Nuclear Non-Chromatin Proteinaceous Structures: Their Role in the Organization and Function of the Interphase Nucleus

J. Cell Set. 44, 395-435 (1980) 295 Printed in Great Britain © Company of BiologitU Limited igSo

NUCLEAR NON-CHROMATIN PROTEINACEOUS STRUCTURES: THEIR ROLE IN THE ORGANIZATION AND FUNCTION OF THE INTERPHASE NUCLEUS

PAUL S. AGUTTER* AND JONATHAN C. W. RICHARDSONf • Department of Biological Sciences, Napier College, Colinton Road, Edinburgh EH10 5DT, Scotland and f Department of Physiology and Pharmacology, University of St Andrews, Bute Medical Buildings, St Andrews, Fife, Scotland

REVIEW ARTICLE: CONTENTS I. INTRODUCTION page 39s (1) Historical background 395 (2) Nomenclature 397

II. NUCLEAR PROTEIN MATRIX AND NUCLEAR GHOSTS 397 (1) Isolation 397 (2) Composition 398 (3) Ultrastructure 401 (4) Enzyme activities associated with the nuclear protein matrix 405 (5) Contractility of the nuclear protein matrix 405 (6) Functions associated with the nucleai protein matrix 408

III. SUBFRACTIONS OF THE NUCLEAR PROTEIN MATRIX 411 (A) The pore-lamina 411 (1) Isolation 411 (2) Composition 413 (3) Ultrastructure 414 (4) The molecular organization of the pore-lamina 417 (B) Other subfractions 419

IV. COMPOSITIONAL AND FUNCTIONAL DIFFERENCES BETWEEN THE PORE-LAMINA AND THE REMAINDER OF THE NUCLEAR PROTEIN MATRIX 42O

V. PROSPECTS FOR FURTHER RESEARCH 422 (1) The role of the nuclear protein matrix in nucleo-cytoplasmic RNA transport 422 (2) Relevance of a knowledge of factors affecting the stability of the intranuclear regions of the matrix to the further development of methods for isolation of the nuclear envelope 423 (3) Fate of the nuclear matrix during mitosis 423

I. INTRODUCTION (1) Historical background Since 1949 there have been several accounts of a 'honeycomb layer' or 'nuclear cortex' in the nuclei of lower eukaryotes (Callan, Randall & Tomlin, 1949; Callan & Tomlin, 1950; Harris & James, 1952; Pappas, 1956; Beams, Tahmisian, Devine & 26-2 396 P. S. Agutter andj. C. W. Richardson Anderson, 1957; Mercer, 1959; Gray & Guillery, 1963; Daniels & Breyer, 1967; Barton, Kisieleski, Wassermann & Mackevicius, 1971). This highly structured layer appears either to separate the inner nuclear membrane from the peripheral chromatin or to structure the heterochromatin region. It extends for up to 300 nm into the nucleoplasm, though this distance varies considerably with cell type, and shows discontinuities in the region of the pore complexes. In the decade 1960-70 a similar, but less extensive highly organized layer of material was described in the nuclei of higher eukaryotes. The structure was termed, alternatively, the 'granular perinuclear layer' (Bruni & Porter, 1965), 'dense lamella' (Kalifat, Bouteille & Delarue, 1967), 'fibrous lamina' (Coggeshall & Fawcett, 1964; Fawcett, 1966; Ghadially, Bhatnager & Fuller, 1972; Cohen & Sundeen, 1976) or 'zonula nucleum limitans' (Mazanek, 1967; Patrizi & Poger, 1967). Davies and his co-workers, in a detailed study of this layer, found that it comprised alternating narrow electron-dense and electron-transparent bands arranged parallel to the envelope, intersected orthogonally by fine electron-dense fibrils (Davies, 1967, 1968; Davies & Small, 1968). Detailed investigation of amphibian oocyte nuclear envelopes revealed, instead of this perinuclear layer, an array of fibres extending from the nuclear face of the pore complexes into the nucleoplasm (Franke & Scheer, 1970). Collectively, these studies suggested that at least some part of the region of the nucleus contiguous with the inner nuclear membrane is structured by a network of fibrils continuous with the pore complexes. Such a structure has since been isolated and termed the 'nuclear pore-lamina fraction' (Aaronson & Blobel, 1974, 1975). Over the years 1942-69, evidence accrued for the existence of a structural network of non-chromatin protein fibrils extending throughout the nucleus (Mayer & Gulick, 1942; Zbarsky & Debov, 1948; Wang et al. 1950; Allfrey, Dally & Mirsky, 1955; Du Praw, 1956; Zbarsky & Georgiev, 1959; Georgiev & Chentsov, 1962; Zbarsky, Dmitrieva & Yermolayeva, 1962; Wang, 1961, 1966; Steel&Busch, 1963; Bernhard & Granbonlan, 1963; Swift, 1963; Holtzman, Smith & Penman, 1966; Kaye & McMaster-Kaye, 1966; Monneron & Bernhard, 1969). Such a network has since been isolated (Berezney & Coffey, 1974a, b) and termed the 'nuclear protein matrix'. The early evidence for the structure depended on its resistance to the procedures used for the solubilization of chromatin: it retained the original shape of the nucleus after extraction with 2 M NaCl and dilute alkali. Corroborating evidence included: (a) the observation that while nuclease treatment did not markedly alter the shape and size of the nucleus, protease treatment quickly resulted in swelling and rupture (Anderson, 1953); and (b) the maintenance of the gross morphology of the nucleus after removal of the nuclear membranes with non-ionic detergents (Bach & Johnston, 1967). Later investigations by Busch and co-workers led to the idea of a structural ribonucleoprotein network, rather than a merely proteinaceous structure, extending throughout the nucleus (Smetana, Steele & Busch, 1963; Steele & Busch, 1966; Narayan, Steele, Smetana & Busch, 1967). Before 1970, therefore, there was a substantial body of literature concerning at least 2 sorts of non-membranous structural elements inside nuclei. It seems reasonable a priori to regard the juxtamembranous layer described in detail by Davies (1968) as Nuclear matrix, ghost and pore-lamina 397 the peripheral portion of the protein or ribonucleoprotein network discussed above. Since 1970, several biochemical studies of these structural elements have been carried out. The present review is devoted to the implications of these studies for an understanding of the organization of nuclei and their functions during interphase.

(2) Nomenclature Before discussion of these studies can be undertaken, clarification of the terminology is essential. Different laboratories naturally develop different systems of nomenclature; the system outlined here is based, as far as possible, on the most frequent current usage. It should be emphasized that our terminology does not distinguish between a structure observed in situ by microscopy and an apparently similar but not necessarily identical structure prepared by subfractionation of isolated nuclei. To make such a distinction would greatly complicate the nomenclature, but its importance in the interpretation of experimental findings should not be overlooked. We use the term nuclear envelope to describe the peripheral structure isolated from nuclei. The nuclear envelope has 4 ultrastructurally distinct subfractions: outer nuclear membrane, inner nuclear membrane, pore complexes and fibrous lamina. Removal of the 2 membranes leaves the ' nuclear pore-lamina fraction' or, more briefly, pore-lamina (see above and section in A, below). Careful removal of the chromatin and the 2 nuclear membranes from whole nuclei leaves a structure which has been called the nuclear protein matrix (see above and section 11, below). This structure includes the pore-lamina: pore complexes inter- connected with fibrils are visible in it. If nuclei are vigorously extracted with detergent and high-ionic-strength media but not treated with nucleases, nuclear ghosts can be banded out on sucrose gradients. The distinction between these nuclear ghosts (which are operationally distinct from nuclear envelopes) and the nuclear protein matrix is discussed in section 11. The nuclear protein matrix appears to contain residual nucleoli as morphologically distinct regions.

II. NUCLEAR PROTEIN MATRICES AND NUCLEAR GHOSTS (1) Isolation The nuclear protein matrix. Berezney, Coffey and co-workers have evolved a procedure for isolating nuclear protein matrix from whole nuclei (Berezney & Coffey 1974a, b, 1975a, b, 1977). Essentially, the method involves: (a) swelling of the nuclei (isolated by a variant of the method of Blobel & Potter, 1966) by overnight storage at 5 °C and treatment with a buffer containing a low concentration (0-2 ITIM) of magnesium ions; (b) solubilization of the chromatin in 2 M NaCl; (c) removal of the nuclear membranes with 1 % (w/v) Triton X-100; and (d) removal of residual nucleic acids with DNase and RNase. More recently, a modified procedure involving omission of step (a) and inclusion of inhibitors of proteolysis in the media has been employed. This modified procedure has been claimed to result in the production of more intact matrices (Berezney & Buchholtz, 1978). This or closely related methods for isolating nuclear protein matrices have been successfully used in other laboratories (Shelton, 398 P. S. Agutter andj. C. W. Richardson Cobbs, Povlishock & Burkat, 1976; Faiferman & Pogo, 1975; Hildebrand, Okinaka & Gurley, 1975; Hodge, Mancini, Davis & Heywood, 1977; Agutter & Birchall, 1979). Faiferman & Pogo (1975) have also isolated the matrix fraction, albeit in a fragmented state, from nuclei disrupted by nitrogen cavitation or in a French pressure cell. The nuclear ghost. Riley, Keller and co-workers have described the isolation of nuclear ghosts by a procedure involving: (a) washing of the nuclei in 1 % Tween 40 and 0-5% (w/v) sodium deoxycholate; (b) disruption of the nuclear contents with 0-5 M MgCl2; and (c) sucrose gradient centrifugation. The nuclear ghosts band on the gradient at 47-52% (w/v) sucrose (Riley, Keller & Byers, 1975; Riley & Keller, 1976a, b, 1978a, b; Keller & Riley, 19760, b).

(2) Composition The nuclear protein matrix. The nuclear protein matrix accounts for about 10-20% of the total nuclear protein, depending on the tissue of origin, and has the overall composition: 98-2% protein, o-i% DNA, 1-2% RNA, 0-5% phospholipid (Berezney & Coffey, 1974a). The protein in preparations from liver and other mammalian tissues comprises 3 major acidic polypeptides with molecular weights as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of 69000, 66000 and 62000. Since molecular weight estimates differ slightly between different laboratories (cf. Berezney & Coffey, 1976; Hildebrand et al. 1975; Buldiaeva, Kuzmina & Zbarsky, 1978), we propose to refer to these 3 polypeptides as P^ P2 and P3 respectively, rather than signify them by any one published set of molecular weight values. It is noteworthy that no histone contamination of the nuclear protein matrix is visible on SDS-PAGE (Berezney & Coffey, 1976). Inclusion of proteolytic inhibitors in the isolation media results in the retention of more high-molecular-weight polypeptides and increase in the DNA content of the matrix to 1-2% (Berezney & Buchholtz, 1978). The possibility that some of these polypeptides are identical with non-histone chromatin proteins deserves further investigation, though there is evidence that the 3 major matrix polypeptides are absent, or virtually absent, from the major chromatin protein classes (see table 3 in Elgin & Weintraub, 1975; see also Berezney & Coffey, 1976). It is possible that the SDS-PAGE band pattern would be different if different nuclear isolation procedures were employed, as is the case with chromatin proteins (Elgin & Weintraub, 1975). The nuclear ghost. The nuclear ghost material contains 71% protein, 14% DNA, 5% RNA and 10% phospholipid (Riley & Keller, 19766). The high phospholipid content suggests that this sort of preparation is more enriched in peripheral, envelope- associated material and less enriched in intranuclear material than the nuclear protein matrix (cf. above). The protein apparently comprises 6 major polypeptides with molecular weights on SDS-PAGE of 67000, 60000, 57500, 56000, 51000 and 46000; it seems likely that the first 3 of these are Plf P2 and P3. Unfortunately, it is not possible to determine the histone content of the nuclear ghosts from the published gel patterns (Riley & Keller, 19766). Nuclear matrix, ghost and pore-lamina 399

?.*&*%**

Fig. i. Electron-microscopic sections through the nuclear protein matrix revealing the internal structural components of the matrix. In A, the structure of an entire matrix is seen. RN, residual nucleolus; IM, internal matrix; RE, residual nuclear envelope layer (lamina). Note the empty spaces surrounding the residual nucleoli and along the periphery of the nucleus (arrows in A). These may correspond to regions previously occupied by the perinucleolar and peripheral condensed chromatin in intact nuclei. At higher magnification (B), close association of the internal matrix with the residual nuclear envelope is evident (white arrows). A residual nuclear pore complex structure is projecting through the residual nuclear envelope layer (black arrows). Regions of the internal matrix structure (enclosed in broken line) resemble clusters of interchromatinic granules seen in both isolated and in situ nuclei (compare nuclear ghost, Fig. 3). (A, XI7OOO;B, X 41000; bars indicate 1 //m for A and 0-4 fim for B.) (From Berezney & Coffey, 1977; Berezney, 1979.) 400 P. S. Agutter and J. C. W. Richardson

Fig. 2. A. High magnification of the nuclear protein matrix in the area of the residual nucleolus. The residual nucleolus, RN, appears continuous with the internal matrix (IM). Empty spaces (arrows) surrounding the residual nucleolus may correspond to regions previously occupied by the perinucleolar condensed chromatin (compare with Fig. i A and B). Note that these empty areas are bordered by the nucleolus and internal matrix structures. B, high-magnification section of a region in the interior of an isolated nucleus. Distinct areas of condensed chromatin, C, are seen in the upper-centre and lower-left regions of the micrograph. The region between these condensed chromatin areas is the interchromatinic matrix which contains electron-dense interchromatinic granules (ig) and less electron-dense fibrous structures (f). c, high magnification of a Nuclear matrix, ghost and pore-lamina 401

