Traditional small-molecule fluorophores are always fluorescent. That attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single userdesignated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steadystate level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t 1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.Cells control their intracellular environment through careful gating of the influx of extracellular material (1,2). To distinguish between molecules to be internalized from those to be excluded, cells use specific interactions with cell-surface proteins, lipids, and carbohydrates. Nonspecific interactions mediated by Coulombic forces can also lead to internalization, often in an unregulated manner (1-5). 1 Such nonspecific interactions can be modulated by increasing, decreasing, or masking the cationic charge on the biomolecule (3,5).Two classes of molecules that exploit high cationicity to effect nonspecific adsorption to the cell surface and internalization are cell-penetrating peptides (CPPs 2 ) and ribonucleases (7-10). CPPs have received considerable attention due to their ability to transport otherwise membrane-impermeable cargo into cells (9,11,12). The detailed mechanism of CPP internalization is unclear, but is known to involve multiple steps. Those steps include binding † This work was supported by Grant CA73808 (NIH). R.J.J. and L.D.L. were supported by Biotechnology Training grant 08349 (NIH).L.D.L. was also supported by an ACS Division of Organic Chemistry Fellowship, sponsored by the Genentech Foundation. The Biophysics Instrumentation Facility was established with grants BIR-9512577 (NSF) and RR13790 (NIH).*To whom correspondence should be addressed: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544. Telephone: 608-262-8588. Fax: 608-262-3453. raines@biochem.wisc.edu.. 1 We prefer to use the term Coulombic rather than electrostatic to describe the for...

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N-and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.