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

Tissue-specific targeting of DNA nanodevices in a multicellular living organism

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology

Self-Assembled Near-Infrared Dye Nanoparticles as a Selective Protein Sensor by Activation of a Dormant Fluorophore

Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine (<b>Sq</b>) dye (λ_abs= 670 nm, λ_em= 700 nm). The dye self-assembles to form nonfluorescent nanoparticles (<i>D</i>_h = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the <b>Sq</b> dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the <b>Sq</b> nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum

Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides

Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver diverse cargos into cells. CPPs access the cytosol either by direct translocation through the plasma membrane or via endocytosis followed by endosomal escape. Both direct translocation and endosomal escape can occur simultaneously, making it non-trivial to specifically study endosomal escape alone. Here we depolarize the plasma membrane and showed that it inhibits the direct translocation of several CPPs but does not affect their uptake into endosomes. Despite good endocytic uptake many CPPs previously considered to access the cytosol via endosomal escape, failed to access the cytosol once direct translocation was abrogated. Even CPPs designed for enhanced endosomal escape actually showed negligible endosomal escape into the cytosol. Our data reveal that cytosolic localization of CPPs occurs mainly by direct translocation across the plasma membrane. Cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs

Passive endocytosis in model protocells

Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters

Tissue-specific targeting of DNA nanodevices in a multicellular living organism

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology

Figure 3

Fig 3b-d: Pearson’s correlation coefficient (PCC) of colocalization (CL) and pixel shift (PS) of cells fluorescently labeled Tf with EE labeled by 3WEE in hMSR1-transfected HEK 293T cells (b), Colocalization of RE tracer and 3WRE  in HEK 293T cells (c), and colocalization of TGN marker and 3WTGN in scFv-furin-transfected HEK 293T cells (d). Fig 3e-f: Plots of pH versus [K+] of RE in HEK 293T cells.  Fig 3h: Plots of pH versus [K+] of EE  in HEK 293T cells.  Fig 3i: Plots of pH versus [K+] of TGN  in HEK 293T cells. </p

Figure 4

Fig 4b:  Pearson’s correlation coefficient (PCC) of colocalization (CL) and pixel shift (PS) between RE tracer and 3WRE  in BMDM cells. Fig 4d:  Plot of pH and [K+] of RE in WT of BMDM cells  Fig 4e:  Plot of pH and [K+] of RE in TWIK2-/- of BMDM cells  </p

Figure 5

Fig 5b: [K+]TGN and pH values in UT cells. Fig 5c: [K+]TGN and pH values in UT cells treated with tetraethyl ammonium chloride (TEA). Fig 5d: [K+]TGN and pH values in WT cells. Fig 5c: [K+]TGN and pH values in WT cells treated with cisapride. </p

Figure 6

Figure 6b:  [K+]RE and pH values of recycling endosomes (REs) in WT cells. Figure 6c:  [K+]RE and pH values of REs in WT cells treated with tetraethyl ammonium chloride (TEA) Figure 6d:  [K+]RE and pH values of REs in UT cells treated with TEA. Figure 6e:  [K+]RE and pH values of REs in WT cells treated with cisapride. Figure 6f:  [K+]RE and pH values of REs in G601S cells. Figure 6g:  [K+]RE and pH values of REs in G601S cells treated with dofetilide overnight and after wash out.  Figure 6i: I-V curves recorded from WT cells and G601S cells in standard extracellular saline. Figure 6j: I-V curves recorded from WT cells and G601S cells in modified extracellular saline mimicking ion gradients across RE membranes. Figure 6k: Plot of peak IKv11.1 refcorded from WT cells and G601S cells in standard extracellular saline. Figure 6l: Plot of peak IKv11.1 recorded from WT cells and G601S cells in modified extracellular saline mimicking ion gradients across RE membranes.</p