Finite element analysis in 3-D models of equine cheek teeth.

Periodontal diseases occur frequently in equine dentistry and excessive strain in biological tissues is assumed to be a predisposing factor in their development. Finite element (FE) analysis enables strains and stresses occurring in the periodontium to be calculated and is a useful tool for testing this hypothesis. The current study aimed to establish reliable 3-D models from equine maxillary and mandibular cheek teeth for use in FE simulations, with particular attention to the detailed construction of the periodontal ligament (PDL). Age-related morphological aspects of the teeth and the periodontium were also considered by constructing different models for three defined age groups. Finally, the biomechanical behaviour of the equine PDL was examined during experimental tooth intrusion (i.e. axial displacement of the tooth into its alveolar socket). The results showed an increase in intrusion with increasing age due to age-related changes in the periodontal elastic properties. The general stress level in PDL and surrounding bone also increased with age. Reliable FE models were established to calculate aetiologically relevant biomechanical effects occurring in the equine periodontium.

Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization.

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil--forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent alpha-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of approximately 7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed.

Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis.

In eukaryotic cells, both soluble transport factors and components of the nuclear pore complex mediate protein and RNA trafficking between the nucleus and the cytoplasm. Here, we investigated whether caspases, the major execution system in apoptosis, target the nuclear pore or components of the nuclear transport machinery. Four nucleoporins, Nup153, RanBP2, Nup214 and Tpr are cleaved by caspases during apoptosis. In contrast, the nuclear transport factors, Ran, importin alpha and importin beta are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation. Also, mRNA accumulates into the nucleus before caspases become active. Microinjection experiments further revealed that early in apoptosis, the nucleus becomes permeable to dextran molecules of 70 kD molecular weight. Redistribution of import factors and mRNA, as well as nuclear permeabilisation, occur prior to caspase-mediated nucleoporin cleavage. Our findings suggest that the apoptotic programme includes modifications in the machinery responsible for nucleocytoplasmic transport, which are independent from caspase-mediated degradation of nuclear proteins.

Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex.

Tpr is a 267-kDa protein of unknown function recently identified as a constitutive component of the nuclear pore complex (NPC2)-attached intranuclear filaments. Secondary structure predictions suggest that the protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. To identify which of Tpr's molecular segments determine its specific intranuclear localization, we have constructed expression vectors encoding various Tpr deletion mutants as well as chimeric combinations of Tpr sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified a short region within Tpr's carboxyterminal domain that is essential and sufficient to mediate nuclear import of Tpr and which can also confer nuclear accumulation of pyruvate kinase. Tpr deletion mutants that contain this nuclear targeting segment, but lack the aminoterminal domain, appear evenly dispersed throughout the nucleus without any noticeable association to the NPC. In contrast, the aminoterminal domain lacking the carboxyterminal region remains located within the cytoplasm, forming aggregate-like structures not associated with the nuclear envelope. However, when tagged to Tpr's short nuclear targeting segment or to the nuclear localization signal of the SV40 large T protein, the aminoterminal domain is imported into the nucleus, where it then associates with the NPC. This association is mediated by shorter molecular segments within the aminoterminal domain which contain clusters of heptad repeats, whereas other regions are dispensable. This assignment of different topogenetic properties to distinct molecular segments of Tpr will now allow the design of future experiments to study the protein's structural properties further and determine its actual function.

Human RanBP3, a group of nuclear RanGTP binding proteins.

A group of novel human Ran-binding proteins, RanBP3, was identified using the yeast two-hybrid system via Ran-mediated interaction with the nucleotide exchange factor RCC1. Several open reading frames, representing putative alternatively spliced products, were established by cDNA cloning. Two of them, RanBP3-a and RanBP3-b, encode nuclear hydrophilic proteins of 499 and 562 amino acid residues. The sequences contain FXFG motifs, characteristic of a subgroup of nucleoporins, and a C-terminal domain showing similarity to the Ran-binding protein RanBP1. These proteins are localized in the nucleus, preferentially bind RanGTP and may be nuclear effectors of the Ran pathway.

Sequence analysis of a nuclear pore complex protein in a lower metazoan: nucleoporin p62 of the coelenterate Hydra vulgaris.

We have isolated cDNA clones and polymerase chain reaction products containing the entire coding region for nuclear pore complex protein p62 of a lower metazoan, the freshwater polyp Hydra vulgaris (Hv), and compared the deduced amino acid (aa) sequence with those of the vertebrate and yeast homologues. The open reading frame defines a protein of 534 aa, corresponding to a molecular mass of 56,072 Da and an isoelectric point (pI) of 5.0. Secondary structure predictions revealed the division into two domains, as previously observed in vertebrate p62: the N-terminal domain (aa 1-338) with a pI of 10.7 contains the evolutionarily conserved repeated pentapeptide motif, XFXFG, known from several nucleoporins, a low content of charged aa (3.25%) and a high degree of hydroxy aa (40.2%). Otherwise, sequence identity between the N-terminal domains of p62 from Hv and various vertebrates is rather low (28-34%). By contrast, the C-terminal domain with a pI of 4.6 is richer in charged aa (36.7%), exhibits heptad repeats typical for alpha-helices organized in coiled-coils and shows a high sequence identity with amphibian (53%) and mammalian p62 (55%). Differences and similarities between p62 of Hv and vertebrates, and between Hv p62 and its putative homologues from the budding yeast, Saccharomyces cerivisiae, and the fission yeast, Schizosaccharomyces pombe, are discussed.

Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments.

Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M(r) approximately 270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515-1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC-attached filaments and discuss its possible functions.

Mediators of nuclear protein import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.

Cytoplasmic annulate lamellae in cultured cells: composition, distribution, and mitotic behavior.

