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Nuclear lamina and organization of nuclear architecture
TIBS 11 - November 1986
Nuclear
lamina
443 and
organization
of
humanreCentlYlaminsdeducedA andfr°mc14,15,cDNAtwoCl°neSof threef°r
nuclear architecture
major lamins (A, B, and C)commonly present in mammalian somatic cells. These two polypeptides were shown to Larry Gerace contain an internal region of ~350 amino acid residues which has strong sequence homology with the a-helical domain The nuclear lamina is a polymeric protein meshwork that lines the nucleoplasmic surface of common to all intermediate-type ilia~enuclearenve~pe.Recentw~rkdem~nstratesthatthelaminai~c~rnp~sed~fintermediate. ment (IF) proteins (for reviews see Refs type filaments, one of the major classes of structural filaments in cells. The lamina may be an 16 and 17; see also Osborn and Weber, importantdeterrninantofln'gherorderchroma~narchitectureduringinterphase, andi~impli- p. 469 of this issue). This conserved ctcated in regulation of nuclear envelope structure during the cell cycle, helical domain of IF polypeptides, flanked by end domains which vary in The nuclear envelope is the membrane specificallyin the nuclear lamina by light size and sequence among different IF system that forms the boundary of the and electron microscopic immunocyto- classes, generates a two-stranded coilednuclear compartment in eukaryotes (for chemistry (Fig. 2). Biochemical data 3 coil with the corresponding region of a reviews see Refs 1 and 2). Its structural suggests that the lamina contains a neighboring subunit to create a40-50 nm components include a double membrane, polymer of these major lamina proteins, rod. The latter forms the backbone of pore complexes and the nuclear lamina termed 'lamins'~z. Since it is a relatively IFs through several hierarchical levels of (Fig. 1). Pore complexes, elaborate insoluble structure, the lamina is com- assembly. In agreement with predictions protein superstructures that occur at monly present in residual 'nuclear of the lamin sequence data, structural regions where the inner and outer matrix'fractions(e.g. Ref. 13), although and biochemical analyses of isolated nuclear membranes are joined to form these fractions often contain other mammalian lamins A and C demonpores, provide aqueous channels across extraction-resistant intranuclear com- strate that these two proteins (as well as the nuclear envelope for nucleocytoplas- ponents as well. lamin B) are rod-shaped dimers comprismic transport. Circumstantial evidence ing two globular heads attached to one suggests that the pore complex may The lamina is a meshwork of end of an ~50 nm tail18 (Fig. 3b). As mediate active vectorial transport of intermediate-typeffdaments deduced from the sequence data, the macromolecules between the nucleus The nuclear lamina has a fibrous struc- coiled-coil a-helical domain of lamins A and cytoplasm, but few biochemical tureZ0,zl, as emphasized long ago in elec- and C is flanked by essentially non-helidetails are known, tron microscopic studies8. However, cal regions at the amino and carboxyl terThe nuclear lamina (for reviews see detailed features of lamina architecture mini of - 4 kDa and -20-30 kDa, Refs 3 and 4) is postulated to provide an were unclear until very recently, when a respectively14,1s. The globular heads architectural framework for the nuclear combination of protein sequence and seen on isolated lamins presumably coyenvelope s and an anchoring site at the structural studies on the nuclear lamins respond to the extensive carboxy-terminuclear periphery for interphase chromo- led to surprising conclusions, nal end domains of these molecules. somes 5-7. In certain eukaryotic cells the The first amino acid sequences were The detailed structure of the lamina lamina can be directly visualized th s/tu by thin-section electron microscopy; it / forms a discrete 30-100 nm thick layer r - ~ ~ Rough endoplosmie refleulum interposed between the inner nuclear Ribosomes" (~ ~ _
membrane and chromafin8. However. in most cells the lamina is a much thinner structure which can bc s e e n only after nudear subf,a~ionation'-". I,~r~uno-
j
~E~C~C~L
~0
uter nuclear membrane
C / ~
\\
(
/I
IL " ~
~ ,°ner nut,ear
lamina is present in nearly all interphase ( nuclei, at least in higher eukaryotes3,4. Insoluble proteinaceous fractions enriched in the lamina can be isolated by ~~--~ ~ I / extracting isolated nuclear envelopes Smoo~h with non-ionic detergents and high salt ~x~ 3) -- ~ ~ / endoplasmic concentrations3,4,1°, 11(Fig. 2),conditions ~ ~ reticulum that solubilize membranes, residual Lamina/ chromatin, and (in some situations3) pore complexes. Depending on the cell type and organism, such fractions contain one to three prominent 60-75 kDa Pore complex Chromosome polypeptides which are immunologically Fig.1. Diagram of the major structural components of the nuclear envelope. The nuclear envelope contains related. These proteins are localized innerand•uternuclearmembranes•whichareperi•dically••inedatnuclearp•rec•mplexes•supram•lecuLarry Gerace is at the Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA.
lar protein structures that provide channels across the nuclear envelope for molecular transport. The outer nuclear membrane is morphologically continuous with rough and smooth endoplasmic reticulum, and has many endoplasmic reticulum-like properties, including bound ribosomes. The nuclear lamina, a meshwork of intermediate-type filaments that lines the nucleoplasmic surface of the inner nuclear membrane, is thought to provide an anchoring site at the nuclear periphery for interphase chromosomes. (~1986. ElsevierSciencePublishersB.V..Amsterdam
0376 5067186/$02,00
444
T I B S 11 - N o v e m b e r 1986
reutilized in nuclei formed during early (a)
( ........ (~~ ) ~
1
2
3
cleavage embry°genesis19'2°'bySUbsequentlY'Lx and stages L n , Lmwhichis °f graduallYbecome expressedreplaced
: .~::,,~::~ ..... t ~
.....
....
at the mid-blastula and gastrula stages of development, respectively19,2°. Interestingly, Lin is also expressed much later in development in selected cell types (neurons and muscle cells) where it is present with L~ and LII2°. During gametogenesis in some vertebrates, extensive changes in lamin expression19 and lamina structure 21 take place, coinciding with dramatic changes in
...... ....
