Günter Giese

Endocytic vesicles move at the tips of actin tails in cultured mast cells.

*Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK ‡Department of Pathology, Centre Medical Universitaire, Rue Michel-Servet 1, CH-1211 Geneva, Switzerland §Max Planck Institute für Medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany †Present address: Vollum Institute, 3181 Sam Jackson Park Road, Portland, Oregon 97201-3098, USA ¶e-mail: almersw@ohsu.edu

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

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)

Second harmonic generation imaging of collagen fibrils in cornea and sclera

Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation (SHG) imaging. SHG imaging revealed that corneal collagen fibrils are regularly packed as a polycrystalline lattice, accounting for the transparency of cornea. In contrast, scleral fibrils possess inhomogeneous, tubelike structures with thin hard shells, maintaining the high stiffness and elasticity of the sclera.

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Estradiol binding to cell surface raises cytosolic free calcium in T cells

The Fura‐2 method is used to examine a possible action of 17β‐estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose‐dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2‐induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2‐BSA‐FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2‐signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.

A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing

Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

4Pi-confocal imaging in fixed biological specimens.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100-140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

Mini−invasive corneal surgery and imaging with femtosecond lasers

Based on the transparency of corneal tissue and on laser plasma mediated non-thermal tissue ablation, near infrared femtosecond lasers are promising tools for minimally invasive intrastromal refractive surgery. Femtosecond lasers also enable novel nonlinear optical imaging methods like second harmonic corneal imaging. The microscopic effects of femtosecond laser intrastromal surgery were successfully visualized by using second harmonic corneal imaging with diffraction limited resolution, strong imaging contrast and large sensing depth, without requiring tissue fixation or sectioning. The performance of femtosecond laser intrastromal surgery proved to be precise, repeatable and predictable. It might be possible to integrate both surgical and probing functions into a single femtosecond laser system.