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Wer als Zellbiologe hatte sich nicht schon immer gewunscht, mit seinem Lichtmikroskop feinste Details in der Zelle aufzulosen? Denn die Struktur einer biologischen Einheit sagt viel uber ihre Funktionsweise und Dynamik aus. Den Strich durch die Rechnung macht nicht die biologische Probe selbst, sondern ein physikalisches Gesetz, welches Ernst Abbe bereits 1873 formulierte [1]. Es besagt, dass die Auflosung eines Weitfeld Lichtmikroskops durch die Beugung fundamental begrenzt ist. Es gibt aber mittlerweile erfolgreiche Ansatze, das Mikroskop und die mikroskopische Abbildung so zu modifizieren, dass eine Auflosung jenseits des Abbeschen Gesetzes moglich wird. Eines dieser Verfahren ist die strukturierte Beleuchtungsmikroskopie (SIM; engl. „Structured Illumination Microscopy”).

Most processes in the eukaryotic cell nucleus are spatially and temporally regulated. To understand the dynamics in the underlying networks in quantitative terms necessitates the employment of technologies that can measure molecular concentrations, interactions and diffusion of proteins with high resolution. Using a combination of the available toolkit offers the potential to get a full picture of the dynamics of nuclear proteins in their most natural setting, the living cell.

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.

The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5′–3′-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5′–3′ direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5′–3′-exonucleolytic activities of the DNase and the polymerase-associated 3′–5′ exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3′–5′ exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.