: ~'.
...........
[aminA%._~.l ~
~
,,~ i
CJ-
~
chromosome otic Mammalian prophase,structure lamins A, during B, and firStcmeiare co-expressed in many different somatic cells3. Lamins A and C have very similar primary structures and in human cells differ only in their non-ct helical end
......... ...........
:
ii
....
Fig. 2. Isolation and immunocytochemical localization of the nuclear lamina. (a) Salt-washed rat liver nuclear envelopes (lane 1) were incubated in 2% Triton X-IO0 and 0.3 ~ KCI and centrifuged at low speed to yield a solubilized fraction (lane 2) and an insoluble pellet which is highly enriched in the nuclear lamina comprisinglaminsA, B, andC (lane3). Samples were then analysed on an SDS gel. Theextructionprocedure completely removes nuclear membranes and pore complexes from the lamina meshwork3. The major non-lamin components in the pellet are contaminating keratin IF polypeptides (arrows in lane 3). (b) The nuclear lamins were localized in isolated rat liver nuclear envelopes using an immunoferritin technique 24and thin-section electron microscopy. Antibody labelling occurs only on the nucleoplasmic surface of the inner nuclear membrane (INM). The outer nuclear membrane (ONM) can be easily distinguished since it is mechanically fragile and periodically removed. Arrows denote pore complexes. Bar = 150 nm.
was recently revealed by electron microscopy of detergent-extracted nuclear envelopes of X e n o p u s oocytesTM. This work showed that the lamina actually comprises long filaments with the same diameter ( - 1 0 nm) as IFs (see also Ref. 11). Furthermore, isolated rat liver lamins A and C can be reconstituted in vitro to form 10-nm filaments18 with the same axial periodicity (25 um) as that described for native X e n o p u s lamina iliaments, and similar to that seen for IF in general (21-23 nm). Thus, the lamins have all of the structural properties that are characteristic of IF proteins, both as individual moieties and when assembled into filamentsa6,17. Together with the sequence data, these results indicate that lamins are genuine IF polypeptides, In X e n o p u s oocytes, the lamina forms a striking meshwork of two approximately, orthogonal sets of 10-rim iliaments ~s (Fig. 3a). Thin-section electron microscopy suggests that the lamina of rat hepatocytes may have a similarstrueturO 0, raising the possibility that a meshwork of orthogonally-oriented IFs is a widespread characteristic of the lamina. The relatively thick (30-100 nm)
d°mains14'15" Lamin A has an additi°nal region to lamin C of - 1 0 kDa. Although lamin B has a distinct tryptic peptide map from lamins A and C22, is structurally homologous to the latter based on biochemical and electron microscopic studies TM(see above). F u n g f l o n a l i n t e r a c l i o n s o f lalnins
The lamina is proposed to have functionally important interactions with the inner nuclear membrane, chromatin and pore complexes5. While all lamins would
be expected to have a structure homo1ogous to IF proteins, it is probable that different lamins in the same organism, lamina in certain cell types8 could consist exhibit partially distinct functions, which of multiple 'layers' of an IF lattice, could relate to their interactions with other macromolecules and to their iliaThe lamin family of polypeptifles ment-forming characteristics. Lamins have been studied biochemiIn cells where multiple lamins are cally in a number of different higher expressed, it is not established whether eukaryotes, including mammals, amphi- all lamins are co-assembled in the same bians, birds, insects and molluscs (for re- filaments, but individual lamins do not ferences see Ref. 4). These proteins have occupy any detectably separate lateral been most extensively analysed in ver- domains (e.g. adjacent to pore corntebrate organisms, where it is apparent plexes) in the lamina of rat liver nuclei23. that lamins form a multigene protein A co-polymer of all three mammalian family whose different members are ex- lamins could most easily account for the pressed in a developmentally regulated coordinate interaction of the lamina with fashion in at least some species, chromatin and membranes, assuming The pattern of lamin expression dur- that separate lamins carry out partially ing early development has been analysed distinct functions involving interactions in detail in X e n o p u s laevis19, 2°. Four dis- with these two structures. tinct lamins with different tryptic peptide The close association of the lamina maps and two-dimensional gel mobilities with the inner nuclear membrane could (Lr--Liv) have been characterized in dif- be important for physically coupling the ferent X e n o p u s tissues4. Both L I and L n dynamics of the lamina to nuclear memare present in many somatic cells, while brane structure. While the detailed Lw occurs in male germ cells and L m is biochemical basis for this interaction is the only detectable lamin of mature not understood, in mammalian cells oocytes. There is a sufficient stockpile of lamin B appears to have a specialized L m assembled in the large nucleus of membrane-binding role: it is more mature oocytes for several thousand resistant to chemical extraction from somatic cell nuclei, and oocyte L m is membranes than lamins A and C24 and
TIBS 11 - November 1986
445
Fig. 3. Visualization of the nuclear lamina and isolated lamins by electron microscopy. (a) A nuclear envelope from a Xenopus oocyte was extracted with Triton X-IO0 to remove membranes and pore cornplexes from the insoluble nuclear lamina, which was then visualized by unidirectional shadowing 18. In wellpreserved areas, the lamina consists of two approximately, orthogonal sets of lO-nm filaments with a crossover spacing of 52 nm. (b) Isolated rat liver lamin A and C dimers were examined after rotary shadowing is. The lamin molecules usually consist of two globular heads attached to one end of a 52 nm rodlike tail. Bars: a = 50Onto, b = 100 nm.
