Frank Wunderlich

Functional testosterone receptors in plasma membranes of T cells

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4+ and CD8+ subsets of T cells are directly revealed with the impeded ligand testosterone‐BSA‐FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone‐BSA conjugate induces a rapid rise (<5s) in[Ca2+]i of Fura‐2‐loaded T cells. This rise reflects influx of extracellular Ca2+ through non‐voltage‐gated and Ni2+‐blockable Ca2+ channels of the plasma membrane. The testosterone‐BSA‐induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti‐AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription‐polymerase chain reactions and Western blotting. AR can be visualized with the anti‐AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H‐R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.—Benten, W. P. M., Lieberherr, M., Giese, G., Wrehlke, C., Stamm, O., Sekeris, C. E., Mossmann, H., Wunderlich, F. Functional testosterone receptors in plasma membranes of T cells. FASEB J. 13, 123–133 (1999)

Membranes of Tetrahymena: III. The effect of temperature on membrane core structures and fatty acid composition of Tetrahymena cells

Abstract Membrane core structures as revealed by the freeze-etch electron microscopy and the fatty acid composition measured by gas-liquid chromatography have been analyzed in Tetrahymena cells exposed to low temperature for varying periods. When cells were grown to mid-log phase at the optimal growth temperature of 28 °C and then chilled to 10 °C, cell division was inhibited. However, within 16 h the cells adapted to the low temperature. Chilling effected drastic structural alterations in the cores of different membrane types (membranes of the pellicula, the alveolar sacs, the endoplasmic reticulum and the nuclei). In all cases, there was a segregation of smooth faces from particle-rich faces in the fracture planes. However, the native membrane state, i.e. like that of cells grown at 28 °C, reappeared when the cells adapted to the low temperature. The total lipids of Tetrahymena cells contained primarily even-numbered fatty acids ranging from C 12 to C 18 , but we also detected appreciable amounts of C 20 acids; this has not been reported before. During the initial phase of chilling, when cell division is inhibited, about 50% of the saturated fatty acids were replaced by unsaturated fatty acids, primarily monoenoic, dienoic and trienoic acids. We conclude that the structural recovery of the membranes in chilled Tetrahymena cells is accomplished by a desaturation of membrane fatty acids. This is discussed with respect to membrane “fluidity”.

Latrophilin-like receptor from the parasitic nematode Haemonchus contortus as target for the anthelmintic depsipeptide PF1022A.

PF1022A belongs to a new class of cyclodepsipeptides with broad anthelmintic activity. Here, we describe a novel target for PF1022A. Using PF1022A ligand immunoscreening of a cDNA library constructed from the parasitic nematode Haemonchus contortus, we identified a 3539 bp cDNA encoding a novel orphan heptahelical transmembrane 110 kDa‐receptor, termed HC110‐ R, similar to the mammalian G‐protein coupled receptor latrophilin. HC110‐R is localized at plasma membranes and in intracellular vesicles of HC110‐R‐transfected HEK‐293 cells. The ligand of latrophilin, a‐latrotoxin (LTX), binds to the extracellular N‐terminal region of HC110‐ R and induces influx of external Ca2+ through Cd2+‐ and nifedipine‐blockable Ca2+ channels. PF1022A also binds to the N‐terminus of HC110‐R and acts as an antagonist to LTX signaling in HC110‐R transfected HEK‐293 cells.

Differential effects of temperature on the nuclear and plasma membranes of lymphoid cells. A study by freeze-etch electron microscopy.

Abstract 1. 1. We have used freeze-fracture electron microscopy to examine the effects of cooling on the core ultrastructure of the plasma and nuclear membranes of normal thymocytes and lymph node cells, as well as concanavalin A-treated thymocytes and mouse lymphoma cells. 2. 2. Chilling below 22 °C produces smooth areas, free of intramembranous particles on both faces of both inner and outer nuclear membranes. This effect is reversible and can be prevented by glutaraldehyde fixation. 3. 3. Plasma membranes, in contrast to the nuclear membranes, exhibit no change in freeze-fracture morphology upon cooling. 4. 4. We hypothesize that the changes observed in the nuclear membranes represent thermotropic lipid phase transitions and that such transitions either do not occur in plasma membranes or are there constrained to very small regions.

