Ueli Aebi

Toward a more complete 3-D structure of the nuclear pore complex

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent knobs that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a fishtrap. These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.

Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope.

Lamina‐associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane, which binds directly to both lamin B1 and chromosomes in a mitotic phosphorylation‐regulated manner. The biochemical and physiological properties of LAP2 suggest an important role in nuclear envelope re‐assembly at the end of mitosis and/or anchoring of the nuclear lamina and interphase chromosomes to the nuclear envelope. We describe the cDNA cloning of LAP2 and characterization of its membrane topology and targeting to the nuclear envelope. The LAP2 cDNA sequence predicts a protein of 452 amino acids, containing a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane‐spanning sequence at residues 410‐433. Immunogold localization of an LAP2 epitope in isolated nuclear envelopes indicates that the large amino‐terminal hydrophilic domain (residues 1‐409) is exposed to the nucleoplasm. By expressing deletion mutants of LAP2 in cultured cells, we have identified multiple regions in its nucleoplasmic domain that promote localization at the nuclear envelope. These data suggest that targeting of LAP2 to the nuclear envelope is mediated by cooperative interactions with multiple binding sites at the inner nuclear membrane.

Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase

The nuclear lamina is an intermediate filament‐type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head‐to‐tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head‐to‐tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N‐ and C‐terminal to the central alpha‐helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head‐to‐tail polymers. The relative importance of N‐ and C‐terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C‐terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N‐terminal site (Ser16‐‐‐‐Ala) rendered head‐to‐tail polymers resistant to disassembly. These results emphasize the importance of the N‐terminal end domain for lamin head‐to‐tail polymerization in vitro, and they demonstrate that phosphorylation‐dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.

Molecular Dissection of the Nuclear Pore Complex

The nuclear pore complex (NPC) is an approximately 120 megadalton (MDa) supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that mediates bidirectional molecular trafficking between the cytoplasm and the nucleus of interphase eukaryotic cells. The structure of the NPC has been studied extensively by electron microscopy (EM), and a consensus model of its basic framework has emerged. Over the past few years, there has been significant progress in dissecting the molecular constituents of the NPC and in identifying distinct NPC subcomplexes. The combination of well-characterized antibodies with different EM specimen preparation methods has allowed localization of several of these proteins within the three-dimensional (3-D) architecture of the NPC. Thus, the molecular dissection of the NPC is definitely on its way to being elucidated. Here, we review these findings and discuss the emerging structural concepts.

Making heads and tails of intermediate filament assembly, dynamics and networks.

Thus far, intermediate filaments (IFs) have been the least understood of the three cytoskeletal filament systems with regard to their structure, assembly, network formation, and dynamics. This picture is now slowly but definitely changing, as recent in vivo and in vitro experiments, including generation of transgenic animals, have yielded important new data shedding light on the following areas: the molecular architecture of IFs; the role of the highly variable end domains during IF assembly and network formation; the factors that govern whether IF proteins are involved in de novo filament formation or are incorporated into a pre-existing IF network; and the effects of post-translational modifications, such as phosphorylation and glycosylation of IF polypeptides, on filament assembly, dynamics and turnover.

Toward the molecular dissection of protein import into nuclei

Transport of proteins, RNAs and ribonucleoprotein particles into and out of the nucleus is essential for many cellular functions to proceed. Recent progress in this area of research has led to the identification of a number of signals and cytosolic factors that mediate the nuclear import of proteins through the nuclear pore complexes. However, as the sites on the nuclear pore complex at which these signals and factors exert their function are still largely unidentified, the molecular mechanisms underlying this nuclear import pathway remain to be elucidated.

The role of the head and tail domain in lamin structure and assembly: Analysis of bacterially expressed chicken Lamin A and truncated B2 lamins

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered headless, tailless, and rod chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.

Has negative staining still a place in biomacromolecular electron microscopy

Transmission electron microscopy of proteins has provided molecular- and in a few cases near-atomic-resolution structural information. In this review, we critically evaluate the potential and the limitations in obtaining molecular resolution, particularly with negatively stained specimens, and put these into perspective with cryomicroscopy of unstained frozen-hydrated and sugar-embedded preparations.

Polymorphism of reconstituted human epidermal keratin filaments: Determination of their mass-per-length and width by scanning transmission electron microscopy (STEM)

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded protofibrils (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.

Studies of the structure of the T4 bacteriophage tail sheath. I. The recovery of three-dimensional structural information from the extended sheath.

Abstract The extended tail sheath of bacteriophage T4 has been used to study the transfer of information from an electron micrograph to the three-dimensional reconstruction obtained from it. Two methods have been developed to assess micrograph images of helical particles and their reconstructions. First, a filter has been designed which eliminates all structure in the image inconsistent with the symmetry and assumed radius of the helical particle. Individual micrographs can therefore be assessed with respect to their consistency with the assumed symmetry and radius, before reconstruction. Second, a map of the root-meansquare deviation of individual reconstructions from their average provides a quantitative measure of the consistency of the individual sets of tail data and allows the regions in the average reconstruction which are most sensitive to differences between the particles to be identified. The averaged reconstruction is used to examine the problems related to resolution and reproducibility of the structural information and to define the extent of the different components of the extended sheath.