selectively remains associated with membrane vesicles after nuclear envelope disassembly during mitosis25. However, a functionallysignificant (although qualitatively different) interaction of lamins A and C with the inner nuclear membrane is also conceivable, Higher order chromosome structure is thought to be important for most aspects of nuclear function, including DNA replication, gene expression, and chromosome segregation during mitosis, Chromatin appears to be closely associated with the lamina in most cell types, and it is plausible that this interaction is important for stabilizing or maintaining certain aspects of higher order chromosome architecture during interphase5-7. Interestingly, three-dimensional reconstructionsof polytene chromosomes in the cells of Drosophila salivary glands show that certain discrete chromosomal sites (containing intercalary heterochromatin) are apposed to the nuclear envelope with high frequency in salivary gland cells26. Thus, laminachromatin interactions are probably non-random (at least in some cells), although the exact sets of chromosomelamina associations in different cell types may depend on the functional state of chromatin (see Ref. 27). In principle the lamina could interact with chromatin by binding to specific or non-specific DNA sequencesr, 7 or to other chromosomal proteins28, but none of these possibilities has been dearly defined. Cell cycle dynamics of the lamina The nuclear envelope undergoes a continuous increase in surface area throughout interphase in growing cells29, and the lamina must correspondingly
undergo growth and/or restructuring, Increase in nuclear surface area may be directly regulated by growth of the lamina filament meshwork. How the lamina meshwork undergoes addition of new subunits is a structurally complex problem, and may involve lengthening of pre-existing filaments by internal or end addition, as well as formation of new filaments. The latter process in particular would be required for maintenanceof a constant cross-over spacing of orthogonal lamina filaments TM (see Fig. 3) upon increase in nuclear surface area. Synthesis and assembly of the lamins has been analysed in a number of cell lines. In CHO (Chinese hamster ovary) cells, the lamins are made continuously throughout interphase3. Following synthesis, the proteins are apparently assembled into a lamina structure fairly rapidly (with half-times ofassemblyof 15 min to 1 h)3,30. In both mammalian and avian cells, lamin A is synthesized as a slightly higher Mr precursor ( - 2 kDa on SDS gels)3,3°,31 which is processed to a molecule with the authentic Mr of lamin A only after assembly into an insoluble structure (presumably the lamina)3,3°. The function of this apparent precursor and its biochemical differences from the mature lamin A are unclear but it is not necessary for targeting lamin A to the lamina, since authentic lamin A assembles normally into the lamina after mitotic disassembly12. The only reversible in vivo postsyntheticmoditicationoflaminsthathasbeen detected is phosphorylation of serine and threonine residues3L In growing interphase cells the lamins have low concentrations of associated phosphate (0.250.4 mol P/tool lamin), and all detectable
phosphate incorporation is associated with assembled lamins32. Considering the apparent involvementof phosphorylation in regulation of mitotic disassembly of the lamina (see below), one possible function of this interphase modification could be to weaken locally laminlamin interactions in the lamina filaments to permit insertion of new lamins and allow filament growth. Dramatic structural reorganization of the lamina occurs during mitosis, when the lamina is reversibly disassembled5,12,33. As seen through the light microscope (Fig. 4), the lamins become dispersed throughout the cytoplasm and lose their association with chromosomes during prophase, concomitant with nuclear envelope disassembly. When the nuclear envelope reforms during telophase, the lamins progressively and completely reassemble around chromosomes. In C H O cells, the disassembled lamins sediment as 4-5S moieties on sucrose gradients 12, suggesting that they are dimers or tetramers after disassembly TM. In Xenopus eggs (arrested in meiotic metaphase), lamin LIIIis also completely disassembled, but in contrast to mammalian lamins, sediments as a 9S moiety on sucrose gradients 19. The mitotic lamins of CHO cells become hyperphosphorylated (2-3 mol P/mol lamin) during disassembly in vivo 12,32 as well as in vitro34,41 and lose their phosphate upon reassembly, suggesting that phosphorylation may be a primary regulator of mitotic structural dynamics of the lamina. Interestingly, lamin hyperphosphorylation may be the manifestation of a putative phosphorylation cascade proposed to occur during prophase35,36 which could effect reorganization of many different cellular structures in a temporally coordinated fashion. The reversible disassembly of the lamina is postulated to regulate the disassembly and reformation of the nuclear envelope during mitosisS.1L According to this scheme, lamina disassembly during prophase provides a necessary (although not sufficient) condition for fragmentation of nuclear membranes into vesicles. Subsequent vesiculation of nuclear membranes presumably occurs by the same mechanisms that lead to generalized fragmentation of the endoplasmic reticulum in mitotic cells37. Conversely, reassembly of the lamina during telophase is proposed to mediate directly the assembly of membranes around chromosomes, with the membraneassociated lamin B serving to target nuclear membrane vesicles to the chromosome surfaces3. Since lamin B is probably the main nuclear envelope
446
T I B S I1 - N o v e m b e r 1986
Fig. 4. Disassembly and reformation of the nuclear lamina during mitosis. Rat tissue culture cells were analysed by immunofluorescence microscopy with antibodies recognizing the lamins. During interphase (a), the lamins are localized exclusively in the nuclear envelope, resulting in a rim-like staining at the nuelear periphery. During prophase as the nuclear envelope is disassembled, the lamins gradually become dispersed throughout the cytoplasm. By prometaphase (b) they have a uniform cytoplasmic distribution and are not associated with the mitotic chromosomes (seen as the stain-excluding areas). The lamins remain disassembled until the nuclear envelope begins to reform during early telophase (c), when they start to associate with surfaces of the separated sister chromosomes (now fused into single masses). Periehromosomal lamin assembly is largely completed by late telophase (d), when a continuous nuclear envelope has been reconstructed. Nuclear rim staining is again apparent in early G1 when chromatin decondensation has occurred (e).
