![]() ![]() Simultaneous bright field and epifluorescence imaging was performed on cells grown on glass coverslips and placed in an Atto Chamber (Molecular Probes) within a thermostatted perfusion holder (Open Perfusion Micro-Incubator Medical Systems, Greenvale, NY) on the stage of a Zeiss Axiovert I35 inverted microscope (Zeiss). The fluorescence emission of this probe, which is quenched by protons in the physiological range, was suitable to monitor the pH of the ER in intact HeLa cells. The modified recombinant toxin was conjugated covalently to 5-(4,6-dichlorotriazinyl) aminofluorescein. Proteins expressing this tetrapeptide are known to be retrieved from the Golgi complex by receptors that ferry their ligands back to the reticulum ( 5). To this end, a recombinant B subunit of Shiga toxin containing the C-terminal ER retrieval signal, KDEL, was used ( 4). It was apparent that enhanced delivery and/or improved retention of the toxins in the endoplasmic reticulum would be required to obtain adequate signals for measurement of pH. This amount was insufficient to measure the pH of the ER. Though Shiga and related toxins are thought to exert their biological effects at the ER ( 2), only a minute fraction of the toxin molecules is normally translocated to the nuclear membrane and peripheral reticulum during the course of hours. The pH of this compartment could be measured optically by covalently attaching -sensitive fluorophores to the B subunit of verotoxin ( 3). The B subunits bind to surface glycolipids and are internalized via a retrograde pathway to the Golgi complex ( 2). coli, are composed of two types of subunits: a cytotoxic A subunit and a homopentamer of B subunits that serve a targeting role. Toxins from Shigella dysenteria and other Shiga-like toxins, such as verotoxin from E. Some success has been obtained recently targeting pH-sensitive probes to more distal elements of the secretory pathway, by using bacterial toxins. The purpose of the work described here was to implement an alternative, spectroscopic method whereby the fluorescence emission of a selectively targeted probe can be used to monitor pH in the ER continuously by ratio imaging. This approach precludes dynamic analysis of H + transport and its regulation. To our knowledge, the only attempts to estimate the pH of the ER were made by quantifying the partition of a permeant weak base by immunoelectron microscopy ( 1). However, the determinants and regulation of the pH within organelles of the secretory pathway, particularly, the endoplasmic reticulum (ER), have remained virtually unexplored, due primarily to our inability to specifically target -sensitive probes to these compartments. ![]() Protein sorting and targeting during secretion, and retrieval of resident chaperones and receptors are dictated by the prevailing pH in the individual compartments of the secretory pathway. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH. The ER was found to be highly permeable to H + (equivalents), so that the prevailing is susceptible to alterations in the cytosolic pH. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. Fluorescence ratio imaging and two independent calibration procedures were applied to determine the pH of the ER in situ. ![]() Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive fluorophore, was used for a two-stage delivery strategy. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by -sensitive probes. The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. ![]()
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