Krishnan Lab

The Krishnan Lab explores the intricate chemistry of organelles, the cellular reaction vessels. Using bionanotechnology, they construct precise chemical maps of organelle lumens to understand how their optimized compositions drive biochemical processes, influencing organelle, cell, tissue, and organism functions. Evolution has sharpened a multitude of nano and molecular-scale biological mechanisms, inspiring bionanotechnology to tackle scientific and engineering challenges. Leveraging nucleic acid structures, this lab develops DNA-based nanodevices for quantitative chemical imaging in living systems to approach fundamental principles governing cellular machinery and advance solutions for diverse fields.

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Yamuna Krishnan

Rui Fu

Junyi Zou

Alok Apan Swatiputra

Sangyoon Lee

Avantika Negi

Sandip Chakraborty

Tara Kedda

JoAnn Tinker

Soyoung Kim

Priyanka Dutta Gupta

Bill Chung

Benton Girdler

Weiyue Wang

Vanity Spruill

Recent

A mechanism of lysosomal calcium entry (2024)

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.

Detecting organelle-specific activity of potassium channels with a DNA nanodevice (2023)

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 maps sodium at single organelle resolution (2023)

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.

A DNA-based voltmeter for organelles (2021)

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.

A pH-correctable DNA-based fluorescent reporter for organellar calcium (2019)

It is extremely challenging to quantitate lumenal Ca²⁺ in acidic Ca²⁺ stores of the cell because all Ca²⁺ indicators are pH sensitive, and Ca²⁺ transport is 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 Ca²⁺ simultaneously, CalipHluor functions as a pH-correctable Ca²⁺ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca²⁺ changes during endosomal maturation and found a surge in lumenal Ca²⁺ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca²⁺ accumulation-an example of a lysosome-specific Ca²⁺ importer in animals. By enabling the facile quantification of compartmentalized Ca²⁺, CalipHluor can expand the understanding of subcellular Ca²⁺ importers.