protein associated with disassembled nuclear membranes, a lamina-based assembly mechanism could ensure that minor nuclear membrane proteins are efficiently sorted to the nuclear envelope during telophase, Direct support for this model was recently obtained from studies with a cell-free nuclear assembly system, where selective immunological depletion of either lamins A and C or lamin B (and associated membranes) from the initial assembly mixtures resulted in extensive inhibition of membrane and pore cornplex assembly around chromosomes 24. In related studies, microinjection of antilamin antibodies into metaphase tissue culture cells, at concentrations that inhibited assembly of many of the lamins around chromosomes, prevented chromosome decondensation at the end of mitosis as. While inhibition of chromatin decondensation in this case m a y b e d u e indirectly to structurally abnormal or incomplete nuclear envelopes, this experiment nevertheless demonstrates that lamina assembly is important for normal nuclear reformation in telophase. Future analysis oflamina functions While there is good evidence that the lamina is important for regulating n u clear structure during mitosis, m o s t mechanistic aspects of this problem are poorly defined at present. Furthermore, little direct information is available o n the role of the lamina in organization of interphase chromatin and the pore complex. A combination of different approaches, particularly detailed biochemical analysis of the lamina, will be required to understand these problems in detail and to delineate additional functional properties of the lamina. The period of mitosis affords a particularly useful situation to study lamina functions as the nucleus is reversibly disassembled
during this period in higher eukaryotes. Recently, cell-free preparations of mitotic mammalian tissue culture cells 24,41 and S e n o p u s eggs 34,39,40 have been observed to support prophase-like nuclear disassembly and telophase-like nuclear reformation. In certain cases, these systems have also been demonstrated to reproduce closely in vivo events related to nuclear lamina dynamics 24,34,41. In the future these systerns should provide powerful tools for analysing the lamina and its structural regulation, as well as many other questions related to nuclear architecture.
Acknowledgements I am grateful to Doug Murphy and Ann Hubbard for helpful comments, and to Ueli Aebi and Alayne Senior for the micrographs in Figs 3 and 4, respectively. References 1 Fry, D. (1976) In Mammalian Cell Membranes (Vol. 2) (G. Jamieson and D. Robinson, eds) pp. 197-265, Butterworth 2 Franke, W., Scheer, U., Krohne, G. and Jarasch, E. (1981) J. Cell Biol. 91, 39s-50s 3 Gerace, L., Comeau, C. and Benson, M. (1984) J. Cell Sci. (Suppl. 1) 137-160 4 Krohne, G. and Benavente, R. (1986) Exp. CeURes. 162, 1-10 5 Gerace, L., Blum, A. and Blobel, G. (1978) J. Celt Biol. 79,546-566 6 Hancock, R. and Hughes, M. (1982) Biol. Cell 44,201-212 7 Lebkowski, J. and Laemmli, U. (1982)J. Mol. Biol. 156, 325-344 8 Fawcett, D. (1966) Am. J. Anat. 119, 129-146 9 Aaronson, R. and Blobel, G. (1975) Proc. Natl Acad. Sci. USA 72, 1007-1011 10 Dwyer, N. and Blobel, G. (1976) J. Cell Biol.
70,581-591 11 Scheer, U., Kartenbeck, J., Trendelenburg, M., Stadler, J. and Franke, W. (1976) J. Cell Biol. 69, 1-18 12 Gerace, L. and Blobel, G. (1980) Cell 19, 277287 13 Berezney, R. and Coffey, D. (1977) J, Cell Biol. 73, 6164537 14 McKeon, F., Kirschner, M. and Caput, D.
(1986) Nature 319,463-468 15 Fisher, D. Z., Chaudhary, N. and Blobel, G. (1986) Proc. Natl Acad. Sci. USA 83, 6450-
6454 16 Weber, K. and Geisler, N. (1985) Annals N Y Acad. Sci. 455,126-143 17 Steinert, P., Steven, A. and Roop, D. (1985) Cell 42, 411-419 18 Aebi, U., Cohn, J., Buhle, L. and Gerace, L. (1986) Nature323, 560-564
19 Benavente, R., Krohne, G. and Franke, W. (1985) Cell 41,177-190 20 Stick, R. and Hausen, P. (1985) Cell 41,191200 21 Stick,R. and Schwarz,H. (1983) Cell 33,949958 22 Shelton,
K., Higgins, L., Cochran, D.,
Ruffolo,J. and Egle, P. (1980)J. Biol. Chem. 255,10978--10983 23 Gerace, L., Ottaviano, Y. and Kondor-Koch, C. (1982) J. Cell Biol. 95,826~37 24 Gerace, L. and Blobel, G. (1982) Cold Spring Harbor Syrup. Quant. Biol. 46,967-978 25 Burke, B. and Gerace, L. (1986) Cell 44, 639652 26 Hochstrasser, M., Mathog, D., Gruenbaum, v., Saumweber,H. and Sedat, J. (1986) J. Cell Biol. 102, 112-123 27 82Bl°bel',8527~529G' (1985) Proc. Natl Acad. Sci. USA 28 McKeon, F., TuffaneUi, F., Kobayashi, S. and Kirschner, M. (1984) Cell 36, 83-92 29 Maul, G., Maul, H., Scogna, J., Lieberman, M., Stein, G., Hsu, B. and Borun, T. (1972) J. Cell Biol. 55,433-447 30 Lehner, C., Furstenberger, G., Eppenberger, H. andNigg, E.(1986)Proc. NatlSci. USA83, 2096-2099 31 Lalibertie, J.,Dagenais, A.,Filion, M.,BiborHardy, V., Simard, R. and Royal, A. (1984) J. Cell Biol. 96, 980-985 32 Ottaviano, Y. and Gerace, L. (1985) J. Biol. Chem. 260,624-632 33 Krohne, G., Franke, W., Ely, S., D'Arcy, A. and Jost, E. (1978) Cytobiologie 18, 22-38 34 Miake-Lye, R. and Kirschner, M. (1985) Cell 41,165-175 35 Wu, M. and Gerhart, J. (1980) Dev. Biol. 79, 465~77 36 Miake-Lye, R,, Newport, J. and Kirschner, M. (1983) J. Cell Biol. 97, 81-91 37 Warren, G. (1985) Trends Biochem. Sci. 10, 439-443 38 Benavente, R. a,d Klohne, G. J. Cell Biol. (in press) 39 Lohka, M. and Masui, Y. (1983) Science 220, 719-721 40 Lohka, M. and Mailer, J. (1985) J. Cell Biol. 101,518-523 41 Suprynowicz, F. and Gerace, L. J. Cell Biol. (in press)
Nuclear
lamina
443 and
organization
of
humanreCentlYlaminsdeducedA andfr°mc14,15,cDNAtwoCl°neSof threef°r
nuclear architecture
