Ennio Lucio Benedetti

THE PLASMA MEMBRANES OF EYE LENS FIBRES. BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION

Abstract Plasma membranes have been isolated from calf eye lens fibre cells. The purified fraction is characterized by the occurrence of a large number of junctional complexes. The cholesterol phospholipid ratio is approximately 0.7, a value in between the values reported for erythrocyte ghosts and liver plasma membranes. Specific membrane protein components have been separated electrophoretically from structural lens proteins.

Association of crystallins with the plasma membrane

A number of newly synthesized structural lens polypeptides specifically associate in vitro with isolated lens plasma membranes. We show that membranes treated with protease causing cleavage of the major membrane protein MP26 are also able to interact with a few lens crystallins, in particular the acidic chains of α -crystallin α A 2 , the β h -characteristic polypeptide β B 1a and some cytoskeletal proteins. While protease-treated urea-washed membranes still interact with these proteins they lose the ability to associate with the membrane protein MP34 synthesized de novo.

Changes in membrane protein pattern in relation to lens cell differentiation

In previous papers we have described that the protein pattern of isolated lens fiber membranes is characterized by a number of polypeptides in different proportions [l-4] . Among these proteins two components with molecular weight of about 26 000 and 34 000 (designated as MP26 and MP34, respectively [2] ) become predominant in the protein pattern of a membrane subfraction, enriched in communicating junctions [3] . This type of junctions which connects the lens fibers is assembled in the elongation zone where the epithelial cells differentiate into the lens fibers [4,5] . Moreover, recent observations revealed that polyribosomes derived from the cortical part of the eye lens are able to direct the biosynthesis of membrane proteins even in heterologous cell-free systems [6] . Under special conditions similar results can be obtained with messenger RNA isolated from these polyribosomes [7] . The aim of the present study was to prepare purified plasma membranes from calf lens epithelium and to establish whether the diffmentiation of lens epithelial cells into fibers is accompanied by the appearance of membrane protein(s) differing from those in the epithelial plasma membranes.

SV40-transformed hamster lens epithelial cells: a novel system for the isolation of cytoskeletal messenger RNAs and their translation products.

Epithelial cells from Syrian hamster lens were cultured and transformed by Simian Virus 40 (SV40). This cell line can be brought into suspension culture and then grows very efficiently. The quantitatively major proteins synthesized by these cells appeared to be the cytoskeletal constituents actin and vimentin. Morphologically the occurrence of the two proteins in the cultured cells is reflected by the presence of actin filaments and intermediate-sized filaments. This was demonstrated by immunofluorescence studies and electron microscopy. A messenger RNA fraction that was isolated from the transformed cells directed almost exclusively the synthesis of actin and vimentin after incubation in a heterologous cell-free system.

The organization of the plasma membrane in mammalian cells.

Abstract The plasma membrane in animal cells consists of a lipid bilayer and of particulate protein-glycoprotein entities which may span the membrane width. The fluidity of the lipid phase and other factors may favor the motion and the rearrangement of the particulate entities. The association within the liquid-like lipid phase of identical or quasi-equivalently related proteins might result either in oligomeric complexes or in bidimensional lattices of repeating particles.

Is the cytoskeleton-plasma membrane complex involved in lens protein biosynthesis?

Calf lens fiber cells contain a population of polyribosomes that direct, at leastin vitro, the synthesis of a specific plasma membrane protein MP26. This protein may serve as a marker in terminal differentiation, since it is absent in the lens epithelium but appears in lens fiber plasma membranes. The MP26 manufacturing polyribosomes are found to be associated with a structural complex in which also the cytoskeleton and plasma membranes participate. They can be released from the complex by treatment with DNAse I. This result presumably reflects the involvement of actin in the linkage of the MP26 synthesizing polyribosomes to the cytoskeleton-membrane complex.

The structural organization of calf retinal photoreceptors derived from freeze-fracture study

Freeze-fracturing and double replica technique provided some evidence that the membranes of the outer segment of calf retina are asymmetric. The intramembranous particles, probably consisting of rhodopsin, exposed by the fracture, are apparently more strongly anchored to one half of the bilayer. These results are consistent with the molecular model of disc membrane suggesting that rhodopsin is associated only with one side of the membrane.

Caveolins and flotillin-2 are present in the blood stages of Plasmodium vivax

Blood stages of Plasmodium vivax induce the development of caveolae and caveola–vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1–3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.

Towards a human crystallin map - Two-dimensional gel electrophoresis and computer analysis of water-soluble crystallins from normal and cataractous human lenses

This paper describes a first approach to establish a master data base of human lens crystallins obtained by computer analysis of standardized two-dimensional lenticular protein patterns. To facilitate the eventual identification of the spots, the major crystallins have been separated into alpha-, beta H-, beta L- and gamma-crystallin fractions by gel filtration. The authors encourage colleague investigators to collaborate in a common effort in order to arrive eventually at a two-dimensional gel data base of all lenticular proteins.

The lens cytoskeleton : intermediate-sized filaments, their biosynthesis and association with plasma membranes

Lens plasma membranes isolated by sucrose density gradient centrifugation in buffers containing K+-and Mg+-ions (cytoskeleton-stabilizing conditions) contain an appreciable amount of intermediate-sized filaments (I F) comprising vimentin as protein subunit. Electron microscopic features of this plasma membrane cytoskeleton complex are suggestive for an intimate association between the IF and the lipid bilayer. On the electron micrograph (Fig. !) the IF can be seen running in different directions. They show frequently end-on attachment to the membrane profile. The close association between membrane leaflets and the filaments is even more impressively demonstrated by freeze-fracture aspects of the complex (1). The latter technique reveals that rodlike structures (IF) are linked to the protoplasmic membrane fracture face. Further evidence for this association is provided by the insertion of newly synthesized vimentin into membranes added to a reticulocyte lysate which has