Lysosomes are multi-functional, sub-cellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimers, Parkinsons and rare lysosomal storage diseases but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome sub-populations with different physical characteristics including dynamics, morphology or cellular localization. Here we chemically resolve lysosome sub-populations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from normal individuals show two major lysosome populations, one of which is lost in primary cells derived from Niemann-Pick disease patients. When patient cells are treated with relevant therapeutic, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

It is extremely challenging to quantitate lumenal Ca 2+ in acidic Ca 2+ stores of the cell because all Ca 2+ indicators are pH sensitive, and Ca 2+ transport coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca 2+ simultaneously, it functions as a pHcorrectable, Ca 2+ reporter. By targeting CalipHluor to the endolysosomal pathway we mapped lumenal Ca 2+ changes during endosomal maturation and found a surge in lumenal Ca 2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis we found that catp-6, a C. elegans homolog of ATP13A2, was responsible for lysosomal Ca 2+ accumulation-the first example of a lysosome-specific Ca 2+ importer in animals. By enabling the facile quantification of compartmentalized Ca 2+ , CalipHluor can expand our understanding of subcellular Ca 2+ importers.

DNA Trojan horse: A DNA icosahedron (black, see scheme) held together with aptamers (red) was used to encapsulate molecular cargo such as fluorescent dextran (green). In the presence of a molecular trigger (gray hexagons), the aptamers fold back leading to opening of the icosahedron and simultaneous release of the encapsulated cargo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.