We have used antibodies against different proteins of the nuclear pore complex to identify cytoplasmic annulate lamellae by immunocytochemistry at the light- and electron-microscopic level in various lines of cultured mammalian cells. Although annulate lamellae (AL) are seldom observed in some cell lines, they occur in large numbers in other lineages, also with cell-type-specific size distributions. AL are especially abundant and prominent in African green monkey kidney epithelial cells of line RC37 and bovine mammary gland epithelial, cells of line BMGE. We have studied the distribution of AL in relation to other organelles by using double-label immunofluorescence microscopy and have demonstrated a significant relationship between AL and rough endoplasmic reticulum. We have further shown that AL are disassembled during prophase and reassembled at the end of mitosis, almost concomitantly with the disassembly and reassembly of the nuclear envelope. Cultured mammalian cells that are rich in AL can therefore be used as suitable models for studies of the biogenesis of these lamellae, which can conveniently be detected by immunofluorescence microscopy.

High content of a nuclear pore complex protein in cytoplasmic annulate lamellae of Xenopus oocytes.

The Xenopus laevis oocyte and egg represent an established model system to study nucleocytoplasmic transport and the assembly of the nuclear envelope (NE) and its pore complexes (PC). PCs, however, are not restricted to the NE but are also known to occur in cytoplasmic annulate lamellae (AL) in a variety of cells, including the Xenopus oocyte. However, the proportion of PCs found in such AL relative to those located in the NE, is unknown. In this study we have analyzed and quantified cytoplasmic AL in the full-grown (stage VI) Xenopus oocyte by immunolocalization at the light and electron microscopic level. Moreover, we have developed a method to enrich AL from enucleated oocytes, and have quantified a PC marker protein, nucleoporin p62, in both cytoplasmic AL and the NE. For this purpose we have used a specific monoclonal antibody (A225) which recognizes an epitope localized between amino acids 251 and 268 of Xenopus p62. We show that the number of PCs and p62 molecules present in AL far exceeds that of the NE. The possible implications of these findings to nucleocytoplasmic transport and nuclear PC (NPC) assembly are discussed.

Immunocytochemistry of annulate lamellae: potential cell biological markers for studies of cell differentiation and pathology.

We have generated specific antibodies recognizing different proteins of the nuclear pore complex and have applied them for the biochemical and microscopical analysis of annulate lamellae (AL). Using the example of the Xenopus laevis oocyte we show that the combined application of such antibodies to different components of the nuclear pore complex allows the unequivocal identification of AL. We propose to use such antibodies in immunostaining light microscopy for detecting AL as a diagnostic marker in a diversity of cell types, including those with pathological alterations in AL frequency and distribution.

Intranuclear filaments containing a nuclear pore complex protein.

Nuclear pore complexes (NPCs) are anchoring sites of intranuclear filaments of 3-6 nm diameter that are coaxially arranged on the perimeter of a cylinder and project into the nuclear interior for lengths varying in different kinds of cells. Using a specific monoclonal antibody we have found that a polypeptide of approximately 190 kD on SDS-PAGE, which appears to be identical to the recently described NPC protein "nup 153," is a general constituent of these intranuclear NPC-attached filaments in different types of cells from diverse species, including amphibian oocytes where these filaments are abundant and can be relatively long. We have further observed that during mitosis this filament protein transiently disassembles, resulting in a distinct soluble molecular entity of approximately 12.5 S, and then disperses over most of the cytoplasm. Similarly, the amphibian oocyte protein appears in a soluble form of approximately 16 S during meiotic metaphase and can be immunoprecipitated from egg cytoplasmic supernatants. We conclude that this NPC protein can assemble into a filamentous form at considerable distance from the nuclear envelope and discuss possible functions of these NPC-attached filaments, from a role as guidance structure involved in nucleocytoplasmic transport to a form of excess storage of NPC proteins in oocytes.

Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro.

Gp62 is a nuclear pore complex glycoprotein of vertebrates containing multiple O-linked N-acetylglucosamine monosaccharides. We have recently shown (Cordes et al., Eur. J. Cell Biol. 55, 31-47 (1991)) that gp62 of mouse and Xenopus laevis is predominantly glycosylated in the amino-terminal half of the molecule. Here we describe in vitro glycosylation in rabbit reticulocyte lysate of gp62 and of individual segments of this protein expressed as recombinant polypeptides in E. coli. Competition experiments between purified polypeptides revealed that gp62 exhibits in vitro at least two types of binding sites for a cytosolic N-acetylglucosaminyltransferase resulting in a sequential glycosylation of the protein. High affinity binding sites for this glycosyltransferase were located in the amino-terminal domain between amino acids 248-341 of mouse gp62, whereas low affinity binding sites were scattered in the entire amino-terminal half. The segment containing amino acids 248-341 was able to compete with in vitro O-glycosylation of gp62 subdomains containing low affinity binding sites. This gp62 segment was also able to compete with O-glycosylation of other unrelated cellular proteins in vitro.

Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region.

cDNA clones for nuclear pore complex glycoprotein p62 of two distantly related species, mouse and Xenopus laevis, were isolated. Antibodies raised against recombinant murine p62 react on protein blots with p62 of both species and decorate pore complexes. Analysis of the predicted protein sequence indicates that vertebrate p62 is organized into two structurally different regions. The entire carboxy-terminal half (86.7% identical amino acids) and the amino-terminal 56 amino acids (62.5% identity) have been highly conserved during evolution. The amino-terminal half contains several penta amino acid repeats and is able to form beta-sheets, whereas the carboxy-terminal half is predominantly organized in alpha-helical structures in part with heptad repeats typical for intermediate filament proteins. p62 of mouse and Xenopus is glycosylated by N-acetylglucosamine additions in the amino-terminal half. The region containing these potential glycosylation sites has been identified.