major lamins (A, B, and C)commonly present in mammalian somatic cells. These two polypeptides were shown to Larry Gerace contain an internal region of ~350 amino acid residues which has strong sequence homology with the a-helical domain The nuclear lamina is a polymeric protein meshwork that lines the nucleoplasmic surface of common to all intermediate-type ilia~enuclearenve~pe.Recentw~rkdem~nstratesthatthelaminai~c~rnp~sed~fintermediate. ment (IF) proteins (for reviews see Refs type filaments, one of the major classes of structural filaments in cells. The lamina may be an 16 and 17; see also Osborn and Weber, importantdeterrninantofln'gherorderchroma~narchitectureduringinterphase, andi~impli- p. 469 of this issue). This conserved ctcated in regulation of nuclear envelope structure during the cell cycle, helical domain of IF polypeptides, flanked by end domains which vary in The nuclear envelope is the membrane specificallyin the nuclear lamina by light size and sequence among different IF system that forms the boundary of the and electron microscopic immunocyto- classes, generates a two-stranded coilednuclear compartment in eukaryotes (for chemistry (Fig. 2). Biochemical data 3 coil with the corresponding region of a reviews see Refs 1 and 2). Its structural suggests that the lamina contains a neighboring subunit to create a40-50 nm components include a double membrane, polymer of these major lamina proteins, rod. The latter forms the backbone of pore complexes and the nuclear lamina termed 'lamins'~z. Since it is a relatively IFs through several hierarchical levels of (Fig. 1). Pore complexes, elaborate insoluble structure, the lamina is com- assembly. In agreement with predictions protein superstructures that occur at monly present in residual 'nuclear of the lamin sequence data, structural regions where the inner and outer matrix'fractions(e.g. Ref. 13), although and biochemical analyses of isolated nuclear membranes are joined to form these fractions often contain other mammalian lamins A and C demonpores, provide aqueous channels across extraction-resistant intranuclear com- strate that these two proteins (as well as the nuclear envelope for nucleocytoplas- ponents as well. lamin B) are rod-shaped dimers comprismic transport. Circumstantial evidence ing two globular heads attached to one suggests that the pore complex may The lamina is a meshwork of end of an ~50 nm tail18 (Fig. 3b). As mediate active vectorial transport of intermediate-typeffdaments deduced from the sequence data, the macromolecules between the nucleus The nuclear lamina has a fibrous struc- coiled-coil a-helical domain of lamins A and cytoplasm, but few biochemical tureZ0,zl, as emphasized long ago in elec- and C is flanked by essentially non-helidetails are known, tron microscopic studies8. However, cal regions at the amino and carboxyl terThe nuclear lamina (for reviews see detailed features of lamina architecture mini of - 4 kDa and -20-30 kDa, Refs 3 and 4) is postulated to provide an were unclear until very recently, when a respectively14,1s. The globular heads architectural framework for the nuclear combination of protein sequence and seen on isolated lamins presumably coyenvelope s and an anchoring site at the structural studies on the nuclear lamins respond to the extensive carboxy-terminuclear periphery for interphase chromo- led to surprising conclusions, nal end domains of these molecules. somes 5-7. In certain eukaryotic cells the The first amino acid sequences were The detailed structure of the lamina lamina can be directly visualized th s/tu by thin-section electron microscopy; it / forms a discrete 30-100 nm thick layer r - ~ ~ Rough endoplosmie refleulum interposed between the inner nuclear Ribosomes" (~ ~ _
membrane and chromafin8. However. in most cells the lamina is a much thinner structure which can bc s e e n only after nudear subf,a~ionation'-". I,~r~uno-
j
~E~C~C~L
~0
uter nuclear membrane
C / ~
\\
(
/I
IL " ~
~ ,°ner nut,ear
lamina is present in nearly all interphase ( nuclei, at least in higher eukaryotes3,4. Insoluble proteinaceous fractions enriched in the lamina can be isolated by ~~--~ ~ I / extracting isolated nuclear envelopes Smoo~h with non-ionic detergents and high salt ~x~ 3) -- ~ ~ / endoplasmic concentrations3,4,1°, 11(Fig. 2),conditions ~ ~ reticulum that solubilize membranes, residual Lamina/ chromatin, and (in some situations3) pore complexes. Depending on the cell type and organism, such fractions contain one to three prominent 60-75 kDa Pore complex Chromosome polypeptides which are immunologically Fig.1. Diagram of the major structural components of the nuclear envelope. The nuclear envelope contains related. These proteins are localized innerand•uternuclearmembranes•whichareperi•dically••inedatnuclearp•rec•mplexes•supram•lecuLarry Gerace is at the Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA.
lar protein structures that provide channels across the nuclear envelope for molecular transport. The outer nuclear membrane is morphologically continuous with rough and smooth endoplasmic reticulum, and has many endoplasmic reticulum-like properties, including bound ribosomes. The nuclear lamina, a meshwork of intermediate-type filaments that lines the nucleoplasmic surface of the inner nuclear membrane, is thought to provide an anchoring site at the nuclear periphery for interphase chromosomes. (~1986. ElsevierSciencePublishersB.V..Amsterdam
0376 5067186/$02,00
444
T I B S 11 - N o v e m b e r 1986
reutilized in nuclei formed during early (a)
( ........ (~~ ) ~
1
2
3
cleavage embry°genesis19'2°'bySUbsequentlY'Lx and stages L n , Lmwhichis °f graduallYbecome expressedreplaced
: .~::,,~::~ ..... t ~
.....
....
at the mid-blastula and gastrula stages of development, respectively19,2°. Interestingly, Lin is also expressed much later in development in selected cell types (neurons and muscle cells) where it is present with L~ and LII2°. During gametogenesis in some vertebrates, extensive changes in lamin expression19 and lamina structure 21 take place, coinciding with dramatic changes in
...... ....
