Higher-order structure of chromatin from resting cells. II. High-resolution computer analysis of native chromatin fibres and freeze-etching of nuclei from rat liver cells

II. H I G H - R E S O L U T I O N COMPUTER ANALYSIS OF N A T I V E C H R O M A T I N FIBRES A N D FREEZE-ETCHING OF NUCLEI FROM RAT LIVER CELLS 0 Temple University, Philadelphia, U.SA., and Consiglio Nazionale delle Ricerche , Genova , Italy C. N.R., Centro di Sludi Chimico Fisici di Macromolecole Sintetiche e Naturali, Genova, Italy Istituto di Elettrotecnica, Sezione di Ingegneria Biofisica ed Elettronica, Universita di Genova, Italy Istituto di Farmacologia, Universita di Genova, Italy Istituto di Anatomia, Universita di Bologna, Italy Interdisciplinary Group of Bios truelure. University of Genova , Italy FROM Non-destructive electron microscopy of native chromatin from rat liver nuclei reveals that the 30 nm fibre is formed of four 11 nm nucleofilaments, arranged in a coiled-coil (or rope-like) conformation. At low ionic strength, native fibres show an alternating pattern of compact and unwound regions. Freeze-etching experiments carried out on the same nuclei are compatible with the existence of periodic attachments of the fibres to the nuclear envelope near the pores in a regular, drapery-like fashion. For the first time, computer image analysis has been applied to electron micrographs of giant chromatin fibres and a few essential geometrical parameters characterizing the conformation of the higher-order structures have been determined. No significant difference has been found between calf thymus and rat liver chromatin. C. NICOLINI1' 6, B. CAVAZZA2' 6, V. T R E F I L E T T I 2 ' 6 , F. P I O L I 2 ' 6, F. B E L T R A M E 3 ' 6 , G. B R A M B I L L A 4 ' 6 , N. MARALDI5 AND E. P A T R O N E ' 2 6 The concept of regular folding of the nucleofilament into a solenoidal (Finch & Klug, 1976) or a two-order superhelical (Nicolini & Kendall, 1977) fibre, approximately 30 nm wide, has been challenged by recent electron-microscopic observations on calf thymus chromatin obtained on a phospholipid monolayer (see accompanying paper), suggesting that the same fibre is formed of four interlinked nucleofilaments. The aim of this paper is to probe further the higher-order chromatin structure in rat liver nuclei as revealed by a combination of 'non-destructive' electron microscopy resolved by a Plumbicon scanner and a computer to a level not obtainable by normal observation, and freeze-etching. The rigorous statistical analysis carried out on the computer-enhanced fibres led to the evaluation of a few relevant parameters, characterizing the higher-order chromatin organization compatible with previously reported observations on interphase nuclei. Furthermore, the branched multifibrillar structure of chromatin is found to be similar in calf thymus and rat liver nuclei. Non-destructive electron microscopy High-resolution photographs of chromatin isolated from intact swollen nuclei of rat liver were obtained by means of a recently introduced electron-microscopic technique involving a phospholipid monolayer (Cavazza et al. 1979). Nuclei were swollen in 1 mM-Tris-HCl (pH8) as described (see accompanying paper). As previously pointed out, the chromatin images obtained are free from distortion or artifacts due to fixation and/or staining, and present a limited, acceptable level of background noise. Rat liver nuclei were isolated using a perfusion method (Nicolini et al. 1982). Isolated rat liver nuclei (Widnell & Tata, 1964) were fixed in 2-5% glutaraldehyde in 0-1 Mphosphate buffer (pH7-2) for 1 h and then rinsed in 015M-phosphate buffer. The nuclear pellet was resuspended in 30% glycerol in distilled water for 30min, frozen in Freon and processed for cleaving and replication in a Balzers 360 M freeze-etch device. Analytical image processing Images were obtained 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 electron microscope (EM) pictures were imaged through a macroepidiascope (final optical magnification = 24) on an European standard TV scanner target, equipped with a Plumbicon tube, which ensures a highly linear transfer function between light intensity and electrical signal. Individual EM pictures, from chromatin on phospholipid monolayer, were obtained as an array of several thousand picture points. 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, also called 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 is fed through a fast A / D conversion group (8 bit, 30 MHz, a monolithic integrated circuit) and each video frame can be stored in real time on a memory according to the format 512x512 pixels, 8 bit 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); the same images were occasionally sent back from the magnetic tape to the fast memory for further processing. The digital video signal originating from the memory is sent through a look-up table system and D / A conversion to black/white and colour TV monitor. Specifically, the look-up tables, being under program control, could allow us, whenever critically needed, to use pseudocolour techniques to enhance the images further. Geometric and densitometric analysis were frequently carried out on selected regions of the fibre images, by means of an interactive procedure involving a variable frame, program controlled for position and XY dimensions. Final images were obtained after subtraction and equalization of the background due to optical and electronic noise, as determined in equivalent regions outside the fibre. At variance with the unwinding process observed for giant chromatin fibres from calf thymus, which are almost invariably found in a compact form several hours after nuclear swelling (see accompanying paper), a pronounced branching of the 30nm strand is observed at the earliest times (a few minutes) in the case of rat liver chromatin. The unwinding of the native fibre, localized in discrete regions defined by fragments of nuclear envelope material (Fig. 1A, B), leads to the subdivision of the 30 nm strand into subfibres of smaller size down to the nucleofilament (11 nm thick), as shown in Fig. 1A, B. On a typical 60 ^m long chromatin fibre, emerging intact following swelling of the nucleus (Fig. 1A, B, C) we have carried out a statistical analysis to determine the size distribution of the branched (Fig. 2A) and unbranched (Fig. 2B) native segments of thefibrelying between the fragments of the nuclear envelope material. The size of these fragments has also been determined (F (...truncated)