(3) Ultrastructure The nuclear protein matrix. The isolated nuclear protein matrix is a network of fibrils which retains the overall shape of the nucleus. The outlines of nucleoli (i.e. residual nucleoli: see section 12, above) are visible, and the fibrils at the periphery of the structure terminate in pore complexes (Berezney & Coffey 1974 a, 1975 a). It seems reasonable to suppose, on the basis of this evidence, that the nuclear protein matrix is a fibrillar network extending throughout and conferring structural stability upon the interphase nucleus, and that the pore complexes represent the specialized termini of the peripheral fibrils of this network. The nuclear protein matrix has been described in Tetrahymena (Wunderlich & Herlan, 1977; Wunderlich, Giese & Bucherer, 1978), livers of mammals other than rat (Comings & Wallack, 1978; Ghosh, Pawletz & Ghosh, 1978), mammalian endometrium (Barrack et al. 1977; Agutter & Birchall, 1979) and lung (Hemminki, 1977; Agutter & Birchall, 1979), cultured mammalian cells (Hildebrand et al. 1975) and avian erythrocytes (Cochran, Cobbs & Shelton, 1975). This suggests that the nuclear protein matrix is a widespread, if not ubiquitous structure in eukaryotes. The nuclear ghost. Nuclear ghosts comprise annuli, possibly the remains of pore complexes; rods of dimensions 260 x 50 nm, some of which link these annuli; one or more apparently amorphous 'dense bodies' (see below); and fine DNase-labile fibrils linking the 260x50 nm rods to other, similar rods (Keller & Riley, 1976 a). DNase treatment leads to the collapse of the structure of the system. This is certainly not the case in the nuclear protein matrix, though one report suggests that it may happen in residual nucleoli (Anteunis, Pouchelet, Gansmuller & Robineaux, 1975). The inconsistency in appearance, composition and susceptibility to DNase between the 2 kinds of preparation presumably results from methodological differences in the isolation procedures; nuclear ghosts are perhaps best regarded as incomplete matrices. Three points seem worth noting here. First, Berezney & Coffey (1974 a, b) used very low centrifugal fields at all stages in their procedure; higher fields led to irreversible aggregation and collapse of the matrix. However, Riley & Keller (1976) used much higher centrifugal fields. Secondly, 0-5 M MgCl2 is known to disrupt the pore-lamina (Shelton, 1976; Dwyer & Blobel, 1976) and might therefore be prejudicial to the integrity of the nuclear ghosts; DNA may be necessary to prevent disruption of their structure by high magnesium concentrations. Third, deoxycholate disrupts the nucleoli (Kirschner, Rush & Martin, 1977; see also section IIIB below), and this may

section through the internal matrix of the nuclear protein matrix. This residual structure consists of electron-dense matrix particles (mp) and matrix fibres (f) which bear a close similarity to the interchromarinic matrix structures of isolated as well as in situ liver nuclei (compare with B and Fig. 3). D, E, high-magnification sections through the residual nuclear envelope layer of the nuclear protein matrix (compare Fig. 9). Distinct residual nuclear pore complex structures are observed, which still retain their charac- teristic annular structure (arrows). Central granules are often visible in tangential sections through the residual pore complex structures (white arrow in E). A, X 92000; B, C, x 105000; D, E, x 144000; bars correspond to o-i /tm. (From Berezney & Coffey, 1977; Berezney, 1979.) 402 P. S. Agutter andj. C. W. Richardson 3A Nuclear matrix, ghost and pore-lamina 403 account for the absence of residual nucleoli from the ghosts. Comparative electron- microscopic studies have suggested that the nuclear ghost is, like the matrix, a widespread structure in eukaryotes (Riley & Keller, 1978 a). The detailed ultrastructure of the nuclear ghost is not quite the same in all cases (Riley et al. 1975; Riley & Keller, 19766), and in HeLa cells at least the morphology changes during the cell cycle (Keller & Riley, 19766; Riley & Keller, 19786). Heavily staining 'dense bodies' (also observed in some matrix preparations; see Hildebrand et al. 1975), are small, disperse, and associated with the periphery of the ghost in the Gx phase, but coalesce to form a single, large, centrally located object during the 5-phase. The appearance in G2 is intermediate between these extremes. This de- scription is interesting in view of observations that nuclei in Gx are more fragile than they are later in the cell cycle, since the ultrastructural changes in HeLa might indicate differences in structural stability of the whole nuclear protein matrix; cf. Schumm & Webb (1972), Feldherr (1968), Mironov, Adler, Sokolov & Shapot (1976). It seems likely that assembly of the nuclear protein matrix is incomplete at telophase and continues through Gv If this assembly process required energy and de novo protein synthesis, then, given that the matrix fibrils terminate in pore complexes, this may explain the observation that inhibitors of oxidative phosphorylation and high concentrations of actinomycin D or of cycloheximide inhibit pore complex formation in Gx but not at other stages of the HeLa cell cycle (Maul, Hsu, Borun & Maul, 1973). Clearly, misconceptions about nuclear protein matrix or nuclear ghost structure could have arisen from artifacts of fixation or staining. For instance, rearrangement of the material caused by 0-5 M MgCl2 or polylysine (Riley & Keller used polylysine-coated grids for their electron-microscopic work) might account for the fact that the 260 x 50 nm rods observed in the nuclear ghost are not observed in whole nuclei. It is feasible that these rods are aggregates of the 2-nm fibrils which have been reported in mammalian liver subnuclear fractions (Comings &Wallack, 1978; Scheer et al. 1976). The high magnesium ion concentration could also account for the absence of the usual octagonally symmetrical subunit structure of the pore complex annuli in nuclear ghosts (Keller & Riley, 1976; cf. also the micrographs in Monneron, Blobel & Palade (1972) and Harris & Agutter (1976)). Glutaraldehyde, the most commonly used fixative, precipitates soluble nuclear components (Skaer & Whytock, 1977a, b), but since the existence of the nuclear protein matrix has been indicated by isolation and chemical characterization as well as by electron microscopy, the suggestion by Skaer & Whytock that the structure is an artifact of fixation seems untenable. Moreover, Herlan, Quevedo & Wunderlich (1978), Brasch & Sinclair (1978) and Ghosh et al. (1978) have presented evidence for the existence of the nuclear protein matrix in vitro.

Fig. 3. Non-membranous HeLa cell nuclear ghost flattened and attached in whole- mount fashion with poly-D-lysine. Rod-like and annular structures with the dimensions indicated in the text are widely distributed throughout the ghosts. The low-magnification micrograph demonstrates the distribution of ghost components, but for more useful inspection of the rod-like and annular structure., refer to the higher magnification (B). Small, dense aggregates of material with radiating connected rod sequences are distributed throughout the ghost shown, A, X4500; B, X 25700. P. S. Agutter and J. C. W. Richardson

Sit;

Fig. 4. A non-membranous HeLa cell nuclear ghost isolated during S-phase (2-5 h after release from the second of 2 sequential thymidine blocks) mounted and stained with uranyl acetate and observed by transmission electron microscopy. Single large central aggregates of material are present in the 5-phase ghosts. Smaller dense aggregates with radiating connected rod sequences are absent. Instead, many of the connected rod sequences radiate from the central aggregates outward. Frequency distribution analysis and other considerations indicate that much of the material of the 'skirt' surrounding the central aggregate originated from the ghost surface, that the central aggregate is internal, and that at least some of the radiating connected rod sequences traverse the distance from the interior to the surface, x 5280. Nuclear matrix, ghost and pore-lamina 405

(4) Enzyme activities associated with the nuclear protein matrix Owing perhaps to the rigour of the procedures used for isolating the matrix, few enzymic activities have been reported in association with the system. Berezney's group have described an endogenous protein kinase (Allen, Berezney & Coffey, 1977); a probable role of this is discussed below (section 116a). Higgins, Yeoman & Shelton (1978) have described an endogenous high-salt-activated protease in avian erythrocyte matrices, probably associated with Yr. Protein P3, and Px itself, are more susceptible to proteolysis by this enzyme than is P2. The significance of this activity is not yet clear; the possibility that it might have a role in the processing of the matrix during nuclear assembly is interesting (Higgins et al. 1978). If this protease is widespread in nuclear protein matrices, it might account for some of the variation in the published molecular weight estimates of Px, P2 and P3 (see section 112, above), although differences in electrophoretic technique are likely to be relevant to this issue. Cytochrome c oxidase has been reported in matrix preparations, possible being a residual inner nuclear membrane component (Berezney & Coffey, 1974a). The presence of cytochrome c oxidase in nuclear envelopes is, however, controversial; it occurs in such low levels that it might easily result from mitochondrial contamination (for details of the contro- versy see: Berezney, Macaulay & Crane, 1972; Berezney & Crane, 1972; Zbarsky, 1972; Franke, 1974a, b\ Jarasch & Franke, 1974, 1977; Kasper, 1974; Wunderlich, Berezney & Kleinig, 1976). It will be interesting to see if other activities, for instance those associated with post-transcriptional RNA processing (see section 116c, below), are reported in the nuclear protein matrix in the future.

(5) Contractility of the nuclear protein matrix The demonstration that Tetrahymena nuclear protein matrices expand in low concentrations (imia) of Ca2+ and Mg2+ in a 3/2 molar ratio and contract reversibly in higher concentrations (5 mM) suggests that the structure exhibits ATP-independent contractility (Comings & Harris, 1976; Wunderlich & Herlan, 1977; Herlan et al. 1978; Wunderlich et al. 1978), and leads to 3 questions: (i) is contractility a universal property of nuclear matrices?; (ii) what in vivo functions might contractility of the nuclear protein matrix (or of parts of the matrix) serve ?; (iii) what factors other than divalent cation concentration might influence the state of expansion of the system ? Clear answers to these questions are not at present available, but interesting possibilities are raised from studies on whole nuclei and from investigations (see section 116, below) of the functions of the matrix. It must be noted that Tetrahymena matrix contains a protein of molecular weight 18000, which has not been described in other systems and seems to be involved in the contractility (Wunderlich & Herlan, 1977). The following sets of observations concerning expansion of whole nuclei are worthy of note. (a) Just as divalent cations influence the volume of the nuclear protein matrix, so they influence the volume of whole nuclei (Leake, Trench & Barry, 1972; Harris & Milne, 1975; Giese, Fromme & Wunderlich, 1979). Up to a point, beyond which the swelling seems to be irreversible, the nuclei can be re-shrunk. Leake et al. (1972) calculated the ion concentrations required for half-maximal swelling of hen erythrocyte 406 P. S. Agutter and J. C. W. Richardson

B Nuclear matrix, ghost and pore-lamina 407 nuclei and obtained values of 0-85 mM for Mg2+, 0-70 mM for Ca2+, 0-42 mM for Mn2+ and 1-65 mM for Ni2+. Monovalent cations also influenced nuclear volume, but much higher concentrations were required; the half-maximal concentration for K+ was 85 mM. The effect of these ions is certainly exerted at an intranuclear site; nuclear envelopes are freely permeable to species of molecular weight less than 500 (Harding & Feldherr, 1959; Feldherr, 1968; Paine, Moore & Horowitz, 1975) and small in- organic cations and amines not only freely enter the nucleus but are concentrated in it (Anderson & Wilbur, 1952; Anderson & Norris, i960; Naora et al. 1962; Kanno & Loewenstein, 1963; Langendorf, Siebert & Nitz-Litzow, 1964; Jones, Johnson, Gupta & Hall, 1979). The matter of reversibility of nuclear swelling was further investigated by Barry & Merriam (1972) and the swelling was found to occur in 2 stages; the first, involving a small volume change, was Mg2+-sensitive and reversible; the second, involving a much larger change, was Mg2+-insensitive and irreversible. The possibility that swelling of the nuclear protein matrix may occur in 2 such dis- crete steps appears not to have been investigated, though it is not improbable that the second stage observed in whole nuclei resulted from an irreversible reorganization, possibly accompanied by markedly increased hydration, of the chromatin. DNA content itself influences nuclear volume (Cavalier-Smith, 1978). (b) Some polyanions bring about the swelling of rat liver nuclei (Anderson & Wilbur, 1951; Kraemer & Coffey, 1970). Heparin and dextran sulphate were effective, while chondroitin sulphate and hyaluronidate were not (Kraemer & Coffey, 1970); polyaspartate of molecular weight 20-35000, but not of lower molecular weight (5500), also caused swelling. Polyanions bind to the nuclear protein matrix (cf. Brown & Coffey, 1971) and have been shown to lead to a radical alteration in chromatin structure (Saiga & Kinoshita, 1976). There is evidence that these processes are associated with the initiation of DNA replication and other nuclear functions (Brown & Coffey, 1971, 1972; Arnold et al. 1972; Coffey, Barrack & Heston, 1974). Smaller molecular weight negatively charged species bring about nuclear swelling; ATP and EDTA are examples (Leake et al. 1972; Chai, Weinfeld & Sandberg, 1978). Pre- sumably these species operate by a chelating action, lowering the free divalent cation concentration (cf. section 116 a, above). (c) Heterologous cytoplasm greatly increases the volume of transplanted nuclei. The mechanism is unclear, but there is evidence that cytoplasmic proteins are important (Harris, 1967; Gurdon & Woodland, 1968; Bolund, Ringertz & Harris, 1969; Flickinger, 1970; Maul et al. 1973). The effect of homologous cytoplasmic proteins on

Fig. 5. Predominant HeLa cell nuclear ghost species present in ghost populations isolated at times corresponding to Gx (14 h after release from a second thymidine block). Instead of a single large central aggregate there are numerous smaller aggregates each with radiating connected rod sequences. Frequency distribution analysis indicates that most of the stainable material was on the surface of the unflattened Gx- phase ghosts. The proximity to the outer ghost margin of some of the small dense aggregates suggests that some or all of these aggregates also were at or near the ghost surface (see text), A, x 3700; B, X 34600. 408 P. S. Agutter andjf. C. W. Richardson the nuclear matrix is at present unknown, but seems well worth studying in view of the importance of specific protein factors in regulating the nucleo-cytoplasmic transport of ribosomal subunits and possibly of mRNA (Schumm & Webb, 1974a, b, 1978; Racevskis & Webb, 1974; Sato, Ishikawa & Ogata, 1977), and in regulating transcription and post-transcriptional RNA processing (Gurdon, 1967; Thompson & McCarthy, 1968; De Bellis, 1969; Merriam, 1969; Yu, Racevskis & Webb, 1972; Schumm & Webb, 1972, 1974a, b, 1978; Schumm, McNamara & Webb, 1973a; Schumm, Morris & Webb, 19736; Seifart, Juhazz & Beneche, 1973; Froehner & Bonner, 1973; Chang &Goldwasser, 1973; Banks, Gilbert & Johnson, 1974; McNamara, Racevskis, Schumm & Webb, 1975; Revel & Groner, 1978; Heinrich, Gross, Northemann & Scheurlen, 1978). This topic will be discussed further in section v below.

