dynamics, distribution
and evolution. Int. Rev. Cytol. (suppl.) 6, 75-186.
MCGHEE, J. D., RAU, D. C , CHARNEY, E. & FELSENFELD, G. (1980). Orientation of the nucleo-
some within the higher order structure of chromatin. Cell 22, 87—96.
NICOLINI, C. (1979). Chromatin structure from Angstrom to micron levels, and its relationship to
mammalian cell proliferation. In Chromatin Structure and Function, part B (ed. C. Nicolini),
pp. 613-666. New York, London: Plenum Press.
NICOLINI, C. (1980). Nuclear morphometry, quinternary chromatin structure and cell growth.
J.'submicrosc. Cytol. 12, 475-505.
NICOLINI, C. (1983). Chromatin structure, from nuclei to genes. A review. Anticancer Res. 3,
63-86.
NICOLINI, C , TREFILETTI, V., CAVAZZA, B., CUNIBERTI, C , PATRONE, E., CARLO, P. & BRAM-
BILLA, G. (1983). Quaternary and quinternary structures of native chromatin-DNA in liver
nuclei: differential scanning calorimetry. Science 219, 176—178.
NICOLJNI, C , CARLO, P., MARTELLI, A., FINOLLO, R. & BRAMBILLA, G. (1982). Viscoelastic
properties of mammalian nuclear DNA. Higher order DNA packing and cell function. J. molec.
Biol. 161, 155-175.
NICOLINI, C , CAVAZZA, B., TREFILETTI, V., MARALDI, N., PIOLI, F., BELTRAME, F., BRAM-
BILLA, G. & PATRONE, E. (1983). Higher-order chromatin structure in rat liver nuclei: highresolution quantitative electron microscopy. J. Cell Sci. 62, 103-115.
OLINS, A. L. & OLINS, D. E. (1979). Stereo electron microscopy of the 25-nm chromatin fibers in
isolated nuclei. J. Cell Sci. 81, 260-265.
Nuclear pores and interphase chromatin
87
SEDAT, J. & MANUELIDIS, L. (1978). A direct approach to the structure of eukaryotic
chromosomes. Cold Spring Harbor Symp. quant. Biol. 42, 331-350.
TON, J. T. & GONZALEZ, R. C. (1974). Pattern Recognition Principles. New York: AddisonWesley Publishing Company.
UNWIN, P. N. & MILLIGAN, R. A. (1982). Large particles associated with the perimeter of the
nuclear pore complex. .7. Cell Biol. 93, 63-75.
WIDNELL, C. & TATA, J. (1964). A procedure for the isolation of enzymatically active rat liver
nuclei. Biochem.J. 92, 313-317.
YOSHIDA, H. & SAGAI, T. (1972). Banding pattern analysis of polymorphic karyotype in the black
rat by a new differential staining technique. Chromosoma 37, 387-398.
(Received 12 December 1983—Accepted, in revised form, 25 April 1984)