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.

The chemistry of next-generation sequencing.

The first large genome fully sequenced by next-generation sequencing (NGS) was that of a bacteriophage using sequencing by synthesis (SBS) as a paradigm. SBS in NGS is underpinned by 'reversible-terminator chemistry'. To grow from proof of concept to being both affordable and practical, SBS needed to overcome a series of challenges, each of which required the invention of new chemistries. These included the design and synthesis of unnatural deoxynucleotide triphosphates (dNTPs), engineering a suitable polymerase, a new surface chemistry and an ingenious molecular solution to neutralize copying errors inherent to all polymerases. In this historical Perspective, we discuss how NGS was developed from Sanger sequencing, highlighting the chemistry behind this technology, which has impacted biology in unprecedented ways.

Detecting organelle-specific activity of potassium channels with a DNA nanodevice.

Cell surface potassium ion (K+) channels regulate nutrient transport, cell migration and intercellular communication by controlling K+ permeability and are thought to be active only at the plasma membrane. Although these channels transit the trans-Golgi network, early and recycling endosomes, whether they are active in these organelles is unknown. Here we describe a pH-correctable, ratiometric reporter for K+ called pHlicKer, use it to probe the compartment-specific activity of a prototypical voltage-gated K+ channel, Kv11.1, and show that this cell surface channel is active in organelles. Lumenal K+ in organelles increased in cells expressing wild-type Kv11.1 channels but not after treatment with current blockers. Mutant Kv11.1 channels, with impaired transport function, failed to increase K+ levels in recycling endosomes, an effect rescued by pharmacological correction. By providing a way to map the organelle-specific activity of K+ channels, pHlicKer technology could help identify new organellar K+ channels or channel modulators with nuanced functions.

A DNA nanodevice for mapping sodium at single-organelle resolution.

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail.

SLC12A9 is a lysosome-detoxifying ammonium - chloride co-transporter.

Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH 4 + ). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal ammonium exporter that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 is a chloride-driven ammonium co-transporter that is central in an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.

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.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

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.

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.

The evolution of organellar calcium mapping technologies.

Intracellular Ca2+ fluxes are dynamically controlled by the co-involvement of multiple organellar pools of stored Ca2+. Endolysosomes are emerging as physiologically critical, yet underexplored, sources and sinks of intracellular Ca2+. Delineating the role of organelles in Ca2+ signaling has relied on chemical fluorescent probes and electrophysiological strategies. However, the acidic endolysosomal environment presents unique issues, which preclude the use of traditional chemical reporter strategies to map lumenal Ca2+. Here, we broadly address the current state of knowledge about organellar Ca2+ pools. We then outline the application of traditional probes, and their sensing paradigms. We then discuss how a new generation of probes overcomes the limitations of traditional Ca2+probes, emphasizing their ability to offer critical insights into endolysosomal Ca2+, and its feedback with other organellar pools.

A lysosome-targeted DNA nanodevice selectively targets macrophages to attenuate tumours.

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

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 leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in Caenorhabditis elegans, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

Quantifying phagosomal HOCl at single immune-cell resolution.

Neutralization of pathogens by phagocytic immune cells requires the biogenesis of a compartmentalized hotspot of reactive species called the phagosome. One of these reactive species is hypochlorous acid (HOCl), produced by the enzyme myeloperoxidase (MPO) after the phagosome fuses with the lysosome. Mapping HOCl during phagosome maturation can report on pathogen killing and offer insights into regulation of MPO activity, mechanisms of resistance and host-pathogen interactions. However, this has been difficult because of a lack of a suitable method to chemically map a transient organelle with pH fluctuations like the phagosome. Here, we detail a protocol for quantifying HOCl dynamics in phagosomes using a fluorescent DNA-based reporter. Compared to traditional methods of visualizing HOCl or measuring MPO activity, this method offers sub-cellular spatial resolution and the capacity to assay HOCl production with single cell resolution.

Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis.

During vesicular acidification, chloride (Cl-), as the counterion, provides the electrical shunt for proton pumping by the vacuolar H+ ATPase. Intracellular CLC transporters mediate Cl- influx to the endolysosomes through their 2Cl-/H+ exchange activity. However, whole-endolysosomal patch-clamp recording also revealed a mysterious conductance releasing Cl- from the lumen. It remains unknown whether CLCs or other Cl- channels are responsible for this activity. Here, we show that the newly identified proton-activated Cl- (PAC) channel traffics from the plasma membrane to endosomes via the classical YxxL motif. PAC deletion abolishes the endosomal Cl- conductance, raises luminal Cl- level, lowers luminal pH, and increases transferrin receptor-mediated endocytosis. PAC overexpression generates a large endosomal Cl- current with properties similar to those of endogenous conductance, hypo-acidifies endosomal pH, and reduces transferrin uptake. We propose that the endosomal Cl- PAC channel functions as a low pH sensor and prevents hyper-acidification by releasing Cl- from the lumen.

A DNA-based voltmeter for organelles.

The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology.

Quantitative Imaging of Biochemistry in Situ and at the Nanoscale.

Biochemical reactions in eukaryotic cells occur in subcellular, membrane-bound compartments called organelles. Each kind of organelle is characterized by a unique lumenal chemical composition whose stringent regulation is vital to proper organelle function. Disruption of the lumenal ionic content of organelles is inextricably linked to disease. Despite their vital roles in cellular homeostasis, there are large gaps in our knowledge of organellar chemical composition largely from a lack of suitable probes. In this Outlook, we describe how, using organelle-targeted ratiometric probes, one can quantitatively image the lumenal chemical composition and biochemical activity inside organelles. We discuss how excellent fluorescent detection chemistries applied largely to the cytosol may be expanded to study organelles by chemical imaging at subcellular resolution in live cells. DNA-based reporters are a new and versatile platform to enable such approaches because the resultant probes have precise ratiometry and accurate subcellular targeting and are able to map multiple chemicals simultaneously. Quantitatively mapping lumenal ions and biochemical activity can drive the discovery of new biology and biomedical applications.

DNA-based fluorescent probes of NOS2 activity in live brains.

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.