Studying Proton Gradients Across the Nuclear Envelope.

Methods in Molecular Biology
Raul Martinez-Zaguilan, Souad R Sennoune

Abstract

The existence of nuclear pore complexes in the nuclear envelope has led to the assumption that ions move freely from the cytosol into the nucleus, and that the molecular mechanisms at the plasma membrane that regulate cytosolic pH also regulate nuclear pH. Furthermore, studies to measure pH in the nucleus have produced contradictory results, since it has been found that the nuclear pH is either similar to the cytosol or more alkaline than the cytosol. However, most studies of nuclear pH have lacked the rigor needed to understand pH regulation in the nucleus. A major problem has been the lack of in situ titrations in the nucleus and cytosol, since the intracellular environment is different in the cytosol and nucleus and the behavior of fluorescent pH probes is different in these environments. Here we present a method that uses the fluorescence of SNARF-1 that labels both cytosol and nucleus. Using ratio imaging microscopy, regions of interest corresponding to the nucleus and cytosol to perform steady-state pH measurements followed by in situ titrations, to correctly assign pH in those cellular domains.

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