Lars-Göran Öfverstedt

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

Nephrin Promotes Cell-Cell Adhesion through Homophilic Interactions

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+)concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.

The Human Skin Barrier Is Organized as Stacked Bilayers of Fully Extended Ceramides with Cholesterol Molecules Associated with the Ceramide Sphingoid Moiety

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skins surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skins lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skins low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barriers robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.

Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits

Structures in situ of individual ribosomes in E. coli have been determined by computer-aided electron microscope tomography using a tilt series of positively stained embedded cellular sections. Amino acid starvation of a bacterial culture, causing a deficiency for aminoacyl-tRNA, induces a spatial separation between the ribosomal subunits compared with ribosomes in exponentially growing cells. Eight ribosomes from each growth condition were aligned to each other, and the two average structures were determined. Comparison of these suggests that the distance between the two subunits increases by approximately 3 nm upon starvation for aminoacyl-tRNA during protein synthesis. Ribosomes in most other states of the translational elongation cycle in exponentially growing cells show a more compact structure than previously realized.

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Structural analyses reveal that oligomerization between cell adhesion molecules in the same membrane is influenced by their interactions across opposing membranes (see also in this issue the accompanying paper by Müller et al.).

Procedures for Three-Dimensional Reconstruction from Thin Sections with Electron Tomography

Electron tomography (ET) is a method for three-dimensional (3-D) reconstruction of single, transparent objects from a series of images (i.e., a tilt series) recorded with a transmission electron microscope (EM) (Fig. 4.1). The method is related to the procedures used in medical tomography. The 3-D reconstructions are usually computed from the digitized tilt series after a radial weighting scheme has been applied to the Fourier-transformed data. The ET method can be used to reconstruct in 3-D any object that is transparent enough for projection imaging with the transmission EM. This means that specimens of biological origin are usually available for ET 3-D reconstruction, whereas, e.g., colloidal gold particles are not. An ET-calculated 3-D map could be based on projections from objects that have been visualized by EM in several different ways, i.e., from stained or unstained objects, objects visualized at different energy loss levels or objects embedded in different media. The combined analysis of the 3-D structure, imaged in different ways, could thus become very informative. The general applicability also means that it is not restricted to symmetrical or regularly arranged objects, nor to objects with preferred orientations on a support grid. In its present state, the ET method allows reproducible 3-D reconstructions of single molecular objects, with a resolution in the range of 5 nm, of complex cellular specimens. For isolated objects, free of interfering cellular substances, a somewhat higher resolution can be achieved. The ET method covers the intermediate resolution range where there is no other physical technique available to analyze single molecular complexes.

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

Using a fuzzy framework for delineation and decomposition of Immunoglobulin G in cryo electron tomographic images

In structural studies of proteins, the first task is to identify the different parts of the protein. We present a robust method using a fuzzy framework for delineating a protein and to identify its parts. The method is used in a study of the immunoglobulin G antibody, individually imaged using cryo electron tomography, with satisfactory results

Analysis of the Assembly of the HIV Core by Electron Microscope Tomography

Both the proteolytic processing of the p55 gag precursor protein of HIV, myristilation of the p17 (MA) protein, and their morphological rearrangement are necessary to generate structurally mature virus particles (Dahlberg, 1988; Haseltine, 1989). This sequence of events would parallell the observations with e.g. Friend murine oncoviruses, also indicating that a late stage of virus maturation would be accompanied by the production of infectious virus (Katoh et al., 1985).

Three-dimensional structure of outer hair cell pillars

Conclusions. Electron tomography was used to generate three-dimensional reconstructions of the pillars that connect the cell membrane with the cytoskeleton of the outer hair cell. Results are consistent with the hypothesis that pillars are important for mechanically linking the membrane with the cytoskeleton. Objective: To make a qualitative assessment of the morphology of the sub-membrane pillars of cochlear outer hair cells. Materials and methods. Guinea pig cochleae were fixed and prepared for electron microscopy using protocols described previously. Sections were imaged on an electron microscope equipped with a goniometer. The specimens were tilted through a range of 120°, and an image was acquired at each tilt angle. Filtered back-projection was used to generate three-dimensional reconstructions. Results. Twelve individual pillars were successfully reconstructed. Pillars often connect to the cell membrane through a thin segment, and to the cytoskeleton through a forking structure that may form a central cavity.