The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

vertebrate lamin filaments in vitro. Therefore, we have used the oocyte system, where exogenously expressed somatic B-type and A-type lamins assemble into filaments. Expression of Btype lamins induces the formation of intranuclear membranes that are covered by single filament layers. LIII filaments appear identical to the endogenous lamina, whereas lamin B2 assembles into filaments that are organized less precisely. Lamin A induces sheets of thicker filaments on the endogenous lamina and significantly increases the rigidity of the nuclear envelope.

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Abstract. By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B~, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B, and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B, decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell-or tissue-specific differences in nuclear architecture.T He nuclear lamina is a filamentous meshwork closely apposed to the nucleoplasmic surface of the inner nuclear membrane (1; for review see reference 15). It is thought to serve a nucleoskeletal role important for nuclear envelope integrity (8,12,28) and interphase chromatin organization (4,14,17,22); this organization in turn may be important for DNA replication and differential gene expression (for references see 6, 9, 17). Lamin proteins have been found in a wide variety of organisms, including insects, amphibia, birds, and mammals (for review see reference 19). The three major mammalian lamins have been designated as lamins A, B, and C (12). Lamin B is implicated in anchoring the lamina to the nuclear membrane, whereas lamins A and C are thought to interact with chromatin (8,13,15). Based on cDNA sequence data (11, 27) as well as direct structural information obtained from electron microscopy (1), members of the lamin protein family were recently shown to constitute a novel class of (nuclear) intermediate filament proteins.With the aid of monoclonal antibodies we have begun to study the composition and assembly of the nuclear lamina in chicken (24,25). In particular, we have recently characterized three structurally distinct chicken proteins that we have termed lamins A, B~, and B2 (25). In chicken embryo R. Stick's present address is Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland.fibroblasts lamins A and B2 predominate, whereas lamin B1 represents a quanttafively minor component (25). By immunological criteria, chicken lamin B, is related to mammalian lamin B, whereas chicken lamin B2 is related to lamin A as well as to a quantitatively minor mammalian protein that had not previously been considered as...

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