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...

Abstract. The sorting of macromolecules within and between membranous organelles is often directed by information contained in protein primary or secondary structure. We show here that absent such structural information, macromolecules internalized by endocytosis in macrophages can be sorted by size. After endocytosis, small solute probes of fluid-phase pinocytosis were recycled to the extracellular medium more efficiently than large solutes. Using macropinosomes pulse labeled with fluorescent dextrans, we examined the ability of organelles to exchange solute contents. Dextran exchange was optimal between organelles of similar age, and small dextrans exchanged more efficiently than large dextrans. Efferent solute movement, from lysosomes or phagolysosomes toward the plasma membrane, occurred through the same endocytic vesicles as afferent movement, toward lysosomes and this movement was solute size dependent. Remarkably, uniform mixtures of different-sized dextrans delivered into lysosomes separated into distinct organelles containing only one dextran or the other. Thus, the dynamics of endosomes and lysosomes were sufficient to segregate macromolecules by size. This intracellular size fractionation could explain how, during antigen presentation, peptides generated by lysosomal proteases recycle selectively from lysosomes to endosomes for association with class II MHC molecules.T HE traffic of macromolecules within and through vesicular organelles is directed both by the dynamics of the organelles and by information contained in the structure of the macromolecules. Light and electron microscopic studies of endocytic organelle dynamics indicate a high degree of communication between endosomes and lysosomes, mediated by vesicle fusion and fission and by microtubule-mediated organelle movements. In most cells, solute internalized by fluid-phase endocytosis moves sequentially through early endosomes and late endosomes into lysosomes, with some percentage of that solute leaving the cell via vesicular recycling. Macromolecules that take other paths carry information in their structure that enhances either retention in endosomes or movement into other compartments. Such sorting information is often contained in protein primary or secondary structure, and works by selectively directing molecules into transport vesicles or by mediating associations with other molecules that are so directed (Sandoval and Bakke, 1994). Some molecular sorting may occur without such signals, however. As antigen-presenting cells, macrophages process many different and essentially unrecognized proteins. Proteins delivered to lysosomes via endocytosis are degraded to peptides, which then recycle to endosomes or to other nonlysosomal compartments, where they associate with MHC class II molecules (Germain, 1994 lysosomes to endosomes indicates a sorting process, in that these small molecules are recycled but the lysosomal enzymes are not. The great variety of peptides that travel this route makes it unlikely that sorting information in their prim...

Cholangiocarcinoma is rising in clinical importance because of increasing incidence, poor prognosis, and suboptimal response to therapy. Recent investigations into the underlying molecular mechanisms involved in cholangiocarcinogenesis and tumor growth have contributed greatly to our understanding of this disease. To review this topic, we discuss the molecular mechanisms in sections reflecting the unique features that allow cancer cells to develop and maintain a growth advantage. Through a better understanding of these mechanisms, improved and more specific diagnostic, therapeutic, and preventative strategies may be developed and hopefully improve the outcome of this devastating disease.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrowderived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.