Brénot F, Aubry L, Martin J B, Satre M, Klein G
Laboratoire de Biologie Cellulaire (UA 1130 du CNRS), Centre d'Etudes Nucléaires de Grenoble, France.
Biochimie. 1992 Sep-Oct;74(9-10):883-95. doi: 10.1016/0300-9084(92)90072-m.
We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).
我们检测了细胞黏菌盘基网柄菌变形虫中各种内体区室的pH值。这是通过荧光法和体内³¹P - NMR方法完成的。将荧光素标记的葡聚糖这种液相标记物投喂给变形虫，以报告其胞吞小泡的平均pH值。在连续的内体区室逐步装载过程中，我们观察到早期酸化，在20℃下30分钟后pH值降至最小值≤5.3，随后当所有内体区室都被液相标记物装载时，pH值升至平均5.8。FITC - 葡聚糖在酸性pH下的荧光强度较弱，妨碍了更详细的研究，因此我们检查了各种膦酸酯化合物作为内体区室潜在的³¹P - NMR pH探针。选择了氨甲基膦酸和2 - 氨基乙基膦酸这两种分子进行这项研究，因为它们的化学位移随pH变化的幅度较大（分别为2和2.5 ppm），且它们的酸性pK值分别为5.5和6.3。它们对盘基网柄菌变形虫的无菌生长和分化程序只有中等毒性（IC50%约为10 mM）。如用1 mM钒酸盐或7.5 mM咖啡因完全抑制它们的进入所显示的那样，这两种氨基膦酸的内化仅通过液相胞饮途径发生，钒酸盐和咖啡因是盘基网柄菌中两种先前已表征的内吞作用抑制剂。我们发现，对用氨基膦酸孵育的变形虫悬浮液进行体内³¹P - NMR检测，可以检测到pH值分别为4.3、5.8 - 6.0和7.3的三个不同的细胞内区室。分析了氨基膦酸进入的动力学，结果使我们能够重建内吞过程中酸化序列的时间进程。这些数据与以下假设一致：在盘基网柄菌变形虫中，膦酸酯先占据高度酸性的早期内体区室（t1/2 = 18分钟；pH 4.3），然后到达酸性较弱的晚期内体/前溶酶体区室（pH 5.8 - 6.0），从那里它们立即被转运并滞留在细胞质（pH 7.3）中。
