CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.     

Establishment and characterization of conditionally immortalized organ of corti cell lines

A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes. Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins.     

Cellular distribution of myosin-V in the guinea pig cochlea

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.     

The structural organization of the pathogenic protozoan Tritrichomonas foetus as seen in replicas of quick frozen,freeze-fractured and deep etched cells

Summary— The quick-freezing and freeze-etching technique was used to analyse the cytoskeleton of Tritrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle. The cytoplasm presented a network of filamentous structures interacting with each other, with the surface of the hydrogenosomes and the nuclear membrane. Two nm wide filamentous structures were found in the luminal space of the Golgi complex, connecting the two faces of each cisterna. The microtubules of the pelta-axostyle system were connected by bridges 30–40 nm long and 10 nm wide, regularly spaced with an interval of 25 nm. The costa is a structure formed by a complex array of filaments and globous structures. It seems to be connected to the recurrent flagellum through a complex network formed by 15 and 10 nm wide filaments which emerge from the peripheral region of the costa and penetrate into the surface projections of the protozoan body to which the recurrent flagellum is attached. Other filaments were seen connecting the surface of these projections with the surface of the flagellum.     

Morphological responses to calcium-induced interaction of phosphatidylserine-containing vesicles.

Structural changes in phospholipid vesicles made of dioleylphosphatidylethanolamine (DOPE)/bovine phosphatidylserine (PS) (1/1, 3/1, 10/1) or of egg phosphatidylcholine (PC)/PS (3/1) and exposed to calcium chloride for various times have been observed by means of video-enhanced light microscopy and freeze-fracture electron microscopy. Calcium induces the formation of large, smooth double-bilayer diaphragms as the spherical vesicles adhere to and deform each other. No subsequent changes are seen with PC/PS vesicles. DOPE/PS vesicles respond to the resultant stress, with about equal probability, by either fusing, through diaphragm rupture, or deflating, by way of volume loss through intact bilayers, even when they contain up to 400 mM sucrose. The diaphragm areas only rarely show the structural destabilization necessary for fusion. The final state is lipid segregated into DOPE hexagonal and Ca-PS lamellar bulk phases with the exclusion of most of the vesicle contents. Results with these and pure PS vesicles studied earlier indicate that the early response of vesicles to calcium chloride is determined by the competing rates at which mechanical stress (bilayer tension and intravesicular pressure) builds up as the vesicles adhere and flatten against each other, and is relieved by vesicle fusion or by volume loss. We attribute the qualitatively different responses of these three lipid systems to their measured differences in adhesion energies and consequent rate of build-up of mechanical stress. Yield to that stress for any one of these lipid systems is not a unique sequence of morphological changes, and so it remains obscure how such a stochastic process could be used in the controlled process of cellular fusion.     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

Membrane vesicles in magnetotactic bacteria

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured magnetotactic bacteria from a marine environment using transmission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magnetospirillum magnetotacticum bacteria. All uncultured magnetotactic bacteria presented membrane vesicles on their surface in addition to an extensive capsular material and an S-layer formed by particles arranged in a hexagonal symmetry. We did not observe any indication of electron-dense precipitation on the surface of these microorganisms. Our results indicate that membrane vesicles are a common characteristic of magneto-tactic bacteria in natural sediments.     

A Collagen Substrate Enhances the Sealing Capacity of Tight Junctions of A6 Cell Monolayers

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 × 10⁻⁷ S/cm for cells grown on collagen and 30.3 × 10⁻⁷ S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands. Received: 18 September 1996/Revised: 17 June 1997     

Cell shape and motility of oligodendrocytes cultured without neurons

Summary Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), were cultured from newborn rat brain and optic nerve to study how they differentiate in vitro in the absence of neurons. By use of galactocerebroside (GC) as a reference marker, the development of the cell phenotype was studied with video-enhanced differential interference contrast microscopy, immunofluorescence and electron microscopy. After a few days in culture, oligodendrocytes extend 5 to 10 main processes that are very rich in microtubules, but they did not stain with a monoclonal antibody reacting with all known classes of intermediate filaments. The number of processes can vary with the substrate on which the cells are grown; fewer processes form on laminin than on polylysine coated glass. Oligodendrocytes, in a fashion similar to that of neurons appear to keep their body immobile while the long processes grow. However, while neurons display motile activities mostly at the end of the cell processes called growth cones, the oligodendrocytes display motile, actin rich filopodia and lamellipodia along the entire length of all processes. The outgrowth of motile processes from oligodendrocytes sometimes occurs preferentially towards neighboring astrocytes. Oligodendrocyte processes display intense bidirectional movement of cytoplasmic organelles. Movement of surface components also occurs since GC molecules cross-linked by antibodies move from the processes towards the cell body. Thus, oligodendrocytes cultured without neurons develop on schedule a complex phenotype similar to their in vivo counterpart. In addition, their processes are capable of specific motile activities which may function in vivo to find the target axon and to transport myelin membrane components at the site of myelin assembly.Abbreviations (CNS) Central nervous system - (DIC) Differential interference contrast - (GC) Galactocerebroside - (GFA) protein Glial fibrillary acidic - (NSE) Neuron-specific enolase