Biology subjects

Brand, G.

Publications and source records attributed to Brand, G..

2 recordsLinked to original sources

bioRxiv2019

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

ABSRACTTracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques such as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) was measured and characterized. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ~30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in subregions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live-cell labeling approaches.

biophysics↗

bioRxiv2019

Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

While the role of Synaptotagmin-1 in living cells has been described in detail, it remains a challenge to dissect the contribution of membrane remodelling by its two cytoplasmic C2 domains (C2AB) to the Ca2+-secretion coupling mechanism. Here, we study membrane remodeling using pairs of optically-trapped beads coated with SNARE-free synthetic membranes. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. A lipid mixing assay with confocal imaging reveals that at low Syt1 concentrations, no hemifusion is observed. Notably, for similar low concentrations of Doc2b hemifusion does occur. Consistently, both C2AB fragments cause a reduction in the membrane bending modulus, as measured by an AFM-based method. This lowering of the energy required for membrane deformation likely contributes to the overall Ca2+-secretion triggering mechanism by calcium sensor proteins. When comparing symmetrical (both sides) and asymmetrical (one side) presence of protein on the membranes, Syt1 favors an asymmetrical but Doc2b a symmetrical configuration, as inferred from higher tether probabilities and break forces. This provides support for the direct bridging hypothesis for Syt-1, while hinting to possible preference for protein-protein (and not protein-membrane) interactions for Doc2b. Overall, our study sheds new light on the mechanism of Ca2+ induced fusion triggering, which is essential for fundamental understanding of secretion of neurotransmitters and endocrine substances.

biophysics↗