A mechanism of lysosomal calcium entry.

Lysosomal calcium (Ca2+) release is critical to cell signaling and is mediated by well-known lysosomal Ca2+ channels. Yet, how lysosomes refill their Ca2+ remains hitherto undescribed. Here, from an RNA interference screen in Caenorhabditis elegans, we identify an evolutionarily conserved gene, lci-1, that facilitates lysosomal Ca2+ entry in C. elegans and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca2+/H+ exchanger, imports Ca2+ pH dependently into lysosomes. Using two-ion mapping and electrophysiology, we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca2+ importer. Defects in lysosomal Ca2+ channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca2+ importers may provide previously unidentified avenues to explore the physiology of Ca2+ channels.

Mild and scalable synthesis of phosphonorhodamines.

Since their discovery in 1887, rhodamines have become indispensable fluorophores for biological imaging. Recent studies have extensively explored heteroatom substitution at the 10' position and a variety of substitution patterns on the 3',6' nitrogens. Although 3-carboxy- and 3-sulfono-rhodamines were first reported in the 19th century, the 3-phosphono analogues have never been reported. Here, we report a mild, scalable synthetic route to 3-phosphonorhodamines. We explore the substrate scope and investigate mechanistic details of an exogenous acid-free condensation. Tetramethyl-3-phosphonorhodamine (phosTMR) derivatives can be accessed on the 1.5 mmol scale in up to 98% yield (2 steps). phosTMR shows a 12- to 500-fold increase in water solubility relative to 3-carboxy and 3-sulfonorhodamine derivatives and has excellent chemical stability. Additionally, phosphonates allow for chemical derivatization; esterification of phosTMR facilitates intracellular delivery with localization profiles that differ from 3-carboxyrhodamines. The free phosphonate can be incorporated into a molecular wire scaffold to create a phosphonated rhodamine voltage reporter, phosphonoRhoVR. PhosRhoVR 1 can be synthesized in just 6 steps, with an overall yield of 37% to provide >400 mg of material, compared to a 6-step, ∼2% yield for the previously reported RhoVR 1. PhosRhoVR 1 possesses excellent voltage sensitivity (37% ΔF/F) and a 2-fold increase in cellular brightness compared to RhoVR 1.