: ~'.
...........
[aminA%._~.l ~
~
,,~ i
CJ-
~
chromosome otic Mammalian prophase,structure lamins A, during B, and firStcmeiare co-expressed in many different somatic cells3. Lamins A and C have very similar primary structures and in human cells differ only in their non-ct helical end
......... ...........
:
ii
....
Fig. 2. Isolation and immunocytochemical localization of the nuclear lamina. (a) Salt-washed rat liver nuclear envelopes (lane 1) were incubated in 2% Triton X-IO0 and 0.3 ~ KCI and centrifuged at low speed to yield a solubilized fraction (lane 2) and an insoluble pellet which is highly enriched in the nuclear lamina comprisinglaminsA, B, andC (lane3). Samples were then analysed on an SDS gel. Theextructionprocedure completely removes nuclear membranes and pore complexes from the lamina meshwork3. The major non-lamin components in the pellet are contaminating keratin IF polypeptides (arrows in lane 3). (b) The nuclear lamins were localized in isolated rat liver nuclear envelopes using an immunoferritin technique 24and thin-section electron microscopy. Antibody labelling occurs only on the nucleoplasmic surface of the inner nuclear membrane (INM). The outer nuclear membrane (ONM) can be easily distinguished since it is mechanically fragile and periodically removed. Arrows denote pore complexes. Bar = 150 nm.
was recently revealed by electron microscopy of detergent-extracted nuclear envelopes of X e n o p u s oocytesTM. This work showed that the lamina actually comprises long filaments with the same diameter ( - 1 0 nm) as IFs (see also Ref. 11). Furthermore, isolated rat liver lamins A and C can be reconstituted in vitro to form 10-nm filaments18 with the same axial periodicity (25 um) as that described for native X e n o p u s lamina iliaments, and similar to that seen for IF in general (21-23 nm). Thus, the lamins have all of the structural properties that are characteristic of IF proteins, both as individual moieties and when assembled into filamentsa6,17. Together with the sequence data, these results indicate that lamins are genuine IF polypeptides, In X e n o p u s oocytes, the lamina forms a striking meshwork of two approximately, orthogonal sets of 10-rim iliaments ~s (Fig. 3a). Thin-section electron microscopy suggests that the lamina of rat hepatocytes may have a similarstrueturO 0, raising the possibility that a meshwork of orthogonally-oriented IFs is a widespread characteristic of the lamina. The relatively thick (30-100 nm)
d°mains14'15" Lamin A has an additi°nal region to lamin C of - 1 0 kDa. Although lamin B has a distinct tryptic peptide map from lamins A and C22, is structurally homologous to the latter based on biochemical and electron microscopic studies TM(see above). F u n g f l o n a l i n t e r a c l i o n s o f lalnins
The lamina is proposed to have functionally important interactions with the inner nuclear membrane, chromatin and pore complexes5. While all lamins would
be expected to have a structure homo1ogous to IF proteins, it is probable that different lamins in the same organism, lamina in certain cell types8 could consist exhibit partially distinct functions, which of multiple 'layers' of an IF lattice, could relate to their interactions with other macromolecules and to their iliaThe lamin family of polypeptifles ment-forming characteristics. Lamins have been studied biochemiIn cells where multiple lamins are cally in a number of different higher expressed, it is not established whether eukaryotes, including mammals, amphi- all lamins are co-assembled in the same bians, birds, insects and molluscs (for re- filaments, but individual lamins do not ferences see Ref. 4). These proteins have occupy any detectably separate lateral been most extensively analysed in ver- domains (e.g. adjacent to pore corntebrate organisms, where it is apparent plexes) in the lamina of rat liver nuclei23. that lamins form a multigene protein A co-polymer of all three mammalian family whose different members are ex- lamins could most easily account for the pressed in a developmentally regulated coordinate interaction of the lamina with fashion in at least some species, chromatin and membranes, assuming The pattern of lamin expression dur- that separate lamins carry out partially ing early development has been analysed distinct functions involving interactions in detail in X e n o p u s laevis19, 2°. Four dis- with these two structures. tinct lamins with different tryptic peptide The close association of the lamina maps and two-dimensional gel mobilities with the inner nuclear membrane could (Lr--Liv) have been characterized in dif- be important for physically coupling the ferent X e n o p u s tissues4. Both L I and L n dynamics of the lamina to nuclear memare present in many somatic cells, while brane structure. While the detailed Lw occurs in male germ cells and L m is biochemical basis for this interaction is the only detectable lamin of mature not understood, in mammalian cells oocytes. There is a sufficient stockpile of lamin B appears to have a specialized L m assembled in the large nucleus of membrane-binding role: it is more mature oocytes for several thousand resistant to chemical extraction from somatic cell nuclei, and oocyte L m is membranes than lamins A and C24 and
TIBS 11 - November 1986
445
Fig. 3. Visualization of the nuclear lamina and isolated lamins by electron microscopy. (a) A nuclear envelope from a Xenopus oocyte was extracted with Triton X-IO0 to remove membranes and pore cornplexes from the insoluble nuclear lamina, which was then visualized by unidirectional shadowing 18. In wellpreserved areas, the lamina consists of two approximately, orthogonal sets of lO-nm filaments with a crossover spacing of 52 nm. (b) Isolated rat liver lamin A and C dimers were examined after rotary shadowing is. The lamin molecules usually consist of two globular heads attached to one end of a 52 nm rodlike tail. Bars: a = 50Onto, b = 100 nm.
