ORAI1, STIM1/2, and RYR1 shape subsecond Ca microdomains upon T cell activation.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca microdomains. Here, we identified a Ca entry component involved in Ca microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca microdomains required ORAI1, STIM1, and STIM2.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM.

Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling.

Red-mediated recombineering of Salmonella enterica genomes.

The mutagenesis of enterobacterial genomes using phage λ Red recombinase functions is a rapid and versatile experimental tool. In addition to the rapid generation of deletions in the genome of Salmonella enterica, variations of the method allow site-directed mutagenesis, generation of reporter fusions, generation of chimeric genes, or transplantation of regulatory elements directly in the chromosome. We describe the application of these approaches with focus on practical aspects and critical steps.