(6) Functions associated with the nuclear protein matrix The matrix necessarily disperses in early prophase, at least in those species in which mitosis is associated with a loss of nuclear structure, and is presumably reassembled during telophase (Ghosh et al. 1978; cf. Robbins & Gonatas, 1964). That it fulfils the role of conferring structural organization on the chromatin (Ghosh & Roy, 1977; cf. also Hay & Revel, 1963; Comings, 1968) seems clear from the fact that chromatin binds so tightly to it: vigorous conditions have to be employed to remove the DNA and and chromatin proteins from the nuclear protein matrix (see section 111, above) and it has a high affinity for DNA, particularly poly(dT) sequences (Comings & Okada, 1976; Comings & Wallack, 1978). In view of this, the A-T-rich chromomeres ('G- bands') may be assembled on the intranuclear region of the matrix (Comings & Wallack, 1978), and the dense bodies reported in nuclear ghosts by Keller & Riley (19766) and Riley & Keller (1978) may be the remains of nucleosome aggregates (Olins & Olins, 1974; Ide, Nakane, Anzai & Andoh, 1975; Cook, Brazell & Jost, 1976), especially in view of the ultrastructural changes in these during S-phase, which may reflect the change in packing density of nucleosomes associated with newly synthesized chromosomal DNA (cf. Hildebrand & Walters, 1976). Details of the structural relationship between matrix and chromatin remain obscure, but the work of Burgoyne, Skinner & Marshall (1978, 1979) is of considerable interest here. These workers studied the penetrability of nuclei and of isolated chromatin by 3H-labelled glycogen of various particle sizes, and also investigated the susceptibility of nuclear DNA to hydrolysis by free and glycogen-bound DNases. Their results indicated that there were at least 2 classes of penetrable spaces in nuclei: (a) of approximate diameter 4 nm; (b) of approximate diameter 11-15 nm. The former, but not the latter class was also found in isolated chromatin. Nucleases attacked DNA only if they could enter the smaller (class a) spaces. In view of these findings, Burgoyne et al. (1978, 1979) suggested that the 4-nm (class a) spaces were enclosed in cubic quasicrystalline arrays of 8 nucleosomes, with the bridging DNA traversing the space. Increased nuclear activity, it is suggested, is associated with expansion of the nucleosomes into the cavities and consequent nuclear swelling (see section 115 and discussion below). Consistent with this view is the observation that the nascent DNA during replication is unusually Nuclear matrix, ghost and pore-lamina 409 DNase-labile (see e.g. Hewish, 1977). Some details of the work must remain doubtful, since (i) the nuclear membranes were torn and thus allowed free entry of 15-nm particles to which the nucleus would normally be impermeable (Paine et al. 1975), hence some other details of nuclear structure may have been abnormal, (ii) incu- bations were carried out at 0 °C in EDTA, and it is possible that the cavity sizes are sensitive both to temperature and to chelating agents (see section 115 b), and (iii) cal- culations of cavity sizes did not take account of the possible dimensions of hydration layers on the nucleosomes. These considerations, while they make the actual calculated values dubious, do not invalidate the general conclusions. The absence of the larger (class b) cavities from isolated chromatin may suggest that these cavities result from the organization imposed on the chromatin in situ by the matrix fibrils, which appear to be well separated (see Fig. 1). Thus, the quasicrystalline nucleosome arrays may be mounted on the matrix fibrils leaving class (b) cavities between the fibril- aggregate complexes. Given this pattern of close association between matrix and nucleosomes, which is consistent with the observations of Ghosh et al. (1978) and others, it is feasible that matrix contractility will affect the organization and activity of individual nucleosomes. In this context, it is noteworthy that recent work has demonstrated that the nuclear protein matrix is not merely a passive structure. There is evidence for its importance in at least 4 nuclear activities. (a) The initiation of DNA replication has been shown to be localized on the matrix by Berezney's group, who have found that the very low percentage of total nuclear DNA recovered in matrix preparations is markedly enriched in newly synthesized material (Berezney & Coffey, 1975a, b; Berezney & Buchholtz, 1978; see also Dvorkin & Vaniushin, 1978). It is interesting to note in this context that the helix-unwinding enzyme purified from mammalian nuclei has a molecular weight in the region of the major matrix polypeptides (Keller, 1975; Champoux & McConaughy, 1976), though other eukaryotic helix-destabilizing proteins of much lower molecular weight are known, and these also stimulate DNA polymerase activities (Herrick, Delius & Alberts, 1976; Herrick & Alberts, 1976; Duguet et al. 1977; Otto, Baynes & Knippers, 1977). An endogenous protein kinase catalyses ATP-dependent phosphorylation of at least one of the matrix proteins prior to the initiation of replication (see section 114, above) and this phosphorylation is necessary for the process (Kleinsmith, Stein & Stein, 1976; Allen et al. 1977). It is noteworthy that nuclear envelopes from the same tissue, namely rat liver, catalyse the ATP-dependent phosphorylation of a polypeptide co-migrating with P3 (Agutter et al. 1979; Lam & Kasper, 1979). This point is discussed further in section m A4 below. A correlation has been noted between the number of nuclear pore complexes and the number of DNA replication sites (Maul, Price & Lieberman, 1971; Maul et al. 1972), and this may reflect the fact that changes in pore complex number during interphase correlate with the development of the nuclear protein matrix (see also Feldherr, 1962; Grasso, Swift & Ackerman, 1972; Coleman, Duggan & Hackett, 1974; Rejthar & Blumajer, 1974, 1975; Svejda, Vrba & Blumajer, 1975; Ono, Ono & Wada, 1976; Berezney & Coffey, 1976; Lodin, Blumajer & Mares, 1978; Hemminiki, 27 CKL44 410 P. S. Agutter andj. C. W. Richardson 1977; Fawcett & Chemes, 1979). The correlation between DNA content of the nuclei and pore complex number does not indicate - as suggested by Schel, Steenbergen, Bekers & Wanka (1978)-that DNA replication is initiated at the pore complexes (see Maul, 1976; Maul & Deaven, 1977; Maul et al. 1972, 1973). In contrast, there is very good evidence from the studies described above that the intranuclear matrix is a site for the initiation of DNA synthesis. In HeLa cells, pore complex formation is rapid in telophase and early in Glt and again during the 5-phase when the nuclear volume also increases. For the remainder of Gx and G2 it is slow. Similar results were obtained with lymphocytes after phytohaemagglutinin stimulation (Maul et al. 1972) and from the plasmodia of Physarutn polycephalum (Schel et al. 1978). Pore complex number thus correlates with the number of interphase chromosome copies and with the nuclear volume in any single cell type; but a comparison of nuclei from a wide range of sources showed no simple overall correlation between pore complex number and nuclear surface area, volume or DNA content (Maul & Deaven, 1977; see also Cavalier-Smith, 1978). Since the pore complexes, however specialized they might be in composition and function (see section in A 4, below) represent the termini of matrix fibrils, the alterations of pore frequency during the cell cycle may be related to the structural differences between nuclear ghosts isolated at different stages of the cycle (Keller & Riley, 1976a; Riley & Keller, 1978a, b). Since DNA replication is initiated on the matrix, and since matrix development parallels the increase in pore complex number, it follows that the pore complexes are structurally attached to the initiation sites and that the numbers of one correlate with the numbers of the other. This may resolve the conflicting evidence about the involvement of the nuclear envelope in the initiation process (see e.g. Lark, Consigli & Minocha, 1966; Alfert & Das, 1969; Kay, Haines & Johnston, 1971; Wise & Prescott, 1973). Expansion of the matrix such as occurs after nuclear transplantation (Harris, 1967; Gurdon & Woodland, 1968) appears to be a prerequisite for the initiation of replication (Graham, Arms & Gurdon, 1966) in such cases (see section 115, above). (b) Following the speculation of Wunderlich et al. (1976) evidence has been obtained independently from several laboratories for the initiation of transcription at the matrix (Todorov, Galkin, Shen & Zheliaborskaia, 1975; Miller, Huang & Pogo, 1978 a, b\ Herman, Weymouth & Penman, 1978; Plekhanova, Gevorkian, Ashirova & Ilragimov, 1978; Agutter & Birchall, 1979) in rat liver. Rapidly labelled HnRNA was found to be highly purified in the matrix fraction. It is therefore possible that the DNA sequences which bind to the matrix fibrils include promotor sequences. During purification of the nuclear protein matrix the RNase treatment was omitted and an inhibitor of proteolysis was added to the media to prevent proteolytic degradation of the informosomes (Miller et al. 1978 a; Agutter & Birchall, 1979; cf. Berezney & Buchholtz, 1978). Once again, the literature contains evidence that nuclear swelling, and hence expansion of the matrix following a transplantation experiment, is followed by increased RNA synthesis (Harris, 1967; Gurdon & Woodland, 1968). (c) It has been suggested that at least some of the stages in post-transcriptional RNA processing (see Revel & Groner (1978) for a recent review of this topic) also take place in association with the nuclear protein matrix fibrils (Berezney & Coffey, 1976, Nuclear matrix, ghost and pore-lamina 411 1977; Wunderlich et al. 1976; see also section 115 c, above), but at present there is no experimental evidence which bears directly on this issue. Of particular interest in this context is the possibility that RNA splicing (Ohtsuki, Groner & Hurwitz, 1977), which a priori would seem to involve a highly structured catalytic system, might occur in association with the matrix fibrils. If the speculation is valid, the matrix fibrils might be viewed as sites of RNA maturation connecting the site of initiation of transcription to the site of RNA export from the nucleus (the pore complex). This model is consistent with the view of the matrix as a ribonucleoprotein network extending throughout the nucleus (Berezney & Coffey, 19746; Smetana et al. 1963; Steel & Busch, 1966; Narayan et al. 1967; Faiferman & Pogo, 1975). It would be valuable to know whether informofer or ribosomal proteins were recovered, even as trace components, in matrix preparations. Treatment of nuclei with a-amanitin leads to contraction of the chromatin and nucleolar material and exposure of the matrix (Brasch & Sinclair, 1978; Ghosh et al. 1978). Recent studies of Puvion-Dutilleul & Bachellerie (1979) suggest that low doses of actinomycin D may have a similar effect on the ultrastructure of Chinese hamster ovary cell nuclei. (d) The nuclear protein matrix from rat endometrium is a specific (high-affinity) binding site of i7/?-oestradiol (Barrack et al. 1977; Agutter & Birchall, 1979). This finding indicates tissue-specificity in the structure, since oestradiol binding is not found in matrix preparations from liver (Barrack et al. 1977) or lung (Agutter & Birchall, 1979). Since oestrogens trigger the transcription of specific genes, the localization of oestradiol binding at the site where transcription is initiated is interesting, but the physiological significance of this binding has not yet been unequivocally established. It is interesting that steroid binding to the matrix is only measurable when the incubation medium includes EDTA, which is known to bring about expansion of the structure (see section 115, above). Again, therefore, expansion may be a prerequisite for the process (cf. Brown & Coffey, 1972; Arnold et al. 1972; Saiga & Kinoshita, 1976).

III. SUBFRACTIONS OF THE NUCLEAR PROTEIN MATRIX (A) THE PORE-LAMINA Isolation of the pore-lamina could not be attempted until methods for isolating ultrastructurally intact nuclear envelopes had been developed. The method of Kay et al. (1972) provides a satisfactory means of preparing envelopes rapidly, and is now widely employed.

(1) Isolation of the pore-lamina Using nuclei isolated from rat liver by the method of Blobel & Potter (1966), Blobel and his co-workers (Aaronson & Blobel, 1974, 1975; Dwyer & Blobel, 1976) prepared nuclear envelopes by a slight modification of the procedure of Kay et al. (1972). Essentially, this procedure involves lysis of the nuclei in Tris or triethanolamine buffer at a high pH (8-5) and containing a low concentration of Mg2+ (o-i HIM), and removal 27-2 4It P. S. Agutter andj. C. W. Richardson of the expanded and disrupted chromatin by DNase treatment. The envelopes are then washed in the same buffer at pH 7-5 and purity of the preparation is ensured by application of a second DNase treatment. Clearly the initial lysis crucially involves dispersal of the matrix. If sufficient expansion of the nuclei is not achieved at this stage (i.e. if the matrix is not disrupted) then satisfactory envelope preparations cannot be obtained. Preparation of the pore-lamina from the envelopes involves (a) removal of the membrane lipid by incubation in 2% (v/v) Triton X-100 for 10 min at o °C, the residual material being pelleted at 20000 g (unlike the nuclear protein matrix (see

Fig. 6. A. Electron micrograph of an isolated nuclear envelope seen in negative stain (2 % ammonium molybdate). The annular ring of the pore complexes is prominent. Material can be seen within many pore complexes, corresponding to the 'central element', x 31000. B. Electron micrograph of isolated nuclear envelopes in thin section ( x 40000). Pore complexes can be seen in transverse section. Although the inner and outer nuclear membranes are often closely apposed, there is always some separation of the membranes. (Richardson, 1979.)

section 111, above), the pore-lamina will not pellet at low centrifuge speeds), and (b) extraction of the pellet (o °C, 10 min) with 2 M NaCl or 0-3 M MgCl2, followed again by recovery by centrifugation at 20000 g. The salt extraction removes most of the residual histones and nucleic acids and leaves only the fibrous lamina, complete with pore complexes. A modified, more rapid procedure for isolation of the pore-lamina has been described (Aaronson, 1977), but the essential features are similar to those outlined above. The similarity between the conditions required to isolate the nuclear protein matrix from whole nuclei, and those required to isolate pore-lamina from nuclear envelopes, is striking (cf. section in, above). Nuclear matrix, ghost and pore-lamina 4**

(2) Composition The pore-lamina contains 2-3 % of the total protein of the nucleus and has the overall composition 95% protein, 3% DNA and 2% RNA (Dwyer & Blobel, 1976). Material of similar composition has been obtained from sheep lung, liver and endometrium, though endometrium pore-lamina tends to be somewhat richer in RNA (Agutter et al. 1978).

AB Fig. 7. SDS electropherogram of nuclear envelope polypeptide fractions stained with Coomassie brilliant blue, A, high-salt washed nuclear envelopes; B, pore-complex lamina fraction of nuclear envelopes extracted with Triton X-100; C, polypeptides extracted from nuclear envelopes by Triton X-100. The major polypeptides Ni, N2 and N3 (mol. wt 70000, 67000 and 58000) are indicated. It can be clearly seen that, although Triton extraction removes more than 95 % of phospholipid from isolated nuclear envelopes (Dwyer & Blobel, 1976), very little protein is extracted by this procedure. Although it may well be that the bulk of nuclear envelope protein resides in the pore-complex lamina fraction, it is also possible that some polypeptides from the inner and outer nuclear membranes precipitate on to the pore complex and their interconnecting lamina during Triton extraction. (Richardson, 1979; Richardson & Maddy, 19806.) 414 P- S. Agutter andj. C. W. Richardson In liver, the protein apparently comprises 3 major polypeptides, which on SDS- PAGE have molecular weights 69000, 68000 and 66000 (Dwyer & Blobel, 1976). Molecular weight estimates vary slightly from laboratory to laboratory as with the matrix polypeptides (section 112, above); we shall refer to these proteins as Nx, N2 and N3 respectively. The question of their identity with P1( P2 and P3 of the matrix is interesting, and will be returned to in section IV; certainly the molecular weights are very similar (Berezney & Coffey, 1974). N3 is removed, along with some trace components and most of the residual nucleic acids, on treatment of the pore-lamina with EDTA (Agutter et al. 1978) or, at least in the case of chicken erythrocyte pore-lamina, with o-i M NaOH (Jackson, 1976). It is virtually absent from envelopes isolated in the presence of heparin (Bornens, 1973; Bornens & Courvalin, 1978; Harris, Agutter & Milne, 1978; Agutter et al. 1978, 1979; see also Shelton & Cochran, 1978). The latter treatment also drastically alters the ultrastructure of the pore complexes and leads to a marked lowering of the envelope nucleoside triphosphatase referred to in section 115 a, above (Agutter et al. 1979). These results are particularly interesting in view of the effects of heparin and EDTA on matrix expansion (see section 1146, above) and the evidence for topological separation of N3 from the other major polypeptides within the lamina (see section 111A4, below). Krohne, Franke & Scheer (19786) have found only one of these 3 major polypeptides (N2) in pore-laminae prepared from amphibian oocytes after microdissection to obtain the nuclear envelope, together with another major polypeptide component of molecular weight 150000. Nx and N3 were found in envelopes and pore-laminae isolated from rat liver in addition to these components. The 150000 material can be seen as a pore-lamina component in other published SDS-gel patterns (e.g. Aaronson & Blobel, 1975) though usually only in trace amounts (e.g. Agutter et al. 1978). This striking difference between the Triturus oocyte pore-lamina and the pore-laminae of mammalian tissues may reflect the difference in extent, i.e. total mass, of the structures and a compositional difference between the pore complex and the lamina fibrils; thus, lung pore-lamina comprises mainly lamina fibrils (Agutter et al. 1978) while amphibian oocyte pore-lamina consists mainly of pore complex material (Krohne et al. 19786). Alternatively it could imply a difference in the purity of preparations. This will be discussed further in the next 2 subsections.