selectively remains associated with membrane vesicles after nuclear envelope disassembly during mitosis25. However, a functionallysignificant (although qualitatively different) interaction of lamins A and C with the inner nuclear membrane is also conceivable, Higher order chromosome structure is thought to be important for most aspects of nuclear function, including DNA replication, gene expression, and chromosome segregation during mitosis, Chromatin appears to be closely associated with the lamina in most cell types, and it is plausible that this interaction is important for stabilizing or maintaining certain aspects of higher order chromosome architecture during interphase5-7. Interestingly, three-dimensional reconstructionsof polytene chromosomes in the cells of Drosophila salivary glands show that certain discrete chromosomal sites (containing intercalary heterochromatin) are apposed to the nuclear envelope with high frequency in salivary gland cells26. Thus, laminachromatin interactions are probably non-random (at least in some cells), although the exact sets of chromosomelamina associations in different cell types may depend on the functional state of chromatin (see Ref. 27). In principle the lamina could interact with chromatin by binding to specific or non-specific DNA sequencesr, 7 or to other chromosomal proteins28, but none of these possibilities has been dearly defined. Cell cycle dynamics of the lamina The nuclear envelope undergoes a continuous increase in surface area throughout interphase in growing cells29, and the lamina must correspondingly
undergo growth and/or restructuring, Increase in nuclear surface area may be directly regulated by growth of the lamina filament meshwork. How the lamina meshwork undergoes addition of new subunits is a structurally complex problem, and may involve lengthening of pre-existing filaments by internal or end addition, as well as formation of new filaments. The latter process in particular would be required for maintenanceof a constant cross-over spacing of orthogonal lamina filaments TM (see Fig. 3) upon increase in nuclear surface area. Synthesis and assembly of the lamins has been analysed in a number of cell lines. In CHO (Chinese hamster ovary) cells, the lamins are made continuously throughout interphase3. Following synthesis, the proteins are apparently assembled into a lamina structure fairly rapidly (with half-times ofassemblyof 15 min to 1 h)3,30. In both mammalian and avian cells, lamin A is synthesized as a slightly higher Mr precursor ( - 2 kDa on SDS gels)3,3°,31 which is processed to a molecule with the authentic Mr of lamin A only after assembly into an insoluble structure (presumably the lamina)3,3°. The function of this apparent precursor and its biochemical differences from the mature lamin A are unclear but it is not necessary for targeting lamin A to the lamina, since authentic lamin A assembles normally into the lamina after mitotic disassembly12. The only reversible in vivo postsyntheticmoditicationoflaminsthathasbeen detected is phosphorylation of serine and threonine residues3L In growing interphase cells the lamins have low concentrations of associated phosphate (0.250.4 mol P/tool lamin), and all detectable
phosphate incorporation is associated with assembled lamins32. Considering the apparent involvementof phosphorylation in regulation of mitotic disassembly of the lamina (see below), one possible function of this interphase modification could be to weaken locally laminlamin interactions in the lamina filaments to permit insertion of new lamins and allow filament growth. Dramatic structural reorganization of the lamina occurs during mitosis, when the lamina is reversibly disassembled5,12,33. As seen through the light microscope (Fig. 4), the lamins become dispersed throughout the cytoplasm and lose their association with chromosomes during prophase, concomitant with nuclear envelope disassembly. When the nuclear envelope reforms during telophase, the lamins progressively and completely reassemble around chromosomes. In C H O cells, the disassembled lamins sediment as 4-5S moieties on sucrose gradients 12, suggesting that they are dimers or tetramers after disassembly TM. In Xenopus eggs (arrested in meiotic metaphase), lamin LIIIis also completely disassembled, but in contrast to mammalian lamins, sediments as a 9S moiety on sucrose gradients 19. The mitotic lamins of CHO cells become hyperphosphorylated (2-3 mol P/mol lamin) during disassembly in vivo 12,32 as well as in vitro34,41 and lose their phosphate upon reassembly, suggesting that phosphorylation may be a primary regulator of mitotic structural dynamics of the lamina. Interestingly, lamin hyperphosphorylation may be the manifestation of a putative phosphorylation cascade proposed to occur during prophase35,36 which could effect reorganization of many different cellular structures in a temporally coordinated fashion. The reversible disassembly of the lamina is postulated to regulate the disassembly and reformation of the nuclear envelope during mitosisS.1L According to this scheme, lamina disassembly during prophase provides a necessary (although not sufficient) condition for fragmentation of nuclear membranes into vesicles. Subsequent vesiculation of nuclear membranes presumably occurs by the same mechanisms that lead to generalized fragmentation of the endoplasmic reticulum in mitotic cells37. Conversely, reassembly of the lamina during telophase is proposed to mediate directly the assembly of membranes around chromosomes, with the membraneassociated lamin B serving to target nuclear membrane vesicles to the chromosome surfaces3. Since lamin B is probably the main nuclear envelope
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T I B S I1 - N o v e m b e r 1986
Fig. 4. Disassembly and reformation of the nuclear lamina during mitosis. Rat tissue culture cells were analysed by immunofluorescence microscopy with antibodies recognizing the lamins. During interphase (a), the lamins are localized exclusively in the nuclear envelope, resulting in a rim-like staining at the nuelear periphery. During prophase as the nuclear envelope is disassembled, the lamins gradually become dispersed throughout the cytoplasm. By prometaphase (b) they have a uniform cytoplasmic distribution and are not associated with the mitotic chromosomes (seen as the stain-excluding areas). The lamins remain disassembled until the nuclear envelope begins to reform during early telophase (c), when they start to associate with surfaces of the separated sister chromosomes (now fused into single masses). Periehromosomal lamin assembly is largely completed by late telophase (d), when a continuous nuclear envelope has been reconstructed. Nuclear rim staining is again apparent in early G1 when chromatin decondensation has occurred (e).
