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The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy.

Cell volume regulation is essential for survival of animal cells. The regulatory volume decrease (RVD) and increase (RVI) are accomplished by water movement driven by KCl efflux and NaCl influx, respectively, mediated by a number of Cl −, K + and cation channels. Persistent cell shrinkage and swelling are major hallmarks of apoptotic and necrotic cell death, respectively, and caused by the apoptotic volume decrease (AVD) and necrotic volume increase (NVI) coupled to impairment of RVI and RVD, respectively. Since the AVD and NVI processes also involve activities of Cl −, K + and cation channels, it is likely that these volume-related ion channels (such as volume-sensitive outwardly rectifying anion channel (VSOR), CFTR, hypertonicity-induced cation channel (HICC), acid-sensitive outwardly rectifying anion channel (ASOR) and TRPM7) play roles in the cell survival-death switching. In fact, our studies in vitro showed that apoptotic and necrotic cell death is induced or rescued by controlling activities of these volume-related ion channels under a variety of conditions, including apoptotic stimulation, excitotoxicity, acidotoxicity, lactacidosis and hypoxia-reoxygenation. Also, ischemia-reperfusion-induced neuronal apoptosis and cardiac necrosis in vivo were found to be rescued by controlling volume-related anion channel activities. Among these volume-related ion channels, the molecular entities of VSOR and ASOR remain elusive. Although TMEM16F (ANO6) and ClC-3 were proposed as the molecular candidates for VSOR and ASOR, respectively, our recent studies excluded these possibilities and showed that they function as distinct-typed Ca 2+-activated and outwardly rectifying chloride channels (CaCC and ORCC), respectively [1–3].

Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Also, the activation pattern of HICCs was not defined yet. We report that in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr, as activators of TRPM2, elicit cation currents that are identical to the osmotic activation of HICCs. Silencing of TRPM2 and of the ecto -enzyme CD38 (as the supposed source of ADPr and cADPr) inhibit HICCand nucleotide-induced currents as well as the osmotic volume response of cells. Quantification of intracellular cADPr and extracellular application of nucleotides reveal, however, that it is the outwardly directed gradient rather than the intracellular activity of ADPr and cADPr triggering TRPM2 and, very likely, this export goes together with a biotransformation of nucleotides. Cloning of TRPM2 identifies the ΔC-splice variant as molecular correlate of the HICC, which is supported by quantification of its Ca 2 + selectivity. Finally, pull-down and FRET/FLIM experiments reveal a close proximity of TRPM2 and CD38 and we propose a transport related nucleotide export via CD38 as novel mechanism of TRPM2 activation.

Hydration plays a key role in cell physiology. We defined the hydration profiles of HeLa and HepG2 cells as a function of hypertonicity and temperature, at high time and spatial resolution using Fluorescence Correlation Spectroscopy (FCS) and Raman Scanning Microscopy (RSM) and the underlying activation energies were determined. With FCS we quantified (1), the diffusion speed and the number of diffusing GFP particles in the cytoplasm and (2), the correlating changes in cell viscosity, under hypertonic conditions. Both parameters were correlated to each other and exhibited a clear dependence on the osmolarity. RSM, on the other hand, allowed us to monitor directly the absolute amount of cell water and to map the hydration of a probe at high spatial resolution. This was instrumental in verifying the FCS measurements and, furthermore, the actual amount of bound water inside the cell could be computed, in a label-free fashion. Our measurements prove the robustness of the cell volume regulatory machinery and define the energy profiles of its activation over a wide osmolarity and temperature range.

During cryo-preservation, cells loose considerable amounts of water and they partial regain volumes when brought back from the cold. Here we applied different levels of hypertonic stress as well as cryo-conditions (–50 °C) to perturb the hydration (volume) of HepG2 cells and to define the activation energies (E as) of aquaporins (AQPs) and HICCs using scanning acoustic microscopy and whole-cell patch-clamp recordings. The E as of regulatory volume increase (RVI) and HICCs showed a defined osmotic profile, whereas activation of AQPs did not depend on hypertonicity. The E as of RVI were significantly decreased after cryo-preservation indicative of a higher capability of cells to regain osmolytes and water, very likely through a stimulation of cation uptake. In sharp contrast, the E as of AQPs were not significantly changed after cryo-preservation, implying a rather permissive role of water channels in the process.

This paper presents and describes the interactions occurring in polyvinyl-alcohol-containing alumina suspensions containing different multivalent cations. The influence of Mg (+II), Ca (+II), Al (+III) and Zr (+IV) cations was studied systematically by means of surface potential, viscosity, sedimentation and adsorption measurement techniques. The results were compared with the results obtained for a monovalent reference (K (+I)). Whereas for Mg (+II), Ca (+II) and Al (+III) the interactions can be explained by compression of the electric double layer or formation of hydroxides, Zr (+IV) shows a unique affinity for polyvinyl alcohol. This interaction yields a significant improvement in suspension flowability and sedimentation stability. Moreover this specific interaction is independent of t he solid surface. Thus the stabilizing effect of polyvinyl alcohol/Zr (+IV) can be combined with the effect of a polyacrylate dispersant.

Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits alpha, beta, and gamma of the epithelial Na+ channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 -> 400 mosM) increased membrane conductance from 75.4 +/- 9.4 to 91.1 +/- 11.2 pS (p

aus der Einführung: 'Virtuelle Lernumgebungen ermöglichen Aus- und Weiterbildung unabhängig von Zeit und Raum, z.B. können Berufstätige während wie auch nach der Arbeitszeit Kurse über ihren PC mit Internetzugang absolvieren und dabei verteilte Lerngruppen und Projektteams bilden. Eine Vielzahl anderer Szenerien virtueller Lernumgebungen sind denkbar. Faktoren, die diese Entwicklung massiv unterstützen, sind der Verbreitungsgrad multimedialer PCs - ca. 7 Mio. Personen über 14 Jahren hatten 1998 in Deutschland beruflich oder privat Zugriff auf Onlinedienste bzw. das Internet - und insbesondere die Dynamik, mit der sich das Wissen in bestimmten Fachgebieten verändert und damit ständige berufliche Weiterbildung erfordert. Die Informations- und Multimediatechnik sind hierfür typische Beispiele. Dieser Entwicklung kann mit klassischen Lehrmethoden und -medien, z.B. Fortbildungsseminaren und dem Lehrbuch, nicht adäquat Rechnung getragen werden. Virtuelle Lernumgebungen bieten deshalb eine zeitgemäße Möglichkeit der kostengünstigen und effizienten Aus- und Weiterbildung.

$\underline{Methods:}$ 1) The voltage-dependent inward current in Paramecium caudatum was studied under voltage clamp-conditions in the presence of Ca$^{++}$, Sr$^{++}$, Ba$^{++}$, M$^{++}$, Co$^{++}$, Mn$^{++}$ and Ni$^{++}$. 2) In contrast to the conventional technique the cells were impaled from above while they were observed from below. In this way straight electrodes could be used that are easier to pre pare than bent electrodes conventionally used. 3) The time required to set up a certain membrane potential could be reduced to 300$\mu$s by coating the electrode-tips and by shielding the sensor-electrode. 4) By depolarizing prepulses that inactivate the Ca$^{++}$-channel and by a subtraction-procedure the Ca$^{++}$- and K$^{+}$-currents duringexcitation could be separated (double pulse-technique). 5) In 'substitution-experiments' Ca$^{++}$ in the outer medium was exchanged by the tested cation. In 'addition-experiments' the Ca$^{++}$-concentration was kept constant while the tested cationwas added. [...]