Bruns, D. | Search bioRxiv

bioRxiv2024

Multicore-fiber microendoscopy for functional cellular in-organ imaging

Microendoscopy enables minimally invasive investigations of organs even within small cavities. Conventional microendoscopy is limited by probe size and often restricted to a single excitation wavelength. We developed and characterized a multichannel microendoscope as thin as 360 {micro}m and recorded functional cellular signals in-situ using custom written software for image processing. The endoscope had an effective resolution of 4.64 {micro}m and resolved subcellular structures of neurons. The system enabled analysis of in-situ calcium responses in murine tracheal brush cells and kidney podocytes. Additionally, ratiometric redox responses were recorded in whole, explanted organs and pancreatic islet culture. The flexibility and simplicity of our approach for imaging a variety of tissues and organs paves the way for in-vivo, longitudinal studies with cellular resolution.

bioengineering↗

bioRxiv2023

Key determinants of the dual clamp/activator function of Complexin

Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115- 134) binds to fusion-promoting SNARE proteins, prevents premature secretion and allows vesicles to accumulate in a release-ready state. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the CTD of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the prefusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1-27) specifically cooperates with synaptotagmin I (SytI), but not with synaptotagmin VII. Expression of CpxII rescues the slow release kinetics of the Ca2+- binding mutant Syt I R233Q, whereas the N-terminally truncated variant of CpxII further delays it. These results indicate that the CpxII NTD regulates mechanisms which are governed by the forward rate of Ca2+ binding to Syt I. Overall, our results shed new light on key molecular properties of CpxII that hinder premature exocytosis and accelerate synchronous exocytosis.

neuroscience↗