As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.

Introduction

Interest in studying cellular pH using pH-sensitive fluorescent dyes has its published roots in the 1970s (1). At the heart of this interest lies the fundamental idea that proper cellular function relies on precise, localized pH modulation within cells. By the late 1980s, studying pH changes within single cells using fluorescent dyes and microscopy had become a reality (2). Since then, further innovation has manifest itself in the form of advances in fluorescent microscopy, such as confocality, and novel or modified pH-sensitive fluorescent dyes (3,4,5). All of these advances have resulted in successful use of pH-sensitive dyes as visible measures of pH within living cells.

For a dye to be useful as an intracellular pH measure, it must, at the very least, possess two major qualities relating to its pH sensitivity. First, the dye needs to exhibit distinct excitation or emission wavelengths reflecting its protonated/unprotonated forms. For this to hold, either the emission or excitation wavelength needs to be nonresponsive or one of these wavelengths must be less responsive (or responsive in the opposite direction of the spectrum) than the other. It is the relationship (or ratio) of the changes in emission or absorption at each wavelength that defines the pH response. An intrinsic advantage to this ratiometric technique of pH detection is the fact that the ratio is derived from the same sample, making factors such as sample volume and all environmental conditions (probe concentration, photobleaching, etc.) inherently normalized. The second fundamental quality that the pH-sensing dye must possess in order to accurately measure pH inside cells is a pKa value within physiological pH range, or within the expected range of the subcellular environment to be studied.

To date, the vast majority of the fluorescent pH-sensitive dyes used in cells that possess the aforementioned qualities have been organic dyes. These dyes are traditionally administered to cells from the external medium. Thus, although proficient indicators of pH change, such dyes act as global indicators. Attempts to achieve subcellular targeting of pH sensors have largely focused on genetic engineering of protein-based pH indicators. Examples of this strategy range from the molecular engineering of green fluorescent protein (GFP) itself (4,5,6,7,8) to the more recent FRET-based approach to sensors, the pHlameleons (9). GFP engineering has resulted in many pH-sensitive sensors such as the pHluorins (6), deGFP4 (5,7), and E²GFP (8). These probes do offer advantages associated with their protein natures and some have physiologic pKa values. However, they are not without drawbacks. For instance, pHluorin pH response requires a dual excitation approach with excitations well within the blue range of the spectrum at 395 and 475 nm. Not only do dual excitation dyes require that cells be exposed to double the amount of light compared with emission-based ones, but pHluorin requires this light to be in the part of the spectrum known to promote both noise from cellular autofluorescence and risks of photodamage. E²GFP, on the other hand, can be used either as a dual excitation or as a dual emission ratio-metric probe, whereas deGFP4, as its name implies, was mutated to perform as a dual emission probe. Although E²GFP has been used successfully, it has been reported to exhibit a more limited dynamic range in the latter case and both it and deGFP4 do still require a blue excitation light. The pHlameleon family of sensors (9) offers advantages including that the excitation light can be more optimal for cell-based imaging. The FRET approach also allows the acceptor moiety to be modulated in order to accommodate differing needs. This inherent flexibility, however, dictates that use of this technology requires an added amount of optimization in comparison to the aforementioned pH sensors.

Our approach to generating a reliable intracellular pH sensor that can be subcellularly targeted and that lacks the previously described drawbacks is to combine a protein-based reporter system with a well-established, organic-based, pH-sensitive dye. Specifically, we have combined HaloTag technology (10) with a member of the benzo[c]xanthene family of dyes, namely 5(and 6-) carboxyseminaphthorhodafluor (SNARF-1) (U.S. Patent no. 4945171). HaloTag is a reporter protein whose sequence is easily fused to a gene of interest. Once expressed, this reporter protein is designed to covalently bind ligands that are composed of two parts: a reactive linker that forms the bond with HaloTag protein and a functional moiety in the form of a fluorescent dye or affinity handle. By attaching such a linker to SNARF-1 and using it with an expressed HaloTag fusion construct, we are able to precisely target this pH sensor subcellularly and measure pH. We chose the SNARF-1 dye because it is a member of a family of pH sensors that continue to be standard in the field since they were first introduced in the early 1990s (11,12). These dyes are widely used for pH measurement because they show distinct emission bands based on protonation, they are conveniently excited at 488 or 515 nm with argon ion lasers, and they exhibit pKa values within physiological range. In addition, with emission wavelengths of 580 nm (acidic max) and 640 nm (basic max), SNARF-1 can be used with other fluorescent sensors, such as the popular fluo-3 calcium sensing dye (13), without interference.