(3) Ultrastructure Electron microscopy of thin sections of isolated pore-lamina shows that its component pore complexes are linked by fibrils which are in turn linked with other fibrils, forming a network (Aaronson, 1977; Aaronson & Blobel, 1974, 1975; Dwyer & Blobel,

Fig. 8. Electron micrograph of disrupted nuclear envelope in negative stain (2 % ammonium molybdate. x 60000). This gives a rare glimpse of the detail of the very fine fibres (arrowed) connecting the pore complexes (circled) and constituting the fibrous lamina. The fibres appear continuous between several pore complexes. Such detail is not easily seen after Triton extraction for the fibres tend to clump. (Richardson, I979-) Nuclear matrix, ghost and pore-lamina 4*5 8 P. S. Agutter andj. C. W. Richardson 1976; Kirschner et al. 1977). In the amphibian oocyte pore-lamina, the fibrillar network is confined to a very narrow band contiguous with the nucleoplasmic face of the inner nuclear membrane (perhaps constituting an integral part of that membrane (Scheer et al. 1976; Krohne et al. 19786)), together with filaments which appear to continue for a short distance into the nucleoplasm from the pore complexes (see also Franke & Scheer, 1970).

Fig. 9. Electron micrograph of the nuclear pore complex-lamina fraction in thin section (x 64500). Several pore complexes are seen in tangential section, connected by a fine, but irregular, fibrous meshwork. The internal structure of the pore complexes is, in general, rather diffuse, but central elements and annular subunita are evident in some pore complexes (ringed). Inset: nuclear pore-lamina fraction in thin section ( x 69000). Pore complexes are clearly identifiable in transverse section. The interconnecting lamina (arrows), when seen in transverse section, appears as a thin and closely compacted layer (Richardson & Maddy, 1980a).

In mammalian tissues, the structure is more extensive, and as suggested in the previous subsection may differ somewhat in composition. Descriptions of the pore- lamina in situ also suggest differences in extent of the structure between different cell types (see e.g. Callan et al. 1949; Callan & Tomlin, 1950; Patrizi & Poger, 1967; Franke & Scheer, 1970, 1974; Cohen & Sundeen, 1976; see also Murray & Davies, 1979). The isolated pore-lamina shows none of the highly ordered peripheral nuclear structure described by Davies and co-workers (Davies, 1967, 1968; Davies & Small, 1968), so it appears to become structurally disorganized during the isolation procedure. Nuclear matrix, ghost and pore-lamina 417 There is evidence that the fibrous lamina determines the distribution of pore complexes (Abelson & Smith, 1970; Maul et al. 1971; Markovics, Glass & Small, 1974; Tiegler & Baerwald, 1972). Sonication of the pore-lamina disrupts and disperses the fibres and the pore complexes, the latter showing considerable mechanical stability (Dwyer & Blobel, 1976; Marshall & Harris, 1979). The same phenomenon has been seen in whole nuclear envelopes (Harris, 1977). Exposure of the pore-lamina to 0-5 M MgCl2 at 37 °C for 10 min has a similar effect (Dwyer & Blobel, 1976), which is noteworthy in view of the conditions for isolation of nuclear ghosts, discussed in section 111, above (Riley & Keller, 1976 a, b). Loss of the fibrous lamina and of components of the pore complexes may therefore account, at least in part, for the reduction of nuclear envelope density observed when the isolated envelope is exposed to in- creasing magnesium chloride concentrations, as in the procedure of Monneron et al. (1972) for envelope isolation (see also Fukushima, Okayama, Takahashi & Hayaishi, 1976). In the light of these ultrastructural studies, particularly those of Franke's group, it seems reasonable to suggest that there are 3 morphologically distinct types of fibres in pore-laminae: (i) pore-interconnecting fibrils in the plane of the inner nuclear membrane or immediately adjacent to its inner surface; (ii) intranuclear fibrils attached to the pore complexes and oriented along an axis more or less perpendicular to the plane of the nuclear envelope; and (iii) intranuclear fibrils structuring the region occupied by the peripheral heterochromatin in the intact nucleus. The extent of the type (iii) fibril region varies with cell type (see section iv, below, for further discussion) and in most cases - amphibian oocytes are exceptional here - this region obscures the other fibril types in electron micrographs. The type (ii) region presumably corresponds to the interruptions in the ordered (type (iii) fibril) heterochromatin region in the neighbourhood of the pore complexes (Davies, 1967, 1968). At present, the evidence per- taining to the molecular topology of this system (see next subsection) cannot be related in any obvious or simple way to this ultrastructure. One possibility here is that the 150000-molecular-weight band of Krohne et al. (19786) represents some portion of the type (i) or (ii) fibrils as well as of the pore complexes, since it predominates in the type (iii)-deficient amphibian oocyte.

(4) The molecular organization of the pore-lamina Several attempts have been made to establish the molecular topology of the pore- lamina with a view to extrapolating this information to the intact nuclear envelope and nucleus. Immunochemical localization (Gerace, Blum & Blobel, 1978; Ely, D'Arcy & Jost, 1978) and surface-labelling procedures (Richardson & Maddy, 1979) have been used. Gerace et al. (1978) raised antibodies to N1( N2 and N3 excised from SDS- polyacrylamide gels. Binding of these antibodies to the pore-lamina was then detected by indirect immunofluorescence and indirect immunoperoxidase labelling. The former technique showed that binding was restricted to the periphery of the nucleus; the latter showed that the antibodies reacted exclusively with the fibrous lamina, not with the pore complexes. Apparently, therefore, not only is the pore-lamina antigenically distinct from all parts of the cytoplasm and from the intranuclear and nucleolar 418 P. S. Agutter and J. C. W. Richardson matrices, but also the fibrous lamina is antigenically distinct from the pore complexes. Ely et al. (1978) showed that pore-laminae from rat liver, rat hepatoma and HeLa cells cross-reacted antigenically. In contrast, specific iodination of the cytoplasmic surface of intact nuclei using immobilized lactoperoxidase has demonstrated that 2 of the major polypeptides (N1 and N2) become labelled and remain labelled after removal of the membranes from the envelope (see section in 1), while the third (N3) can be labelled only after disruption of the nuclei and isolation of the envelopes. This strongly suggests that Nx and N2 are localized in the pore complex, while N3 is located on the nucleoplasmic side of the envelope, probably in the fibrous lamina (Richardson & Maddy, 1979). On the basis of the specific activities of Nx and N2 these polypeptides are either less accessible to iodination from outside the nucleus than are some of the minor polypeptide components of the pore complex (of which more than 14 can be identified (Richardson & Maddy, 1979)) or they are to be found in both the pore complex and the fibrous lamina; but it is clear that they are localized differently from N3. N3 is relatively easily separated from the rest of the pore-lamina (Agutter et al. 1978; Krohne et al. 19786), and Krohne et al. (19786) suggest that it may be part of the matrix, contaminating most pore-lamina preparations. One possible explanation for the conflict between the immunochemical and surface labelling results is that the polypeptides are not concentrated in the pore complex in an immunologically reactive form; this view has been put forward by Gerace et al. (1978). It is noteworthy that pore complex proteins are highly conserved and it might therefore be difficult to raise antibodies to them (Franke, 1970). Antigen masking, for example by the nuclear skeletal RNA, could also account for the negative results. It is also likely that Nx, N2 and N3 possess a common antigen, since antibodies to all 3 polypeptides were found to cross-react strongly on Ouchterlony double-diffusion analysis (Gerace et al. 1978). There are indeed reports that the nuclear protein matrix P^ P2 and P3 in avian erythrocytes are related in primary structure (Shelton, Cobbs & Cochran, 1978) and have very similar isoelectric points (Shelton & Egle, 1979), and this may apply to the major pore-lamina polypeptides (cf. Shelton & Cochran, 1978). In view of this, further immunological and protein chemical studies on proteolytic fragments of the major polypeptides would be interesting. The similarities in molecular weights, primary structure and immunological behaviour of the 3 polypeptides may suggest that they have a common origin; the small differences between them on SDS-PAGE could, for instance, result from post-translational modification; alternatively, they might be the result of gene duplication. Thus binding of antibodies to N1; N2 and N3 to the fibrous lamina does not provide unequivocal evidence about the organization of these polypeptides at the nuclear surface. Another diffi- culty with the immunochemical work is that, in view of the complexity of the SDS- PAGE pattern in the neighbourhood of the three major polypeptides (see fig. 2 in Aaronson & Blobel (1975)), excision of single polypeptide species from a gel is an extremely difficult procedure. At present, therefore, the overall view of the topology of the pore-lamina must be that Nj and N2 are present in the pore complexes along with many minor and more highly exposed polypeptides, and that N3 is present in the fibrous lamina. Whether Nuclear matrix, ghost and pore-lamina 419

IS^ and N2 are also present in the fibrous lamina remains uncertain, though this seems likely in view of the evidence that the pore complexes are assembled within the fibrous lamina region (Kirschner et al. 1977). In this context it is interesting to consider recent evidence (Agutter et al. 1979) concerning the nucleoside triphosphatase located in the pore complexes (Yasuzumi &Tsubo, 1966; Yasuzumiei al. 1967) and believed to be involved in nucleocytoplasmic RNA transport (Agutter et al. 1976). This enzyme is rapidly inactivated by treatment of intact nuclei with agarose-immobilized trypsin, and therefore presumably has its active site on the cytoplasmic face of the pore complex. However, it specifically catalyses y^P-ATP-dependent phosphorylation of a polypeptide co-migrating with N3 on SDS-PAGE, and this phosphorylated peptide is an intermediate in ATP hydrolysis (Agutter et al. 1979). It therefore appears that the y-phosphate of ATP is transferred to a polypeptide located on or near the opposite (nucleoplasmic) surface to the active site of the enzyme. This suggests that ATP hydrolysis is associated with a dramatic conformation change within the pore complex and, possibly, the fibrous lamina. Such a change might be expected in association with the transport of large ribonucleoprotein complexes to the cytoplasm. Certainly, the observation that the pore-complex-bound parts of the lamina (type (ii) fibrils - see section in A 3, above) prevent egress of infecting SV40 particles from nuclei (Maul, 1976), while obviously not preventing entry of the viruses, clearly illustrates that the pore complex is not merely a passive filter. There is evidence that intranuclear virus particles become firmly associated with some part of the nuclear protein matrix (Chin & Maizel, 1977; Hodge et al. 1977; Kellermayer, Jobet & Szuce, 1978; Deppert, 1978).

(B) OTHER SUBFRACTIONS The nuclear protein matrix contains residual nucleoli and other intranuclear protein structures (see section 113, above) as well as the pore-lamina, but the isolation of these other subfractions is in principle much more difficult. The pore-lamina has been successfully isolated by virtue of its close association with the nuclear membranes, which presumably impart a high degree of stability to it. Matrix elements in the nucleoli and other intranuclear regions lack such a buffer against the rigours of isolation. Berezney & Coffey (1976) isolated nucleoli by the procedures of Busch (1967) and subjected them to the conditions used in isolation of the nuclear protein matrix from whole nuclei (Berezney & Coffey, 1976 a, b). The polypeptides of the residual nucleoli were examined and found to comprise 2 of the major polypeptides of the matrix (P1 and P2) while P3 was deficient. Several other polypeptides were also present. The possibility that some of these are chromatin or ribosomal proteins has not been investigated. While this must be classed as preliminary observation, since no details are available on the organization of the residual nucleoli, it is clear that once again an ultrastructurally distinct region of the nuclear matrix differs in composition from the remainder. However, the extent and physiological significance of the difference are not certain as yet. Kirschner et al. (1977) removed the membranes from the whole nuclei with Triton, and then removed the pore-lamina using a mixture of 420 P. S. Agutter andj. C. W. Richardson i % Triton X-ioo and i % sodium deoxycholate. It is conceivable that the intranuclear matrix material could be isolated from the remainder of these nuclei by the method of Berezney & Coffey (1974 a, b). Interestingly, the nueleoli were destroyed by the combined detergent treatment, suggesting that the stability of these organelles is prejudiced by deoxycholate (Triton alone does not have this effect). Since deoxycholate was used in the isolation of nuclear ghosts (Riley & Keller, 1976 a, b; Riley et al. 1975), this observation presumably accounts for the absence of residual nueleoli from such preparations (see section 113, above).