Membranes in Tetrahymena: I. The cortical pattern

The membrane pattern of the Tetrahymena cortex is investigated using thin-sectioning and freeze-etching electron microscopy. The plasma and alveolar membranes are different with respect to their thin-sectioning and freeze-etching appearance. Freeze-etching reveals new structural elements, i.e. specific arrangements of particles differently sized and differently disposed in the pellicular, ciliary, and mucocyst membranes. These membrane-associated particles apparently correlated to specific organelles are discussed with respect to their functions.

Structural transformation of the nuclear matrix in situ

Abstract A striking difference in structure can be revealed between the nuclear matrix isolated from the macronuclei of normal Tetrahymena cells and the nuclear matrix of cells pretreated with 50 μg/ml actinomycin D for 2 h. The latter matrix-type shows residual giant fusion-nucleoli, which are formed in the macronuclei of actinomycin-treated cells. Both nuclear matrix types, however, exhibit an identical qualitative protein pattern in 15% SDS-gel electrophoretograms, with only minor changes in the staining properties of a few peptides. These data support the view that the nuclear matrix represents the residual equivalent to an in situ existing nuclear protein framework, which must be regarded not as a rigid, but rather as a dynamic flexible framework.

Influence of nuclear membrane lipid fluidity on nuclear RNA release

Abstract The temperature response of nuclear membrane lipid fluidity and nuclear RNA release is investigated in macronuclei isolated from Tetrahymena cells grown at 28 °C. Electron spin resonance (ESR) using 5-doxylstearic acid as a spin label detects that the lipid fluidity of nuclear membranes decreases, with falling temperatures, biphasically with a discontinuity at ~17 °C. In the same temperature range, a discontinuity occurs in the RNA release from [ 3 H]uridine-prelabelled macronuclei. Nuclei treated with 0.3% Triton X-100, however, show a linear decrease in RNA release upon temperature lowering. These findings are compatible with the view that the nuclear membrane lipid fluidity, inter alia, can modulate nucleocytoplasmic RNA-transport.

Structural transformation of the phagosomal membrane in Tetrahymena cells endocytosing latex beads

A model system with a high phagosomal membrane turnover has been developed: During a 45-min period Tetrahymena cells endocytoze 186 latex beads (diameter: 2.02 μm) per average cell; 166 of these beads are then exocytozed in the course of the following 145 min. During the endocytotic phase an average cell is approximated to fabricate 1200 μm2 phagosomal membrane. Freeze-etch electronmicroscopy reveals that both fracture faces of the nascent phagosomal membrane are associated with the typical 85 Å-particles in approximately equal numbers. Mature phagosomal membranes, however, show an unequal particle distribution. Smooth areas, smooth areas bordered with a fracture rim, and particle-associated depressions up to a diameter of 130 nm can be observed especially on fracture faces of mature phagosomes in the endocytotic phase. These are discussed with respect to membrane fusion.

Effect of Temperature on Nuclear Membranes and Nucleo-Cytoplasmic RNA-Transport in Tetrahymena Grown at Different Temperatures

SummaryThe effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined inTetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18°C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below ∼15 and ∼12°C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18°-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes ofTetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively “open” into a relatively “closed” state thus causing the observed decrease in RNA-transport.

The macronuclear envelope of Tetrahymena pyriformis GL in different physiological states: I. Quantitative structural data

Abstract The pore diameters and the pore frequencies, i.e. number of pores per square micron, of isolated nuclear envelopes were evaluated in four different physiological states of Tetrahymena pyriformis GL (1. logarithmic phase, 2. stationary phase, 3. heat-synchronized cultures at the end of the heat-synchronization treatment, and 4. heat-synchronized cultures at the division maximum). Evidence is presented that a correlation exists between the pore diameters and the pore frequencies: The smaller the average pore diameter of an envelope piece the higher is its pore frequency. As a consequence of this phenomenon there results a constancy of the ratio of pore area to the nuclear surface for all the four investigated different states. Whereas the pore diameters of the T. pyriformis macronucleus vary within the range reported for other cell material, the pore frequencies reach unusually high values up to 190 pores/μ 2 .