Review: the dynamics of the nuclear lamins during the cell cycle-- relationship between structure and function.

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.

Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

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 alpha, 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.

Pathway of incorporation of microinjected lamin A into the nuclear envelope.

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.

Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD.

A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.

Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins.

Four proteins of Mr approximately equal to 60,000, 65,000, 67,000, and 70,000 coisolate with the major intermediate filament (IF) structural proteins of BHK-21 cells. These proteins are keratin-like, they form distinctive paracrystals in vitro, and they are concentrated at the nuclear surface. Since these properties indicate similarities with the nuclear lamins, we have prepared conventional fractions of BHK-21 nuclei from which the same type of paracrystal is obtained. Furthermore, biochemical and immunological data demonstrate that the lamins are identical to the Mr 60,000-70,000 proteins found in IF preparations, and both sets of proteins are similar to keratin. These results suggest that an IF-like protein network is present in the nuclear lamina. We speculate that in some unknown way this network connects to the cytoplasmic IF network that courses from the juxtanuclear region to the cell surface. These proposed interconnecting networks may form part of the infrastructure of cytoskeletal-nuclear matrix-connecting links involved in signal transmission between the nuclear and cytoplasmic compartments of eukaryotic cells.

Intermediate filament networks: organization and possible functions of a diverse group of cytoskeletal elements.

Immunofluorescence and electron microscopic observations demonstrate that intermediate filaments (IF) form cytoplasmic networks between the nucleus and cell surface in several types of cultured cells. Intermediate filaments interact with the nuclear surface, where they appear to terminate at the level of the nuclear envelope. From this region, they radiate towards the cell surface where they are closely associated with the plasma membrane. On the basis of these patterns of IF organization, we suggest that IF represent a cytoskeletal system interconnecting the cell surface with the nucleus. Furthermore, IF also appear to interact with other cytoskeletal components including microtubules and microfilaments. In the former case microtubule-IF interactions are seen in cytoplasmic regions between the nucleus and the cell membrane, whereas microfilament-IF interactions occur in the cortical cytoplasm. IF also appear to be cross-linked to each other; especially in the case of the IF bundles that occur in epithelial cells. In order to determine the molecular and biochemical bases of the organizational state of IF we have developed procedures for obtaining IF-enriched 'cytoskeletons' of cultured cells. In these preparations IF-nuclear and IF-cell surface associations are retained. Thus, these preparations have enabled us to begin to study various IF-associated structures (e.g. desmosomes) and associated proteins (IFAPs) using biochemical and immunological methodologies. To date, the results support the idea that IF and their associated proteins may comprise the cell type specific molecular infrastructure that is involved in transmitting and distributing information amongst the major cellular domains; the cell surface/extracellular matrix, the cytoplasm and the nuclear surface/nuclear matrix.

A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells.

Native intermediate filament (IF) preparations from the baby hamster kidney fibroblastic cell line (BHK-21) contain a number of minor polypeptides in addition to the IF structural subunit proteins desmin, a 54,000-mol-wt protein, and vimentin, a 55,000-mol-wt protein. A monoclonal antibody was produced that reached exclusively with a high molecular weight (300,000) protein representative of these minor proteins. Immunological methods and comparative peptide mapping techniques demonstrated that the 300,000-mol-wt species was biochemically distinct from the 54,000- and 55,000-mol-wt proteins. Double-label immunofluorescence observations on spread BHK cells using this monoclonal antibody and a rabbit polyclonal antibody directed against the 54,000- and 55,000-mol-wt proteins showed that the 300,000-mol-wt species co-distributed with IF in a fibrous pattern. In cells treated with colchicine or those in the early stages of spreading, double-labeling with these antibodies revealed the co-existence of the respective antigens in the juxtanuclear cap of IF that is characteristic of cells in these physiological states. After colchicine removal, or in the late stages of cell spreading, the 300,00-mol-wt species and the IF subunits redistributed to their normal, highly coincident cytoplasmic patterns. Ultrastructural localization by the immunogold technique using the monoclonal antibody supported the light microscopic findings in that the 300,000-mol-wt species was associated with IF in the several physiological and morphological cell states investigated. The gold particle pattern was less intimately associated with IF than that defined by anti-54/55 and was one of non-uniform distribution along IF, being clustered primarily at points of proximity between IF, where an amorphous, proteinaceous material was often the labeled element. Occasionally, "bridges" of label were seen extending outward from such clusters on IF. Gold particles were infrequently bound to microtubules, microfilaments, or other cellular organelles, and when so, IF were usually contiguous. During multiple cycles of in vitro disassembly/assembly of the IF from native preparations, the 300,000-mol-wt protein remained in the fraction containing the 54,000- and 55,000-mol-wt structural subunits, whether the latter were in the soluble state or pelleted as formed filaments. In keeping with the nomenclature developed for the microtubule-associated proteins (MAPs), the acronym IFAP-300K (intermediate filament associated protein) is proposed for this molecule.

