Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.

Fluorescence microscopy revolutionized cell biology and changed requirements for dyes towards higher brightness, novel capacities, and specific targets. With the need for multiplexing assays in high-throughput methodologies, surface staining gained particular interest because it allows rapid application of exogenous stains to track cellular identity in mixed populations. Indeed, the last decade has enriched the toolbox of general lipid stains, fluorescent lipid analogues, sugar-binding lectins, and protein-specific antibodies enabling the first rationally designed plasma membrane-specific dyes. Still, multiple challenges exist, and the unique properties of each dye must be considered when selecting a staining approach for a specific application. Recent advances are also promising that future dyes will provide ultimate brightness and photostability in diverse colors and reduced sizes for high-resolution imaging.