IV. COMPOSITIONAL AND FUNCTIONAL DIFFERENCES BETWEEN THE PORE- LAMINA AND THE REMAINDER OF THE NUCLEAR PROTEIN MATRIX Isolation of the nuclear envelope entails removal from the nucleus of the chromatin and of the intranuclear matrix and of residual nueleoli. The fact that the fibrous lamina survives an isolation procedure such as that of Kay, Fraser & Johnston (1972) or Harris & Milne (1974) suggests either (a) that the pore-lamina and intranuclear parts of the matrix are different in composition and/or organization and are, therefore, different in stability, or (6), as suggested in section IIIB above, that the fibrous lamina is a region of the matrix stabilized by virtue of its close association with the nuclear membranes. Nuclear envelope and pore-lamina preparations may, of course, be contaminated to an unpredictable extent by intranuclear matrix material. None of these possibilities is incompatible with the early studies described in section 1. The ultrastructural similarity between the pore-lamina and the nuclear protein matrix and the similarities in their composition as revealed in the SDS-PAGE studies described in preceding sections (Berezney & Coffey, 1974a, 1975 a, b, 1976; Aaronson & Blobel, 1974, 1975; Dwyer & Blobel, 1976) seem inconsistent with the first possibility. More- over, the fibrillar component isolated from Triturus oocyte nuclear envelopes by re- homogenization and flotation on a sucrose density gradient (Krohne et al. 19786) produced aggregates similar in appearance to nuclear protein matrix preparations. However, evidence has accrued which suggests that there are important differences between the peripheral and internal parts of the in vivo matrix. Two pieces of this evidence have already been mentioned. (1) There is a polypeptide of molecular weight 150000 in at least some pore-lamina preparations (Krohne et al. 19786) but this is not evident as a major component of nuclear protein matrix preparations (Berezney & Coffey, 1974a). Since pore-lamina material forms only a small percentage of the total matrix, then if this polypeptide were restricted to the peripheral part of the nuclear matrix its virtual absence from the matrix would be expected. In view of the argument that nuclear ghosts are enriched in peripheral material, however (see section 112, above), the absence of this 150000 band from SDS gels of ghost material (Riley & Keller, 1976a) is rather surprising. (2) Immunological differences between the fibrous lamina and the rest of the matrix have been shown using antibodies to N1; N2 and N3 (Gerace et al. 1978) or to N2 alone (Krohne et al. 19786; Ely et al. 1978). These studies (detailed in section Nuclear matrix, ghost and pore-lamina 421 in 4, above) indicate that whatever the apparent compositional similarities between nuclear protein matrix and pore-lamina preparations, there are marked differences at least in the organization of the polypeptides. Clearly, co-migration on SDS-PAGE is not an adequate criterion for claiming that 2 polypeptides are identical. There are several minor components with molecular weights close to those of Nx, N2 and N3, and there is no evidence that these major bands are in fact single polypeptides; spectrin from mammalian erythrocytes, for example, runs as two bands on SDS-PAGE, but several N-termini have been detected in each of these bands (Dunn, McBay & Maddy, 1975)- In addition, there seem to be functional differences between the pore-lamina and the remainder of the matrix. Three pieces of evidence are relevant here. (1) Autoradiographic studies indicate that the DNA replication fork is not associated with the fibrous lamina region (Williams & Ockey, 1970; Huberman, Tasi & Deich, 1973; Comings & Okada, 1973; Wise & Prescott, 1973; Fakan & Hancock, 1974), and this conclusion is consistent with the observation that there is no enrichment of DNA polymerase activity in nuclear envelope preparations (Kay et al. 1971). (2) Unlike the nuclear protein matrix (see section n 6 b, above), the pore-lamina from rat liver is not associated with rapidly labelled HnRNA (Agutter & Birchall, 1979). This suggests that the intranuclear part of the matrix is a site for the initiation of transcription, but the fibrous lamina is not. (3) Similarly, the pore-lamina from rat endometrium contains virtually no specific i7/?-oestradiol-binding sites, in contrast to the nuclear protein matrix (Agutter & Birchall, 1979). It may be argued that this difference is an operational artifact: that the oestradiol-binding sites are lost or destroyed during isolation of the nuclear envelope. However, exposure of the isolated nuclear protein matrix to the conditions used for envelope isolation, e.g. 1 mM HCOg for 2 h at 4 °C (Harris & Milne, 1974) or 10 mM Tris-HCl, pH 8-5, for 30 min at 20 °C (Kay et al. 1972), does not prevent specific binding of the steroid, though it does lead to extensive fragmentation of the structure. These functional differences are, perhaps, not surprising if the pore-lamina material in vivo is associated with the peripheral heterochromatin, where transcription does not occur to any marked extent, while the remainder of the nuclear matrix material is associated with the transcriptionally active euchromatin (Frenster et al. 1963). In view of the evidence reviewed here, we suggest that the type (iii) fibrils of the fibrous lamina and the intranuclear matrix are differently organized areas of the nuclear protein matrix, the latter, but not the former, being the site of initiation and control of much nucleic acid synthesis and processing in the interphase nucleus. Thus, the differences in extent of the type (iii) region of the lamina revealed by ultrastructural studies on different cell types might reflect differences in cellular activity; in cells such as amphibian oocytes, in which many genes are being transcribed rapidly, this region is of small extent (Franke & Scheer, 1970, 1974; Scheer et al. 1976; Krohne et al. 19786); in many protozoa, it is more extensive (Callan et al. 1949; Callan & Tomlin, 1950). Shelton and co-workers have not distinguished between pore-lamina and nuclear protein matrix in their studies on avian erythrocyte nuclei (Shelton et al. 1976; 422 P. S. Agutter andj. C. W. Richardson Cochran et al. 1975; Higgins et al. 1978; Shelton & Cochran, 1978; Shelton et al. 1978) but here there is so little metabolic activity unless the cells are activated as in the studies of Bolund et al. (1969) and Carlsson, Moore & Ringertz (1973), that we might on the argument above expect most if not all of the matrix to be lamina. The studies carried out by Shelton's group into the self-assembling properties of Plt P2 and P3 (or N1( N2 and N3 - the identification is not clear) have included an investigation of the role of the readily oxidizable sulphydryl groups in these polypeptides (Shelton & Cochran, 1978) and may help to elucidate the mechanism of assembly of the nuclear matrix in vitro. Hopefully, similar studies on other cell types will clarify the question of differences in organization between the different regions of the matrix.

V. PROSPECTS FOR FURTHER RESEARCH In the course of this review of studies on nuclear protein matrix, nuclear ghost and pore-lamina preparations and their relevance to an understanding of the structure and functions of the interphase nucleus, several problems which are the foci of current research have been identified. These include (a) the explanation of the marked differences between nuclear protein matrix preparations and nuclear ghosts, (b) the question of whether DNA and/or RNA contribute to the structural integrity of the matrix, (c) the enzymology of subfractions of the matrix, (d) the mechanisms of control of matrix contractility in vivo and the relevance of these to control of nuclear function, (e) the question of the reality of the differences in composition and organization between different subfractions of the structure, (/) the molecular topology of each subfraction, (g) the localization of particular binding sites, and (h) the dynamics of the interrelations between the different subfractions. Evaluation of the findings to date suggests at least 3 other potentially fruitful areas for further investigation:

(1) The role of the nuclear protein matrix in nucleo-cytoplasmic RNA transport It is generally agreed that mature ribonucleoproteins enter the cytoplasm through the pore complexes. Since these are continuous with the rest of the nuclear protein matrix, the dynamics of intranuclear parts of the structure could be important in regulating the functional size of the pore complex and macromolecule transport. Whether functionally significant alterations of the pore complex occur and, if they do, whether they are reflected in ultrastructural changes, are controversial questions. Faberge" (1974) has suggested that marked conformation changes can occur, involving a change from octagonal to tetragonal symmetry. However, Kirschner et al. (1977) failed to find any evidence to support Faberge^s hypothesis in their scanning electron- microscopic studies. Willison & Rajaraman (1977) and Willison & Johnston (1978) observed some abnormally large pore complexes in actively growing yeast cells, but Severs & Jordan (1978) were unable to confirm these observations. The studies of Maul et al. (1971), Tiegler & Baerwald (1972), Markovics et al. (1974) and Kirschner et al. (1977) clearly indicate that disruption of the fibrous lamina during experimental manipulations, for instance during the preparation of specimens for electron microscopy, could readily give rise to artifactual modifications of pore complex ultra- Nuclear matrix, ghost and pore-lamina 423 structure. Ultrastructural studies such as those of Faberge" (1974), Willison & Rajaraman (1977) and Willison & Johnston (1978) must therefore be interpreted with caution. Nevertheless, these considerations do not exclude the possibility of functional changes, including changes in transport capacity, of the pore complex, which are not necessarily reflected in changes in ultrastructure. Wunderlich et al. (1978) have shown that the nuclear protein skeleton in Tetra- hymena can expand and contract without altering the ultrastructure of the pore complexes. The nuclear membranes (and, presumably, the fibrous lamina) are stretched, with consequent alterations in membrane lipid fluidity. Studies in the same laboratory have shown that the fluidity of nuclear membrane lipids influences nucleo-cytoplasmic RNA transport (Nagel & Wunderlich, 1972; Herlan, Giese & Wunderlich, 1979). It follows that the contractility of the matrix exerts at least an indirect influence on transport through the pore complex; this emphasizes the growing importance of an understanding of the control of contractility in vivo for an understanding of nuclear function in general (cf. Burgoyne et al. 1978, 1979, and section 116).

(2) Relevance of a knowledge of factors affecting the stability of the intranuclear region of the matrix to the further development of methods for isolation of the nuclear envelope Among the plethora of published nuclear envelope isolation procedures (see e.g. Franke, 1974 a, b; Fry, 1976, and Harris & Agutter, 1976, for reviews), there is a wide methodological range, but one feature which by definition all these procedures share is the dispersal at some stage of the nuclear contents, including the intranuclear portion of the matrix. Examination of the stability of the isolated matrix under the various conditions used for envelope isolation may explain some hitherto mysterious features of these procedures. For instance, it may explain why at neutral pH (a) bi- carbonate lyses nuclei effectively (Harris & Milne, 1974), phosphate much less effectively (resulting in a very low yield of envelope (cf. Zbarsky, 1972; Zbarsky et al. 1969)), and other electrolyte solutions of low ionic strength hardly at all; (b) lysis is rapid in Tris buffer at pH 8-5, but not at pH 7-5 (Kay et al. 1972); (c) nuclei isolated in phosphate buffer at pH 6-o, but not those isolated in Tris buffer at pH 7-5, give rise to nuclear envelopes the structural integrity of which is crucially dependent on the presence of DNA (Agutter, 1972). In addition, it could lead to the design of improved procedures for the isolation of ultrastructurally and enzymologically intact envelopes. It seems likely that information of this kind may become available as a result of studies on the effects of electrolytes on matrix expansion and contraction.

(3) Fate of the nuclear matrix during mitosis The investigation of factors affecting the stability of the matrix might also elucidate the mechanism underlying the dissolution of nuclear structure which accom- panies mitotic prophase in most species. Two sets of observations are of considerable interest here. First, Gerace et al. (1978) have shown that the pore-lamina antigens (see sections in A 4 and IV2, above) become distributed throughout the cytoplasm during mitosis and are not associated 424 P. S. Agutter and J. C. W. Richardson to any important extent with the condensed chromosomes. Second, Laemmli and co- workers (Adolph, Cheng & Laemmli, 1977a; Adolph, Cheng, Paulson & Laemmli, 19776; Laemmli et al. 1977; Paulson & Laemmli, 1977) have shown that metaphase chromosomes depleted of histones and most of their non-histone chromatin proteins retain the ultrastructure of the daughter chromatids, the DNA being attached, probably via specific sequences, to a 'protein scaffold'. This scaffold retains its structure when the majority of the DNA is removed from it; as in the case of the nuclear protein matrix, therefore, it is possible that DNA is not an essential structural component. SDS-PAGE reveals at least 3 major polypeptides in the scaffold, but whether any of these corresponds to Plt P2 or P3 is at present unclear; Prescott & Goldstein (1968) showed that 95 % of the nuclear acidic proteins are distributed in the cytoplasm during mitosis (see also Gerace et al. 1978). One of the major scaffold polypeptides has a molecular weight close to that of the tubulin monomers (55000); it is therefore possibly a residual fragment of the mitotic spindle. If this is so, then studies of the molecular organization of the scaffold should elucidate the nature of the contact between the spindle fibrils and the chromatids. There are some indications that DNA is necessary for the post-mitotic reassembly of the nuclear envelope, hence possibly of the nuclear protein matrix (Peterson & Berns, 1978) and the apparent involvement of the Golgi apparatus in the reassembly process (Peterson & Berns, 1978) might suggest that protein glycosylation is important. Endogenous glycosyltransferases have been described in chromatin-depleted nuclei of Saccharomyces cerevisiae (Palamarozyk & Janozura, 1977), Dictyostelium and rat liver (Richard, Martin & Louisot, 1975), and in several isolated nuclear envelope preparations (Kawasaki & Yamashina, 1972; Scheer et al. 1976; Franke, 19746, 1977; Tamulevicius, Streffer, Roscic & Hubert, 1979). In addition, there are reports that glucosamine can be incorporated into proteins of the fibrous lamina (Mancini, Heywood & Hodge, 1973) and that glucosamine and sialic acid can be incorporated into the nuclear protein matrix (Wunderlich et al. 1976). The study of the nuclear protein matrix is still in its infancy, and no doubt there will be significant advances in understanding of its behaviour - possibly with reference to the problems discussed in this section - during the next few years. The apparent ubiquity of this structure and its involvement in a wide range of nuclear activities strongly suggest that such advances will be of crucial importance for our further understanding of the structure and function of the interphase nucleus.

The authors are indebted toDrs A. J.L. Agutter (Department of English Language, University of Edinburgh), A. H. Maddy (Department of Zoology, University of Edinburgh), U. Scheer (German Cancer Research Centre, Heidelberg) and I. Thomson (Department of Biological Sciences, Napier College, Edinburgh) for their advice and criticism of the draft manuscript. We are also very grateful to Drs R. Berezney (Division of Cell and Molecular Biology, State Uni- versity of New York) and J. M. Keller (Department of Biochemistry, Chicago Medical School) for their generosity in supplying Figs. 1-5, and to the Editorial Boards of J. Cell Biol. and J. Cell Sci. for permission to reproduce these micrographs. Nuclear matrix, ghost and pore-lamina 425

REFERENCES AARONSON, R. P. (1977). Isolation of nuclear proteins associated with the nuclear pore complex and nuclear peripheral lamina of rat liver. Meth. Cell Physiol. 16, 317-342. AARONSON, R. P. & BLOBEL, G. (1974). On the attachment of the pore complex. J. Cell Biol. 62, 746-754- AARONSON, R. P. & BLOBEL, G. (1975). Isolation of nuclear pore complexes in association with a lamina. Proc. natn. Acad. Sci. U.S.A. 72, 1007-1011. ABELSON, H. T. & SMITH, G. H. (1970). Nuclear pores: the pore—annulus relationship in thin section. J. Ultrastruct. Res. 30, 558-588. ADOLPH, K. W., CHENG, S. M. & LAEMMLI, U. K. (1977a). Role of non-histone proteins in metaphase chromosome structure. Cell 12, 805-816. ADOLPH, K. W., CHENG, S. M., PAULSON, J. R. & LAEMMLI, U. K. (19776). Isolation of a protein scaffold from mitotic HeLa cell chromosomes. Proc. natn. Acad. Sci. U.S.A. 74, 4937-494i- AGUTTER, P. S. (1972). The isolation of the envelopes of rat liver nuclei. Biochim. biophys. Acta 255, 397-401. AGUTTER, P. S. & BIRCHALL, K. (1979). Functional differences between mammalian nuclear matrix and pore-lamina preparations. Expl Cell Res. 124, 453-460. AGUTTER, P. S., BIRCHALL, K., CLARK, J. E., HOTSON, J. A. & PORTEOUS, E. R. (1978). Nuclear envelopes and pore-laminae from a variety of mammalian tissues. Biochem. Soc. Trans. 6, 1177-1179. AGUTTER, P. S., COCKRILL, J. B., LAVINE, J. E., MCCALDLN, B. & SIM, R. B. (1979). Properties of mammalian nuclear envelope nucleoside triphosphatase. Biochem. jf. 181, 647-658. AGUTTER, P. S., MCARDLE, H. J. & MCCALDIN, B. (1976). Evidence for involvement of nuclear envelope nucleoside triphosphatase in nucleocytoplasmic translocation of ribonucleoproteins. Nature, Lond. 263, 165-167. ALFERT, M. & DAS, N. K. (1969). Evidence for control of the rate of nuclear DNA synthesis by the nuclear membrane in eucaryotic cells. Proc. natn. Acad. Sci. U.S.A. 63, 123-128. ALLEN, S. L., BEREZNEY, R. & COFFEY, D. S. (1977). Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei. Biochem. biophys. Res. Commun. 75, m-116. ALLFREY, V. G., DALLY, M. M. & MIRSKY, A. E. (1955). Some observations of protein meta- bolism in chromosomes of non-dividing cells. J. gen. Physiol. 38, 415-424. ANDERSON, N. G. (1953). Studies on isolated cell components. VI. The effects of nucleases and proteases on rat liver nuclei. Expl Cell Res. 5, 361-374. ANDERSON, N. G. & NORRIS, C. B. (i960). The effect of amines on the structure of isolated nuclei. Expl Cell Res. 19, 605-618. ANDERSON, N. G. & WILBUR, K. M. (1951). Studies on isolated cell components. III. The release of a nuclear gel by heparin. J. gen. Physiol. 34, 647-656. ANDERSON, N. G. & WILBUR, K. M. (1952). Studies on isolated cell components. IV. The effect of various solutions on the isolated rat liver nucleus. J. gen. Physiol. 35, 781-796. ANTEUNIS, A., POUCHELET, M., GANSMULLER, A. & ROBINEAUX, R. (1975). Ultrastructural demonstration of deoxyribonuclease-sensitive elements in fibrillar zones of the nucleoli of L929 cell interphasic nuclei. C.r. hebd. Sianc. Acad. Sci., Paris 281, 901-903. ARNOLD, E. A., YAWN, D. H., BROWN, D. G., WYLLEE, R. C. & COFFEY, D. S. (1972). Structural alteration in isolated rat liver nuclei after removal of template restrictions by polyanions. J. Cell Biol. S3, 737-757- BACH, M. K. & JOHNSTON, H. G. (1967). The isolation and characterization of a novel micro- some fraction from washed and detergent-treated nuclei of HeLa cells by the use of solutions containing deoxyribonucleic acid. Biochemistry, N.Y. 6, 1916-1933. BANKS, S. P., GILBERT, B. E. & JOHNSON, T. C. (1974). Stimulation of RNA synthesis in isolated mouse brain nuclei by a brain cytosol factor. Life Sci. 14, 303-310. BARRACK, E. R., HAWKINS, E. F., ALLEN, S. L., HICKS, L. L. & COFFEY, D. S. (1977). Concepts related to salt resistant oestradiol receptors in rat uterine nuclei: nuclear matrix. Biochem. biophys. Res. Commun. 79, 829-836. BARRY, J. M. & MERRIAM, R. W. (1972). Swelling of hen erythrocyte nuclei in cytoplasm from Xenopus eggs. Expl Cell Res. 71, 90—96. 28 CEL 44 P. 5. Agutter andj. C. W. Richardson

