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 skin's surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skin's 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 skin's low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barrier's robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.

The polypeptide growth factor, hepatocyte growth factor͞scatter factor (HGF͞SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF͞SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryoelectron microscopy, we show that conversion of pro(single-chain)-HGF͞SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF͞SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF͞SF contact the face of the seven-blade ␤-propeller domain of MET harboring the loops connecting the ␤-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite ''b'' face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF͞SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF͞SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF͞SF-MET signaling.cell signaling ͉ plasminogen ͉ serine proteinases ͉ kringle ͉ x-ray scattering H epatocyte growth factor͞scatter factor (HGF͞SF) (1-6) are vertebrate-specific polypeptide growth factors with a domain structure related to that of plasminogen (7). Interest in HGF͞SF and its receptor MET (8) stems from unique biological roles in embryogenesis (9-11), tissue regeneration (12, 13), and cancer (14). These activities have led to a strong interest in the structure of the molecules as this knowledge may underpin the development of MET-based therapeutics.HGF͞SF consists of six domains: an N-terminal domain (n), four copies of the kringle domain (k1-k4), and a C-terminal domain (sp) structurally related to the catalytic domain of serine proteinases (Fig. 1A). The factor is synthesized as a precursor protein (pro-or single-chain HGF͞SF) and is proteolytically processed to a two-chain form by cleavage of the linker connecting the k4 and sp domains ( Fig. 1 A and B). Single-chain HGF͞SF binds MET (15, 16) but is unable to induce biological responses, for example, dispersion of MDCK cell colonies, even at concentrations 100-fold higher than two-chain HGF͞SF (Fig. 1 C-E).MET is also synthesized as a single-chain precursor that is cleaved by furin yielding an N-terminal ␣-chain and a C-terminal ␤-chain. The MET ectodomain consists of two moieties: the large, N-terminal sema domain, which is responsible f...

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.

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. Podocyte foot processes cover the outer aspect of the glomerular capillaries in an interdigitating manner. The slit between adjacent foot processes contains a highly ordered thin structure referred to as slit diaphragm (SD).The SD is thought to act as the ultimate albumin-excluding ultrafilter critical in the formation of primary urine.

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.