protein associated with disassembled nuclear membranes, a lamina-based assembly mechanism could ensure that minor nuclear membrane proteins are efficiently sorted to the nuclear envelope during telophase, Direct support for this model was recently obtained from studies with a cell-free nuclear assembly system, where selective immunological depletion of either lamins A and C or lamin B (and associated membranes) from the initial assembly mixtures resulted in extensive inhibition of membrane and pore cornplex assembly around chromosomes 24. In related studies, microinjection of antilamin antibodies into metaphase tissue culture cells, at concentrations that inhibited assembly of many of the lamins around chromosomes, prevented chromosome decondensation at the end of mitosis as. While inhibition of chromatin decondensation in this case m a y b e d u e indirectly to structurally abnormal or incomplete nuclear envelopes, this experiment nevertheless demonstrates that lamina assembly is important for normal nuclear reformation in telophase. Future analysis oflamina functions While there is good evidence that the lamina is important for regulating n u clear structure during mitosis, m o s t mechanistic aspects of this problem are poorly defined at present. Furthermore, little direct information is available o n the role of the lamina in organization of interphase chromatin and the pore complex. A combination of different approaches, particularly detailed biochemical analysis of the lamina, will be required to understand these problems in detail and to delineate additional functional properties of the lamina. The period of mitosis affords a particularly useful situation to study lamina functions as the nucleus is reversibly disassembled
during this period in higher eukaryotes. Recently, cell-free preparations of mitotic mammalian tissue culture cells 24,41 and S e n o p u s eggs 34,39,40 have been observed to support prophase-like nuclear disassembly and telophase-like nuclear reformation. In certain cases, these systems have also been demonstrated to reproduce closely in vivo events related to nuclear lamina dynamics 24,34,41. In the future these systerns should provide powerful tools for analysing the lamina and its structural regulation, as well as many other questions related to nuclear architecture.
Acknowledgements I am grateful to Doug Murphy and Ann Hubbard for helpful comments, and to Ueli Aebi and Alayne Senior for the micrographs in Figs 3 and 4, respectively. References 1 Fry, D. (1976) In Mammalian Cell Membranes (Vol. 2) (G. Jamieson and D. Robinson, eds) pp. 197-265, Butterworth 2 Franke, W., Scheer, U., Krohne, G. and Jarasch, E. (1981) J. Cell Biol. 91, 39s-50s 3 Gerace, L., Comeau, C. and Benson, M. (1984) J. Cell Sci. (Suppl. 1) 137-160 4 Krohne, G. and Benavente, R. (1986) Exp. CeURes. 162, 1-10 5 Gerace, L., Blum, A. and Blobel, G. (1978) J. Celt Biol. 79,546-566 6 Hancock, R. and Hughes, M. (1982) Biol. Cell 44,201-212 7 Lebkowski, J. and Laemmli, U. (1982)J. Mol. Biol. 156, 325-344 8 Fawcett, D. (1966) Am. J. Anat. 119, 129-146 9 Aaronson, R. and Blobel, G. (1975) Proc. Natl Acad. Sci. USA 72, 1007-1011 10 Dwyer, N. and Blobel, G. (1976) J. Cell Biol.
70,581-591 11 Scheer, U., Kartenbeck, J., Trendelenburg, M., Stadler, J. and Franke, W. (1976) J. Cell Biol. 69, 1-18 12 Gerace, L. and Blobel, G. (1980) Cell 19, 277287 13 Berezney, R. and Coffey, D. (1977) J, Cell Biol. 73, 6164537 14 McKeon, F., Kirschner, M. and Caput, D.
(1986) Nature 319,463-468 15 Fisher, D. Z., Chaudhary, N. and Blobel, G. (1986) Proc. Natl Acad. Sci. USA 83, 6450-
6454 16 Weber, K. and Geisler, N. (1985) Annals N Y Acad. Sci. 455,126-143 17 Steinert, P., Steven, A. and Roop, D. (1985) Cell 42, 411-419 18 Aebi, U., Cohn, J., Buhle, L. and Gerace, L. (1986) Nature323, 560-564
19 Benavente, R., Krohne, G. and Franke, W. (1985) Cell 41,177-190 20 Stick, R. and Hausen, P. (1985) Cell 41,191200 21 Stick,R. and Schwarz,H. (1983) Cell 33,949958 22 Shelton,
K., Higgins, L., Cochran, D.,
Ruffolo,J. and Egle, P. (1980)J. Biol. Chem. 255,10978--10983 23 Gerace, L., Ottaviano, Y. and Kondor-Koch, C. (1982) J. Cell Biol. 95,826~37 24 Gerace, L. and Blobel, G. (1982) Cold Spring Harbor Syrup. Quant. Biol. 46,967-978 25 Burke, B. and Gerace, L. (1986) Cell 44, 639652 26 Hochstrasser, M., Mathog, D., Gruenbaum, v., Saumweber,H. and Sedat, J. (1986) J. Cell Biol. 102, 112-123 27 82Bl°bel',8527~529G' (1985) Proc. Natl Acad. Sci. USA 28 McKeon, F., TuffaneUi, F., Kobayashi, S. and Kirschner, M. (1984) Cell 36, 83-92 29 Maul, G., Maul, H., Scogna, J., Lieberman, M., Stein, G., Hsu, B. and Borun, T. (1972) J. Cell Biol. 55,433-447 30 Lehner, C., Furstenberger, G., Eppenberger, H. andNigg, E.(1986)Proc. NatlSci. USA83, 2096-2099 31 Lalibertie, J.,Dagenais, A.,Filion, M.,BiborHardy, V., Simard, R. and Royal, A. (1984) J. Cell Biol. 96, 980-985 32 Ottaviano, Y. and Gerace, L. (1985) J. Biol. Chem. 260,624-632 33 Krohne, G., Franke, W., Ely, S., D'Arcy, A. and Jost, E. (1978) Cytobiologie 18, 22-38 34 Miake-Lye, R. and Kirschner, M. (1985) Cell 41,165-175 35 Wu, M. and Gerhart, J. (1980) Dev. Biol. 79, 465~77 36 Miake-Lye, R,, Newport, J. and Kirschner, M. (1983) J. Cell Biol. 97, 81-91 37 Warren, G. (1985) Trends Biochem. Sci. 10, 439-443 38 Benavente, R. a,d Klohne, G. J. Cell Biol. (in press) 39 Lohka, M. and Masui, Y. (1983) Science 220, 719-721 40 Lohka, M. and Mailer, J. (1985) J. Cell Biol. 101,518-523 41 Suprynowicz, F. and Gerace, L. J. Cell Biol. (in press)