BARTON, A. D.( KISEELESKI, W. E., WASSERMANN, F. & MACKEVICIUS, F. (1971). Experimental modification of structure at the periphery of the liver cell nucleus. Z. Zellforsch. mikrosk. Anat. "5. 299-306. BEAMS, H. W., TAHMISIAN, T. N., DEVINE, R. & ANDERSON, E. (1957). Infrastructure of the nuclear membrane of a gregarine parasitic in grasshoppers. Expl Cell Res. 13, 200-204. BEREZNBY, R. (1979). The nuclear protein matTU. In The Cell Nucleus, vol. 8 (ed. H. Busch), pp. 413-455. New York: Academic Press. BEREZNEY, R. & BUCHHOLTZ, L. A. (1978). Nuclear matrices isolated with protease inhibitors: evidence for an increased association of newly-replicated DNA. J. Cell Biol. 79, 130 a. BEREZNEY, R. & COFFEY, D. S. (1974a). Identification of a nuclear protein matrix. Biochem. biophys. Res. Commun. 60, 1410-1417. BEREZNEY, R. & COFFEY, D. S. (19746). Identification of a nuclear structural network in rat liver nuclei. Fedn Proc. Fedn Am. Socs exp. Biol. 33, 1395. BEREZNEY, R. & COFFEY, D. S. (1975a). Nuclear protein matrix: association with newly- synthesised DNA. Science, N.Y. 189, 291-293. BEREZNEY, R. & COFFEY, D. S. (19756). The nuclear matrix: association with rapidly-labelled DNA. Fedn Proc. Fedn Am. Socs exp. Biol. 34, 494. BEREZNEY, R. & COFFEY, D. S. (1976). The nuclear protein matrix: isolation, structure and functions. Adv. Enz. Reg. 14, 63-100. BEREZNEY, R. & COFFEY, D. S. (1977). Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73, 616-637. BEREZNEY, R. & CRANE, F. L. (1972). Characterization of electron transport activity in bovine liver nuclear membranes. J. biol. Chem. Z47, 5562-5568. BEREZNEY, R., MACAULAY, L. K. & CRANE, F. L. (1972). The purification and biochemical characterization of bovine liver nuclear membranes. J. biol. Chem. 247, 5549—5561. BERNHARD, W. & GRANBOULAN, N. (1963). The fine structure of the cancer cell nucleus. Expl Cell Res., Suppl. 9, 19-53. BLOBEL, G. & POTTER, V. R. (1966). Nuclei from rat liver: isolation method that combines purity with high yield. Science, N.Y. 154, 1662-1665. BOLUND, L., RINGERTZ, N. R. & HARRIS, H. (1969). Changes in the cytochemical properties of erythrocyte nuclei reactivated by cell fusion. J. Cell Set. 4, 71—87. BORNENS, M. (1973). Action of heparin on nuclei; solubilization of chromatin enabling the isolation of nuclear membranes. Nature, Lond. 244, 28-30. BORNENS, M. & COURVALIN, J. D. (1978). Isolation of nuclear envelopes with polyanions. J. Cell Biol. 76, 191-206. BRASCH, K. & SINCLAIR, G. D. (1978). The organization, composition and matrix of hepatocyte nuclei exposed to alpha-amanitin. Virchows Arch. (Zell Path.) 27, 193-204. BROWN, D. G. & COFFEY, D. S. (1971). Release of nuclear template restrictions by specific polyribonucleotides. Science, N.Y. 171, 176-178. BROWN, D. G. & COFFEY, D. S. (1972). Effects of polyinosinic acid on the deoxyribonucleic acid template activity of isolated nuclei and soluble chromatin from rat liver. J. biol. Chem. Ml, 7674-7683. BRUNI, C. & PORTER, K. R. (1965). The fine structure of the parenchymal cell of normal rat liver. Am. J. Path. 46, 691-729. BULDIAEVA, T. V., KUZMINA, S. N. & ZBARSKY, I. B. (1978). Protein composition of matrix and residual proteins of rat liver and hepatoma-27 nuclei. Doklady Acad. Nauk. SSSR 241, 1161- 1164. BURGOYNE, L. A. & SKINNER, J. D. (1979). Probing the free space within rat and chicken chromatin with active and passive probes. J. Cell Sci. 37, 85—96. BURGOYNE, L. A., SKINNER, J. D. & MARSHALL, A. (1978). Analysis of the penetrable space within the nucleus. J. Cell Sci. 31, i-n. BUSCH, H. (1967). Isolation and purification of nucleoli. Meth. Enz. 12A, 448-464. CALLAN, H. G., RANDALL, J. R. & TOMLIN, S. G. (1949). An electron microscopic study of the nuclear membrane. Nature, Lond. 163, 280. CALLAN, H. G. & TOMLIN, S. G. (1950). Experimental studies on amphibian oocyte nuclei. I. Investigation of the structure of the nuclear membrane by means of the electron microscope. Proc. R. Soc. B 137, 367-378. Nuclear matrix, ghost and pore-lamina 427 CARLSSON, S. A., MOORE, G. P. M. & RINGERTZ, N. R. (1973). Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons. Expl Cell Res. 76, 234-241. CAVALIER-SMITH, T. (1978). Nuclear volume control by nucleoskeletal DNA selection for cell volume and cell growth rate, and the solution of the DNA C-value paradox. J. Cell Sci. 34, 247-278. CHAI, L. S., WEINFELD, H. & SANDBERG, A. A. (1978). Effects of divalent-cation chelators and chloramphenicol on the spatial relationship of the nuclear envelope to chromatin in micro- nuclei of Chinese hamster cells. J. supramolec. Struct. 9, 459-472. CHAMPOUX, J. J. & MCCONAUGHY, B. L. (1976). Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry, N. Y. 15, 4638-4642. CHANG, C. S. & GOLDWASSER, E. (1973). On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis. Devi Biol. 34, 246-254. CHIN, W. W. & MAIZEL, J. W. (1977). The polypeptides of adenovirus. VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction. Virology 76, 79—89. COCHRAN, D. L., COBBS, C. S. & SHELTON, K. R. (1975). Oligomeric structural proteins of the cell nucleus. J. Cell Biol. 75, 151a. COFFEY, D. S., BARRACK, E. R. & HESTON, W. D. W. (1974). The regulation of nuclear DNA template restrictions by acidic polymers. Adv. Enz. Reg. 12, 219—266. COGGESHALL, R. E. & FAWCETT, D. W. (1964). The fine structure of the central nervous system of the leech Hirudo medicinalis. J. Neurophysiol. 27, 229—289. COHEN, A. H. & SUNDEEN, J. R. (1976). The nuclear fibrouslamin a in human cells: studies on its appearance and distribution. Anat. Rec. 186, 471—476. COLEMAN, S. E., DUGGAN1, J. & HACKETT, R. L. (1974). Freeze-fracture study of changes in nuclei isolated from ischaemic rat kidney. Tissue & Cell 6, 521-534. COMINGS, D. E. (1968). The rationale for an ordered arrangement of chromatin in the interphase nucleus. Am. jf. Hum. Genet. 20, 440-460. COMINGS, D. E. & HARRIS, D. C. (1976). Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei. J. Cell Biol. 70, 440-452. COMINGS, D. E. & OKADA, T. A. (1973). DNA replication and the nuclear membrane. J. molec. Biol. 75, 609-618. COMINGS, D. E. & OKADA, T. A. (1976). Nuclear proteins. III. The fibrillar structure of the nuclear matrix. Expl Cell Res. 103, 341-360. COMINGS, D. E. & WALLACK, A. S. (1978). DNA-binding properties of nuclear matrix proteins. Jf. Cell Sci. 34, 233-246. COOK, P. R., BRAZELL, I. A. & JOST, E. (1976). Characterization of nuclear structures containing superhelical DNA. y. Cell Sci. 22, 303-324. DANIELS, E. W. & BREYER, E. P. (1967). Ultrastructure of the giant amoeba Pelomyxa polustris. y. Protozool. 14, 167-175. DAVIES, H. G. (1967). Fine structure of heterochromatin in certain cell nuclei. Nature, Lond. 214, 208-210. DAVIES, H. G. (1968). Electron microscope observations on the organization of heterochromatin in certain cells, y. Cell Sci. 3, 129-150. DAVIES, H. G. & SMALL, J. V. (1968). Structural units in chromatin and their orientation on nuclear membrane. Nature, Lond. 217, 1122-1125. DE BELLIS, R. H. (1969). Cytoplasmic factors controlling DNA synthesis in isolated nuclei. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 599. DEPPERT, W. (1978). Simian virus-40 specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U- antigen determinants, y. Virol. 26, 165-178. DUGUET, M., Soussi, T., ROSSIGNOL, J. M., MECHALI, M. & DE RECONDO, A. M. (1977). Stimulation of rat liver a and /? type DNA polymerases by an homologous DNA-unwinding protein. FEBS Letters, Amsterdam 79, 160-164. DUNN, M. J., MCBAY, W. & MADDY, A. H. (1975). The N-terminal heterogeneity of EDTA- extractable erythrocyte membrane proteins. Biochim. biophys. Acta 386, 107-119. 28-2 428 P. 5. Agutter and J. C. W. Richardson Du PRAW, E. J. (1956). The organization of nuclei and chromosomes in honeybee embryonic cells. Proc. natn. Acad. Sci. U.S.A. 53, 161-168. DVORKIN, V. M. & VANIUSHIN, B. F. (1978). Replication and reassociation kinetics of nuclear matrix DNA from regenerating rat liver. Biokhimiya 43, 1648-1652. DWYER, N. & BLOBEL, G. (1976). A modified procedure for the isolation of a pore complex- lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581-591. ELGIN, S. G. & WEINTRAUB, H. (1975). Chromosomal proteins and chromatin structure. A. Rev. Biochem. 44, 725-774. ELY, S., D'ARCY, A. & JOST, E. (1978). Interaction of antibodies against nuclear envelope- associated proteins from rat liver nuclei with rodent and human cells. Expl Cell Res. 116, 325-33I- FABERGE, A. C. (1974). The nuclear pore complex: its free existence and an hypothesis as to its origin. Cell Tiss. Res. 151, 403-415. FAIFERMAN, I. & POGO, A. O. (1975). Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope. Biochemistry, N. Y. 14, 3808-3816. FAKAN, S. & HANCOCK, R. (1974). Localization of newly-synthesized DNA in a mammalian cell as visualized by high resolution autoradiography. Expl Cell Res. 83, 95-102. FAWCETT, D. W. (1966). On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. Am. J. Anat. 119, 129-146. FAWCETT, D. W. & CHEMES, H. E. (1979). Changes in distribution of nuclear pores during differentiation of the male germ cells. Tiss. & Cell 11, 147-162. FELDHERR, C. M. (1962). The nuclear annuli as pathways for nucleocytoplasmic exchange. J. Cell Biol. 14, 65-72. FELDHERR, C. M. (1968). Nucleocytoplasmic exchanges during early interphase. J. CellBiol. 39, 49-54- FELDHERR, C. M. (1969). A comparative study of nucleo-cytoplasmic interactions. J. Cell Biol. 43, 841-845. FLICKINGER, C. J. (1970). The fine structure of the nuclear envelope in amoebae: alterations following nuclear transplantation. Expl Cell Res. 6o, 225-236. FRANKE, W. W. (1970). On the universality of nuclear pore complex structure. Z. Zellforsch. mikrosk. Anat. 105, 405-429. FRANKE, W. W. (1974a). Nuclear envelopes. Phil. Trans. R. Soc. B 268, 67-93. FRANKE, W. W. (19746). Structure, biochemistry and functions of the nuclear envelope. Int. Rev. Cytol., Suppl. 4, 72-236. FRANKE, W. W. (1977). Structure and function of nuclear membranes. Biochem. Soc. Symp. 43, 125-135. FRANKE, W. W., KEENAN, T. W., STADLER, J., GENZ, R., JARASCH, E. D. & KARTENBECK, J. (1976). Nuclear membranes from mammalian liver. 7. Characteristics of highly purified nuclear membranes in comparison with other membranes. Cytobiologie 13, 28-56. FRANKE, W. W. & SCHEER, U. (1970). The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. I. The mature oocyte. J. Ultrastruct. Res. 30, 288-316. FRANKE, W. W. & SCHEER, U. (1974). Structure and functions of the nuclear envelope. In The Cell Nucleus, vol. 1 (ed. H. Busch), pp. 279-347. New York: Academic Press. FRENSTER, J. H., ALLFREY, V. G. & MIRSKY, A. E. (1963). Repressed and active chromatin isolated from interphase lymphocytes. Proc. natn. Acad. Sci. U.S.A. 50, 1026-1032. FROEHNER, S. C. & BONNER, J. (1973). Ascites tumour ribonucleic acid polymerases. Isolation, characterization and factor stimulation. Biochemistry, N.Y. 12, 3064-3071. FRY, D. J. (1976). The isolation of nuclear envelopes. In Subcellular Components (ed. G. D. Birnie), pp. 59-105. London: Butterworth. FUKASHIMA, M., OKAYAMA, H., TAKAHASHI, U. & HAYAISHI, O. (1976). Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope. J. Biochem., Tokyo 80, 167-176. GERACE, L., BLUM, A. & BLOBEL, G. (1978). Immunochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. J. Cell Biol. 79, 546-566. Nuclear matrix, ghost and pore-lamina 429 GEORGIEV, G. P. & CHENTSOV, J. S. (1962). The structural organization of nucleolochromo- 8omal ribonucleoproteins. Expl Cell Res. 27, 570-572. GHADIALLY, F. N., BHATNAGER, R. & FULLER, J. (1972). Waxing and waning of nuclear fibrous lamina. Arch. Path. 94, 303-307. GHOSH, S., PAWLETZ, N. & GHOSH, I. (1978). Cytological identification and characterization of the nuclear matrix. Expl Cell Res. in, 363-371. GHOSH, S. & ROY, S. (1977). Orientation of interphase chromosomes as detected by giemsa C-bands. Chromosoma 61, 49-63. GLESE, G., FROMME, I. & WUNDERLICH, F. (1979). Suppression of thermotropic lipid clustering in Tetrahymena nuclear membranes upon Ca1+/Mg1+-induced membrane contraction. Eur. J. Biochem. 95, 275-285. GRAHAM, C. F., ARMS, K. & GURDON, J. B. (1966). The induction of DNA synthesis by egg cytoplasm. Devi Biol. 14, 349-381. GRASSO, J. A., SWIFT, H. & ACKERMAN, G. A. (1972). Observations on the development of erythrocytes in mammalian foetal liver. J. Cell Biol. 14, 235—254. GRAY, E. G. & GUILLERY, R. W. (1973). On nuclear structure in the central nerve cord of the leech Hirudo medicinalis. Z. Zellforsch. mikrosk. Anat. 59, 738-745. GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclear DNA synthesis in frogs' eggs. Proc. natrt. Acad. Sci. U.S.A. 58, 545-552. GURDON, J. B. & WOODLAND, H. R. (1968). The cytoplasmic control of nuclear activity in animal development. Biol. Rev. 43, 233-267. HARDING, C. V. & FELDHERR, C. M. (1959). Semipermeability of the nuclear membrane in the intact cell. J. gen. Physiol. 42, 1155-1162. HARRIS, H. (1967). The reactivation of the red cell nucleus. J. Cell Sci. 2, 23-32. HARRIS, J. R. (1977). Fractionation of the nuclear envelope. In Methodological Surveys in Bio- chemistry, vol. 6: Membranous Elements and Movement of Molecules (ed. E. Reid), pp. 245- 250. Chichester: Horwood. HARRIS, J. R. & AGUTTER, P. S. (1976). The isolation and characterization of the nuclear envelope. In Biochemical Analysis of Membranes (ed. A. H. Maddy), pp. 132-173. London: Chapman & Hall. HARRIS, J. R., AGUTTER, P. S. & MILNE, J. F. 1,1978). Ultrastructural and biochemical studies on avian erythrocyte plasma membrane and nuclear envelope. Micron 9, 117-125. HARRIS, J. R. & MILNE, ]. F. (1974). A rapid procedure for the isolation and purification of rat liver nuclear envelope. Biochem. Soc. Trans. 2, 1251-1253. HARRIS, J. R. & MILNE, J. F. (1975). The behaviour of isolated cell nuclei when divalent cations are removed. .7. Physiol., Lond. 251, 23P-24P. HARRIS, P. & JAMES, T. W. (1952). Electron microscope study of the nuclear membrane of Amoeba proteus in thin section. Experientia 8, 384-385. HAY, E. D. & REVEL, J. P. (1963). The fine structure of the DNP component of the nucleus. J. Cell Biol. 16, 29-51. HEINRICH, P. C, GROSS, V., NORTHEMANN, W. & SCHEURLEN, M. (1978). Structure and function of nuclear ribonucleoprotein complexes. Rev. Physiol. Biochem. Pharmac. 81, 101-134. HEMMINKI, K. (1977). Labelling of histones and non-histones in lung nuclear matrix and chromatin fractions. Hoppe-Seylers Z. physiol. Chem. 358, 1123-1131. HERLAN, G., GEESE, G. & WUNDERLICH, F. (1979). Influence of nuclear membrane lipid fluidity on nuclear RNA release. Expl Cell Res. 118, 305-310. HERLAN, G., QUEVEDO, R. & WUNDERLICH, F. (1978). Structural transformations of nuclear . matrix in situ. Expl Cell Res. 115, 103-110. HERMAN, R., WEYMOUTH, L. & PENMAN, S. (1978). Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei. J. Cell Biol. 78, 663-674. HERRICK, G. & ALBERTS, B. (1976). Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus. J. biol. Chem. 251, 2124-2132. HERRICK, G., DELIUS, H. & ALBERTS, B. (1976). Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus. J. biol. Chem. 251, 2142-2146. HEWISH, D. (1977). Features of replicating and non-replicating chromatin in chicken erythro- blasts. Nucleic Acids Res. 4, 1881-1890. 43° P. S. Agutter andj. C. W. Richardson HIGGINS, L. L., YEOMAN, L. C. & SHELTON, K. R. (1978). A nuclear structural protein proteolytic activity. J. Cell Biol. 79, 132a. HILDEBRAND, C. E., OKINAKA, R. T. & GURLEY, L. R. (197S). Existence of a residual nuclear protein matrix in cultured Chinese hamster cells. J. Cell Biol. 67, 169a. HILDEBRAND, C. E. & WALTERS, R. A. (1976). Rapid assembly of newly-synthesized DNA into chromatin subunits prior to joining of small DNA replication intermediates. Biochem. biophys. Res. Commun. 