STED nanoscopy in living cells using Fluorogen Activating Proteins.

We demonstrate the effectiveness of a genetically encoded Malachite Green (MG) binding fluorogen activating protein (FAP) for live cell stimulated emission depletion nanoscopy (STED). Both extracellular and intracellular FAPs were tested in living cells using fluorogens with either membrane expressed FAP or as an intracellular FAP-actin fusion. Structures with FWHM of 110-122nm were observed.

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation.

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved.

Anatomy and dynamics of a supramolecular membrane protein cluster.

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules.

The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane.

In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required.