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 Organelle contents acidify as they progress through the endosomal-lysosomal pathway (1), but the nature of the progression is not well understood. In addition, little is known about this degradative pathway in neuronal cells (2, 3). To study the spatiotemporal organization of this dynamic process in neurons requires a noninvasive method for measuring organelle pH in live cells. Most studies of intracellular pH have focused on the cytoplasm (e.g., refs. 4-9; for review, see ref. 10), and considerably less work has addressed the pH of organelles within living cells (11)(12)(13). On the latter subject, the extant literature suffers from the use of pH probes that are nonquantitative, subject to a variety of artifacts, impossible to calibrate accurately, or restricted to use in fixed cells. Here we present an accurate quantitative method for determining intraorganelle pH in living cells that is ideal for studies of the endosomal-lysosomal pathway in neurons and other cell types.Our work employs the fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) that has six properties essential for studies of intraorganelle pH: (i) It lacks toxicity orThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.interference with normal cellular functions at the experimental concentrations. (ii) It has a pKa near neutrality and high pH resolution in the physiologic range. (iii) It responds rapidly to changes in pH. (iv) It is a ratiometric indicator, allowing quantitative measurements regardless of organelle size or probe concentration. (v) It can be calibrated within live cells, for precise and accurate quantification. (vi) It is hydrophilic and membrane impermeant, allowing easy loading into endosomes by fluid-phase endocytosis but preventing leakage across intracellular membranes. The latter property is particularly important in studies of neurons, since a marker endocytosed at the axonal tip must be retained within organelles for a relatively long period while the organelles undergo retrograde transport over considerable distances. HPTS has been recognized as a useful probe for dynamic pH measurements of membrane-bound a...

The motor protein kinesin is implicated in organelle movement toward the plus ends of microtubules, but little is known about its interaction with organelle membranes or about the physiological role of the phosphorylation of kinesin and its associated protein kinectin seen in neurons in vivo (Hollenbeck, P. J. (1993) J. Neurochem. 60, 2265-2275). Here we have demonstrated that the kinesin heavy chain (KHC), light chain, and kinectin isolated from chick brain or sympathetic neurons exist in several isoelectric forms. Metabolic labeling followed by phosphatase treatment showed that these are phosphoisoforms, and that phosphorylation is reversible in vitro. To assess the capability of phosphorylation to regulate kinesin's state and/or activity, we performed 32P and 35S pulse-chase experiments with neuronal cultures and determined that kinesin-associated phosphate turns over 3-4 times faster than the proteins themselves. When the phosphoisoform distributions for different kinesin pools were analyzed, it was found that membrane-associated KHC contained predominantly the most highly phosphorylated isoform, while soluble kinesin consisted of less phosphorylated KHC isoforms. Nerve growth factor-induced neurite outgrowth in PC12 cells was found to increase significantly kinesin's 32P specific activity while doubling the relative abundance of the most highly phosphorylated KHC isoform. These results demonstrate that the phosphorylation state of kinesin is closely coupled to its organelle binding and to the magnitude of organelle transport in the cell. We propose that the phosphorylation state of kinesin and associated proteins may regulate motility via association with organelle membranes and, specifically, that KHC phosphorylation induces membrane association.

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