73, 157-163. HODGE, L. D., MANCINI, P., DAVIS, F. M. & HEYWOOD, P. (1977). Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle. J. Cell Biol. 72, 194-208. HOLTZMAN, E., SMITH, I. & PENMAN, S. (1966). Electron microscopic studies of detergent- treated HeLa cell nuclei, jf. molec. Biol. 17, 131-135. HUBERMAN, J. A., TASI, A. & DEICH, R. A. (1973). DNA replication sites within nuclei of mammalian cells. Nature, Lond. 241, 32-36. IDE, T., NAKANE, M., ANZAI, K. & ANDOH, T. (1975). Supercoiled DNA folded by non-histone proteins in cultured mammalian cells. Nature, Lond. 258, 445-450. JACKSON, R. C. (1976). Polypeptides of the nuclear envelope. Biochemistry, N. Y. 15, 5641-5651. JARASCH, E. D. & FRANKE, W. W. (1974). Is cytochrome oxidase a constituent of nuclear membranes? J. biol. Chern. 249, 7245-7254. JARASCH, E. D. & FRANKE, W. W. (1977). Absence of cytochrome oxidase in nuclear membranes from hepatocytes. Expl Cell Res. 109, 450-454. JONES, R. T., JOHNSON, R. T., GUPTA, B. L. & HALL, T. A. (1979). The quantitative measure- ment of electrolyte elements in nuclei of maturing erythrocytes of chick embryo using electron-probe X-ray microanalysis. J. Cell Sci. 35, 67-85. KALIFAT, S. R., BOUTEILLE, G. & DELARUE, J. (1967). Etude ultrastructurale de la lamelle dense observee au contact de la membrane nucleaire interne. J. Microscopie 6, 1019-1026. KANNO, U. & LOEWENSTEIN1, W. R. (1963). A study of the nucleus and cell membranes of oocytes with an intra-cellular electrode. Expl Cell Res. 31, 149-166. KASPER, C. B. (1974). Chemical and biochemical properties of the nuclear envelope. In The Cell Nucleus, vol. 1 (ed. H. Busch), pp. 349-383. New York: Academic Press. KAWASAKI, T. & YAMASHINA, I. (1972). Isolation and characterization of glycopeptides from rat liver nuclear membrane. J. Biochem., Tokyo 72, 1517-1525. KAY, R. R., FRASER, D. & JOHNSTON, I. R. (1972). A method for the rapid isolation of nuclear membranes from rat liver. Eur. J. Biochem. 30, 145-154. KAY, R. R., HAINES, M. E. & JOHNSTON, I. R. (1971). Late replication of the DNA associated with the nuclear membrane. FEBS Letters, Amsterdam 16, 233-236. KAYE, J. S. & MCMASTER-KAYE, R. J. (1966). The fine structure and chemical composition of nuclei during spermatogenesis in the house cricket. J. Cell Biol. 31, 159-179. KELLER, J. M. &RILEY, D. E. (1976a). Nuclear ghosts: a nonmembranous structural component of mammalian cell nuclei. Science, N.Y. 193, 399-401. KELLER, J. M. & RILEY, D. E. (19766). Cell cycle alterations in the morphology of ghosts isolated from HeLa cell nuclei, jf. Cell Biol. 70, 174a. KELLER, W. (1975). Characterization of purified DNA-relaxing enzyme from human tissue culture cells. Proc. rtatn. Acad. Sci. U.S.A. 72, 2550-2554. KELLERMAYER, M., JOBET, K. & SZUCE, G. (1978). Inhibition of intranuclear transport of SV40- induced T antigen in heterokaryons. Cell Biol. int. Rep. 2, 19-24. KIRSCHNEB, R. H., RUSLI, M. & MARTIN, T. E. (1977). Characterization of the nuclear envelope, pore complexes and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy. J. Cell Biol. 72, 118-132. KLEINSMITH, L. J., STEIN, J. & STEIN, G. (1976). Dephosphorylation of nonhistone proteins specifically alters the pattern of gene transcription in reconstituted chromatin. Proc. natn. Acad. Sci. U.S.A. 73, 1174-1178. KRAEMER, R. J. & COFFEY, D. S. (1970). The interaction of natural and synthetic polyanions with mammalian nuclei. II. Nuclear swelling. Biochim. biophys. Acta 224, 568-578. KROHNE, G., FRANKE, W. W.,ELY, S., D'ARCY, A. & JOST, E. (1978a). Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies Nuclear matrix, ghost and pore-lamina 431 against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material. Cytobiologie 18, 22-38. KROHNE, G., FRANKE, W. W. & SCHEKR, U. (1978&). The major polypeptides of the nuclear pore complex. Expl Cell Res. 116, 85-102. LAEMMLI, U.K., CHENG, S. M., ADOLPH, K. W., PAULSON, J. R., BROWN, J. & BAUMBACH, W. A. (1977). Metaphase chromosome structure: role of non-histone proteins. Cold Spring Harb. Syntp. quant. Biol. 42, 351-360. LAM, K. S. & KASPER, C. B. (1979). Selective phosphorylation of a nuclear envelope polypeptide by an endogenous protein kinase. Biochemistry, N.Y. 18, 307-311. LANGENDORF, H., SIEBERT, G. & NITZ-LITZOW, D. (1964). Participation of rat liver nuclei in movements of sodium. Nature, Lond. 204, 888-890. LARK, K. G., CONSIGLI, R. A. & MINOCHA, H. C. (1966). Segregation of sister chromatids in mammalian cells. Science, N.Y. 154, 1202-1204. LEAKE, R. E., TRENCH, M. E. & BARRY, J. M. (1972). Effect of cations on the condensation of hen erythrocyte nuclei and its relation to gene activation. Expl Cell Res. 71, 17-26. LODIN, Z., BLUMAJER, J. & MARES, V. (1978). Nuclear pore complexes in cells in the developing mouse cerebral cortex. Acta histochem. 63, 74-79. MANCINI, P., HEYWOOD, P. & HODGE, L. D. (1973). Synthesis and fate of selected polypeptides of the inner nuclear envelope of HeLa S3 cells. J. Cell Biol. 59, 214a. MARKOVICS, J., GLASS, L. & MAUL, G. G. (1974). Pore patterns on nuclear membranes. Expl Cell Res. 85, 443-451. MARSHALL, P. & HARRIS, J. R. (1979). Isolation of nuclear pore complexes from Triton X-100 extracted rat liver nuclear envelope. Biochem. Soc. Trans. 7, 928-929. MAUL, G. G. (1976). Fibrils attached to the pore complex prevent egress of SV40 particles from the infected nucleus. J. Cell Biol. 70, 714-719. MAUL, G. G. & DEAVEN, L. (1977). Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content. J. Cell Biol. 73, 748-760. MAUL, G. G., MAUL, H. M., SCOGNA, J. E., LIEBERMAN, M. W., STEIN, G. S., HSU, B. Y.-L. & BORUN, T. W. (1972). Time sequence of nuclear pore formation in phytohaemagglurinin- stimulated lymphocytes and in HeLa cells during the cell cycle. J. Cell Biol. 55, 433- 447- MAUL, G. G., PRICE, J. W. & LIEBERMAN, M. W. (1971). Formation and distribution of nuclear pore complexes in interphase. J. Cell Biol. 51, 405-418. MAUL, H. M., HSU, B. Y.-L., BORUN, T. M. & MAUL, G. G. (1973). Effect of metabolic inhibitors on nuclear pore formation during the HeLa S3 cell cycle. J. Cell Biol. 59, 669- 676. MAYER, D. T. & GULICK, A. (1942). The nature of the proteins of cellular nuclei. J. biol. Chem. 146, 433-44°- MAZANEK, K. (1967). Presence de la 'zonula nucleum limitans' dansquelques cellules humaines. J. Microscopie 6, 1027-1032. MCNAMARA, D. J., RACEVSKIS, J., SCHUMM, D. E. & WEBB, T. E. (1975). Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport. Biochem. J. 147, 193-197. MERCER, E. J. (1959). An electron microscope study of Amoeba proteus. Proc. R. Soc. B 150, 216-232. MERRIAM, R. W. (1969). Movement of cytoplasmic proteins into nuclei induced to enlarge and initiate DNA or RNA synthesis. J. Cell Set. 5, 333-349- MILLER, T. E., HUANG, C.-Y. &POGO, A. O. (1978a).Rat liver nuclear skeleton and ribonucleoprotein complexes containing Hn RNA. J. Cell Biol. 76, 675-691. MILLER, T. E., HUANG, C.-Y. & POGO, A. O. (19786). Rat liver nuclear skeleton and small molecular weight RNA species. J. Cell Biol. 76, 692-704. MIRONOV, N. M., ADLER, V. V., SOKOLOV, N. A. &SHAPOT, V. S. (1976). Changes in matrix properties of chromarin during aminoazocarcinogenesis. Biull. Eksp. Biol. Med. 81, 363-365. MONNERON, A. & BERNHARD, W. (1969). Fine structural organization of the interphase nucleus in some mammalian cells, jf. Ultrastruct. Res. 27, 266-288. MONNERON, A., BLOBEL, G. & PALADE, G. E. (1972). Fractionation of the nucleus by divalent cations. J. Cell Biol. 55, 104-125. 432 P. S. Agutter andj. C. W. Richardson MURRAY, A. B. & DAVES, H. G. (1979). Three-dimensional reconstruction of the chromatin bodies in the nuclei of mature erythrocytes from the newt Triturus cristatus: the number of nuclear-envelope attachment sites. J. Cell Sci. 35, 59-66. NAGEL, W. C. & WUNDERLICH, F. (1977). Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport in Tetrahymena grown at different temperatures. J. Membr. Biol. 32, 151-164. NAORA, H., NAORA, N., IZAWA, M., ALLFREY, V. G. & MIRSKY, A. E. (1962). Some observations on differences in composition between the nuclei and cytoplasm of the frog oocyte. Proc. natn. Acad. Sci. U.S.A. 48, 853-857. NARAYAN, K. S., STETU.E, W. J., SMETANA, K. & BUSCH, H. (1967). Ultrastructural aspects of the ribonucleoprotein network in nuclei of Walker tumour and rat livers. Expl Cell Res. 46, 65-77- OHT3UKI, K., GRONER, Y. & HURWITZ, J. (1977). Isolation and purification of double-stranded ribonuclease from calf thymus. J. biol. Chem. 252, 483-491. OLINS, A. L. & OLINS, D. L. (1974). Spheroid chromatin units (nu bodies). Science, N.Y. 183, 33O-332. ONO, J., ONO, M. & WADA, O. (1976). Amino acid incorporation by nuclear membrane fraction of rat liver. Life Sci. 18, 215-222. OTTO, B., BAYNES, M. & KNIPPERS, R. (1977). A single-strand-specific DNA-binding protein from mouse cells that stimulates DNA polymerase. Eur. J. Biochem. 73, 17-24. PAINE, P. E., MOORE, L. C. & HOROWITZ, S. B. (1975). Nuclear envelope permeability. Nature, Land. 254, 109-114. PALAMAROZYK, G. & JANOZURA, E. (1977). Lipid mediated glycosylation in yeast nuclear membranes. FEBS Letters, Amsterdam 77, 169-172. PAPPAS, G. D. (1956). The fine structure of the nuclear envelope of Amoeba proteus. J'. biophys. biochem. Cytol. 2, Suppl. 431-434. PATRIZI, G. & POGER, M. (1967). The ultrastrucrure of the nuclear periphery. J. Ultrastruct. Res. 17, 127-136. PAULSON, J.R. & LAEMMLI, U.K. (1977). The structure of histone-depleted metaphase chromosomes. Cell 12, 817-828. PETERSON, S. P. & BERNS, M. W. (1978). Chromatin influence on the function and formation of the nuclear envelope shown by laser-induced psoralen photoreaction. J. Cell Sci. 3a, 197- 213- PLEKHANOVA, L. S., GEVORKIAN, Ah. G., ASHIROVA, Z. K. & ILRAGIMOV, A. I. (1978). Bio- synthesis of RNA in the nuclear structure of wilted cotton plant leaves. Biokhimiya 43, 407- 412. PRESCOTT, D. & GOLDSTEIN, L. (1968). Proteins in nucleo-cytoplasmic interactions. III. Re- distributions of nuclear proteins during and following mitosis in Amoeba proteus. J. Cell Biol. 39. 4O4-4I4- PUVION-DUTILLEUL, F. & BACHELLERDE, J. P. (1979). Ribosomal transcriptional complexes in subnuclear fractions of Chinese hamster ovary cells after short-term actinomycin D treatment. J. Ultrastruct. Res. 66, 190-199. RACEVSKIS, J. & WEBB, T. E. (1974). Processing and release of ribosomal RNA from isolated nuclei: analysis of the ATP-dependence and cytosol-dependence. Eur.jf. Biochem. 49,93-100. REJTHAR, A. & BLUMAJER, J. (1974). Difference in density of nuclear pores in normal and malignant fibroblasts of Syrian hamster. Neoplasma 21, 479-482. REJTHAR, A. & BLUMAJER, J. (1975). Difference in density of nuclear pores in normal and malignant cells. Scripta med. 49, 123-129. REVEL, M. & GRONER, Y. (1978). Post-transcriptional and translational controls of gene ex- pression in eucaryotes. A. Rev. Biochem. 47, 1079-1126. RICHARD, M., MARTIN, A. & LOUISOT, P. (1975). Evidence for glycosyltransferases in rat liver nuclei. Biochem. biophys. Res. Comrnun. 64, 108-114. RICHARDSON, J. C. W. (1979). Studies in the Molecular Organization of Nuclear Envelope Unpublished Ph.D. Thesis, University of Edinburgh. RICHARDSON, J. C. W. & MADDY, A. H. (1979). The use of immobilized lactoperoxidase as a probe of the molecular organization of the nuclear envelope. Biochem. Soc. Trans. 7, 685-686. Nuclear matrix, ghost and pore-lamina 433 RICHARDSON, J. C. W. & MADDY, A. H. (1980a). The polypeptides of rat liver nuclear envelope. I. Examination of nuclear pore complex polypeptides by solid-state lactoperoxidase labelling. J. Cell Sci. 43, 253-267. RICHARDSON, J. C. W. & MADDY, A. H. (19806). The polypeptides of rat liver nuclear envelope. II. Comparison of rat liver nuclear membrane polypeptides with those of the rough endo- plasmic reticulum. J. Cell Sci. 43, 269-277. RILEY, D. E. & KELLER, J. M. (1976a). The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells. Biochim. biopkys. Acla 444, 899-911. RILEY, D. E. & KELLER, J. M. (19766). The ultrastructure of ghosts isolated from HeLa cell nuclei. Fedn Proc. Fedn Am. Socs exp. Biol. 35, 1449. RILEY, D. E. & KELLER, J. M. (1978a). Cell-cycle dependent changes in non-membranous nuclear ghosts from HeLa cells. J. Cell Set. 29, 129-146. RILEY, D. E. & KELLER, J. M. (19786). The ultrastructure of non-membranous nuclear ghosts. jf. Cell Sci. 32, 249-268. RILEY, D. E., KELLER, J. M. & BYERS, B. (1975). The isolation and characterization of nuclear ghosts from cultured HeLa cells. Biochemistry, N.Y. 14, 3005-3013. ROBBINS, E. & GONOTAS, N. K. (1964). The ultrastructure of a mammalian cell during the mitotic cycle. J. Cell Biol. 21, 429-463. SAIGA, J. & KINOSHITA, S. (1976). Changes of chromatin structure induced by acid muco- polysaccharides. Expl Cell Res. 102, 143-152. SATO, T., ISHIKAWA, K. & OGATA, K. (1977). Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro. Biochim. biophys. Ada 474, 549—561. SCHEER, U., KARTENBECK, J., TRENDELENBURG, M. F., STADLER, J. & FRANKS, W. W. (1976). Experimental disintegration of the nuclear envelope: evidence for pore connecting fibrils. J. Cell Biol. 69, 1-18. SCHEL, J. H. N., STEENBERGEN, L. C. S., BEKERS, A. G. M. & WANKA, F. (1978). Change in the nuclear pore frequency during the nuclear cycle of Physarum polycephalum. J. Cell Sci. 34, 225-232. SCHUMM, D. E., MCNAMARA, D. J. & WEBB, T. E. (1973a). Cytoplasmic proteins regulating messenger RNA release from nuclei. Nature, New Biol. 245, 201-202. SCHUMM, D. E., MORRIS, H. P. & WEBB, T. E. (19736). Cytosol-modulated transport of mess- . enger RNA from isolated nuclei. Cancer Res. 33, 1821-1828. SCHUMM, D. E. & WEBB, T. E. (1972). Transport of informosomes from isolated nuclei of regenerating rat liver. Biochem. biophys. Res. Commun. 48, 1259-1265. SCHUMM, D.E. & WEBB, T. E. (1974a). Differential effects of plasma fractions from normal and tumour-bearing rats on nuclear RNA restriction. Nature, Lond. 256, 508-509. SCHUMM, D. E. & WEBB, T. E. (19746). The in vivo equivalence of a cell-free system for RNA processing and transport. Biochem. biophys. Res. Commun. 58, 354—360. SCHUMM, D. E. & WEBB, T. E. (1978). Effect of adenosine 3':5'-monophosphate and guanosine 3':5'-monopho8phate on RNA release from isolated nuclei. J. biol. Chem. 253, 8513-8517. SEIFART, K. H., JUHAZZ, P. D. & BENECHE, B. J. (1973). A protein factor from rat liver tissue enhancing the transcription of native templates by homologous RNA polymerase B. Eur. J. Biochem. 33, 181-191. SEVERS, N. J. & JORDAN, E. G. (1978). Nuclear pores: can they expand and contract to regulate nucleo-cytoplasmic exchange? Experientia 34, 1007-1011. SHELTON, K. R. (1976). Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein. Biochim. biophys. Acta 455, 973-982. SHELTON, K. R., COBBS, C. S. & COCHRAN, D. L. (1978). Similar polypeptides constitute the major group of oligomeric structural nonhistone proteins in the cell nucleus. Fedn Proc. Fedn Am. Socs exp. Biol. 37, 1978. SHELTON, K. R., COBBS, C. S., POVLISHOCK, J. T. & BURKAT, R. K. (1976). Nuclear envelope fraction proteins: isolation and comparison with the nuclear protein of the avian erythrocyte. Archs Biochem. Biophys. 174, 177-186. SHELTON, K. R. & COCHRAN, D. L. (1978). In vitro oxidation of intrinsic sulphydryl groups yields polymers of the two predominant polypeptides of the nuclear envelope fraction. Biochemistry, N.Y. 17, 1212-1216. SHELTON, K. R. & EGLE, P. M. (1979). Analysis of the nuclear envelope polypeptides by isoelectric focusing and electrophoresis. Biochem. biophys. Res. Commun. 90, 425-430. 434 P- S. Agutter andj. C. W. Richardson

