Overly C C, Lee K D, Berthiaume E, Hollenbeck P J
Department of Neurobiology, Harvard Medical School, MA 02115, USA.
Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3156-60. doi: 10.1073/pnas.92.8.3156.
Organelle acidification is an essential element of the endosomal-lysosomal pathway, but our understanding of the mechanisms underlying progression through this pathway has been hindered by the absence of adequate methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately determining the pH of endocytic organelles in live cells. In this report, we demonstrate that the ratiometric fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous available probe for such pH measurements. To measure intraorganelle pH, cells 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 calibration is critical for accurate measurement of pH values: standard curves generated in vitro yielded artifactually low organelle pH values. Calibration was unaffected by the use of culture medium buffered with various buffers or different cell types. By using this technique, we show that both acidic and neutral endocytically derived organelles exist in the axons of sympathetic neurons in different steady-state proportions than in the cell body. Furthermore, we demonstrate that these axonal organelles have a bimodal pH distribution, indicating a rapid acidification 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 or organelle pH along axons, with the relative frequency of acidic organelles increasing with proximity to the cell body.
细胞器酸化是内体 - 溶酶体途径的一个基本要素，但由于缺乏用于定量细胞器内pH值的适当方法，我们对该途径进展的潜在机制的理解受到了阻碍。为了解决神经元中的这个问题，我们开发了一种直接定量方法，用于准确测定活细胞中内吞细胞器的pH值。在本报告中，我们证明比率荧光pH指示剂8 - 羟基芘 - 1,3,6 - 三磺酸（HPTS）是用于此类pH测量的最有利可用探针。为了测量细胞器内pH值，通过HPTS的内吞摄取对细胞进行标记，计算每个细胞器在450nm和405nm激发波长下的荧光发射强度比率（F450 / 405），并使用F450 / 405与pH的标准曲线将比率转换为pH值。正确的校准对于准确测量pH值至关重要：体外生成的标准曲线会产生人为偏低的细胞器pH值。校准不受使用各种缓冲液缓冲的培养基或不同细胞类型的影响。通过使用这种技术，我们表明，与细胞体相比，交感神经元轴突中存在酸性和中性内吞衍生的细胞器，其稳态比例不同。此外，我们证明这些轴突细胞器具有双峰pH分布，表明它们成熟过程中有一个快速酸化步骤，可将一部分细胞器的平均pH值降低2个pH单位，而在稳态时几乎没有中间pH值的细胞器。最后，我们证明了沿轴突存在细胞器pH的空间梯度，酸性细胞器的相对频率随着靠近细胞体而增加。