The organizational fate of intermediate filament networks in two epithelial cell types during mitosis.

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology.

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000-70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.

Practices, strategies, and motivations in treatment of rheumatoid arthritis.

National Analysts conducted primary research with rheumatologists--specifically, two panel discussions, 25 in-depth telephone interviews, and a mail survey of conference (Auranofin Symposium and Workshop) participants--to examine current treatment practices and to probe the rationale and motivations underlying treatment strategies in rheumatoid arthritis. The research identified important areas of consensus in drug perceptions, therapeutic approaches, and disagreements. Physicians differ regarding the minimum time they wait after diagnosing rheumatoid arthritis before initiating remittive therapy, some beginning immediately and others waiting six months or longer. Younger physicians are quicker to initiate remittive treatment than their older colleagues, but both younger and older practitioners are initiating remittive therapy earlier than in the past. Some noteworthy differences between hospital-based and office-based practitioners were discerned with respect to factors that figure in their decisions to initiate remittive therapy. Differences were also found among physicians in the way they pose drug options to their patients; "authoritarian," "libertarian," and "guided democracy" were names given to the three styles identified. In general, however, physicians report that patients are more directly involved in treatment selection than previously, a trend that may in part be due to the use of more aggressive treatment strategies than in the past and a desire to share the psychologic burden of those decisions. Findings suggest that gold compounds will continue to be a mainstay first-line disease-modifying agent in the treatment of rheumatoid arthritis but that there may be less reluctance to use other agents as physicians become increasingly familiar and comfortable with alternative options, especially penicillamine and immunosuppressive agents.

Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells.

We have shown, by indirect immunofluorescence microscopy using an antiserum against the mouse keratin subunit K2 and by electron microscopy, that transformed (PAM) and primary (PME) mouse epidermal cells possess extensive networks of IF bundles. Following trypsinization and replating of PAM cells, IF bundles are seen to move as a continuous network from a perinuclear zone into the peripheral cytoplasmic regions. In PAM cells lysed in high-ionic-strength solutions containing Triton X-100 and DNAase-1, IF bundles appear to be closely associated with nuclear envelope remnants and, in some cases, appear to be attached to nuclear pore complexes. PME cells cultivated in low Ca2+-containing medium possess perinuclear birefringent arrays of IF bundles. Within 2 hours of switching the cells to normal Ca2+ levels, the PME IF bundle network moves towards and establishes contact with the cell surface as desmosomes form. Live cells observed by phase contrast and fixed cells observed by immunofluorescence microscopy demonstrate that desmosomes can be distinguished as dark bands separating neighboring cells. There is little difference between the major proteins seen in SDS-polyacrylamide gel profiles of isolated IF bundle networks from PME cells before and after the Ca2+ switch. Therefore, a reorganization of relatively insoluble membrane-associated protein following the Ca2+ switch may be involved in desmosome formation. The isolated IF networks from PAM cells differ in protein composition compared to the PME IF networks. This may be related to the greatly reduced number of desmosomes in PAM cells. The IF bundle system in epidermal cells appears to be involved in shape formation, shape maintenance, the establishment of desmosomes, nuclear centration, and cell-cell contact.

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells.

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.