SHELTON, K. R., LINDSEY, S. W., COBBS, C. S., POVLISHOCK, J.T. & VANDENBERG, R. D. (1975). Isolation of nuclear structural proteins. J. Cell Biol. 67, 395 a. SKAER, R. J. & WHYTOCK, S. (1977a). Chromatin-like artifacts from nuclear sap. J. Cell Set. 26, 301-310. SKAER, R. J. & WHYTOCK, S. (19776). The fixation of nuclei in glutaraldehyde. J. Cell Set. 27, 13-22. SMETANA, K., STEELE, W. J. & BUSCH, H. (1963). A nuclear ribonucleoprotein network. Expl Cell Res. 31, 198—201. STEELE, W. J. & BUSCH, H. (1963). Studies on the acidic nuclear proteins of the Walker tumour and the liver. Cancer Res. 23, 1153-1163. STEELE, W. J. & BUSCH, H. (1966). Studies on the ribonucleic acid component of the nuclear ribonucleoprotein network. Biochim. biophys. Acta 129, 54-67. SVEJDA, J., VRBA, M. & BLUMAJER, J. (1975). Freeze-etch study of occurrence of nuclear pores in normal and tumour cells. Neoplasnra 22, 385-390. SWIFT, H. (1963). Cytochemical studies of nuclear fine structure. Expl Cell Res., Suppl. 9, 54-67- TAMULEVICIUS, P., STREFFER, C, ROSCIC, O. & HUBERT, E. (1979). Localization of oxidised nicotinamide-adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope. Biochem. J. 178, 467-474. THOMPSON, L. R. & MCCARTHY, B. J. (1968). Stimulation of nuclear DNA and RNA synthesis by cytoplasmic extracts in vitro. Biochem. biophys. Res. Commun. 30, 166—172. TLEGLER, D. J. & BAERWALD, R. J. (1972). A freeze-etch study of clustered nuclear pores. Tissue Sf Cell 4, 447-456. TODOROV, I. N., GALKIN, A. P., SHEN, R. A. & ZHELIABOVSKAIA, S. M. (1975). Change in the RNA synthesizing ability of isolated nuclei and in the matrix activity of rat liver chromatin during cycloheximide suppression of protein synthesis. Doklady Acad. Nauk SSSR 223, 1488-1491. WANG, T. Y. (1961). Saline soluble proteins of isolated thymus nuclei. Biochim. biophys. Ada 49. 230-244. WANG, T. Y. (1966). Solubilization and characterization of the residual proteins of the cell nucleus.^. 610/. Chem. 241, 2913-2917. WANG, T. Y., KIRKHAM, W. R., DALLAM, R. D., MAYER, D. T. & THOMAS, L. E. (1950). Acidic proteins of cellular nuclei. Nature, Lond. 165, 974-975. WILLIAMS, C. A. & OCKEY, C. H. (1970). Distribution of DNA replicator sites in mammalian nuclei after different methods of cell synchronization. Expl Cell Res. 63, 365-372. WILLISON, J. H. M. & JOHNSTON, G. C. (1978). Altered nuclear pore diameters in Gi-arrested cells of the yeast Saccharomyces cerevisiae. J. Bad. 136, 318-323. WILLISON, J. H. M. & RAJARAMAN, R. (1977). 'Large' and 'small' nuclear pore complexes: the influence of glutaraldehyde. J. Microscopy 109, 183-192. WISE, G. E. & PRESCOTT, D. M. (1973). Initiation and continuation of DNA replication are not associated with the nuclear envelope in mammalian cells. Proc. natn. Acad. Set. U.S.A. 70, 714-717. WUNDERLICH, F., BEREZNEY, R. & KLEINIG, H. (1976). The nuclear envelope: an interdisci- plinary analysis of its morphology, composition and functions. In Biological Membranes (ed. D. Chapman & D. F. H. Wallach), pp. 241-333. London & New York: Academic Press. WUNDERLICH, F., GIESE, G. & BUCHERER, C. (1978). Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins? Expl Cell Res. 111, 479— 490. WUNDERLICH, F. & HERLAN, G. (1977). A reversibly contractile nuclear matrix: its isolation, structure and composition. J. Cell Biol. 73, 271-278. YASUZUMI, G., NAKAI, Y., TSUBO, I., YASUDA, M. & SUGIOKA, T. (1967). The fine structure of nuclei as revealed by electron microscopy. IV. The intranuclear inclusion formation in Leydig cells of aging human testes. Expl Cell Res. 45, 261-276. YASUZUMI, G. & TSUBO, I. (1966). The fine structure of nuclei as revealed by electron microscopy. III. Adenosine triphosphatase activity in the pores of the nuclear envelope of mouse choroid plexus epithelial cells. Expl Cell Res. 43, 281-292. Nuclear matrix, ghost and pore-lamina 435 Yu, L. C, RACEVSKIS, J. & WEBB, T. E. (1972). Regulated transport of ribosomalsubunit s from regenerating rat liver nuclei in a cell-free system. Cancer Res. 32, 2314-2321. ZBARSKY, I. B. (1972). Nuclear envelope isolation. In Methods in Cell Physiology, vol. 5 fed. D. M. Prescott), pp. 167-198. New York: Academic Press. ZBARSKY, I. B. & DEBOV, S. S. f 1948). Proteins of cell nuclei. Doklady Akad. Nauk SSSR 63, 795-798. ZBARSKY, I. B., DMITRJEVA, N. P. & YERMOLAYEVA, L. P. (1962). On the structure of tumour cell nuclei. Expl Cell Res. 27, 573-576. ZBARSKY, I. B. & GEORGIEV, G. P. (1959). Cytological characteristics of protein and nucleo- protein fractions of cell nuclei. Biochim. biophys. Acta 33, 301-302. ZBARSKY, I. B., PEREVOSHCHIKOVA, K. A., DELEKTORSKAYA, L. N. & DELEKTORSKY, V. V. (1969). Isolation and biochemical characterization of the nuclear envelope. Nature, Lond. 221, 257— 259- (Received 5 November 1979)