3 years ago

Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study

Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study
A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5–6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.

Publisher URL: www.sciencedirect.com/science

DOI: S1084952117300149

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