QUANTITATIVE MEASUREMENT OF INTRAORGANELLE PH IN THE ENDOSOMAL LYSOSOMAL PATHWAY IN NEURONS BY USING RATIOMETRIC IMAGING WITH PYRANINE

Organelle acidification is an essential element of the endosomal-lysos omal pathway, but our understanding of the mechanisms underlying progr ession through this pathway has been hindered by the absence of adequa te methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately d etermining the pH of endocytic organelles in live cells. In this repor t, we demonstrate that the ratiometric fluorescent pH indicator 8-hydr oxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous avail able probe for such pH measurements. To measure intraorganelle pH, cel ls were labeled by endocytic uptake of HPTS, the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm ( F450/405) was calculated for each organelle, and ratios were converted to pH values by using standard curves for F450/405 vs. pH. Proper cal ibration is critical for accurate measurement of pH values: standard c urves generated in vitro yielded artifactually low organelle pH values . Calibration was unaffected by the use of culture medium buffered wit h various buffers or different cell types. By using this technique we show that both acidic and neutral endocytically derived organelles exi st in the axons of sympathetic neurons in different steady-state propo rtions than in the cell body. Furthermore, we demonstrate that these a xonal organelles have a bimodal pH distribution, indicating a rapid ac idification step in their maturation that reduces the average pH of a fraction of the organelles by 2 pH units while leaving few organelles of intermediate pH at steady state. Finally, we demonstrate a spatial gradient of organelle pH along axons, with the relative frequency of a cidic organelles increasing with proximity to the cell body.