Single Organelle Resolution Research Articles

Quantitative Chemical Imaging of Organelles.

ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca2+ into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.

One size does not fit all: Lysosomes exist in biochemically and functionally distinct states.

Single-organelle resolution approaches have the potential to advance our knowledge of the heterogeneity of lysosome function. Challenging population-based models, we propose a "lysosome states" concept that links single lysosomes to function.

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.

Analysis of Mitochondria by Single-Organelle Resolution.

Cellular organelles are highly specialized compartments with distinct functions. With the increasing resolution of detection methods, it is becoming clearer that same organelles may have different functions or properties not only within different cell populations of a tissue but also within the same cell. Dysfunction or altered function affects the organelle itself and may also lead to malignancies or undesirable cell death. To understand cellular function or dysfunction, it is therefore necessary to analyze cellular components at the single-organelle level. Here, we review the recent advances in analyzing cellular function at single-organelle resolution using high-parameter flow cytometry or multicolor confocal microscopy. We focus on the analysis of mitochondria, as they are organelles at the crossroads of various cellular signaling pathways and functions. However, most of the applied methods/technologies are transferable to any other organelle, such as the endoplasmic reticulum, lysosomes, or peroxisomes.

Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells.

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials (ENMs) which gain cellular entry via active endocytic processes. A recent emphasis on routes of ENM uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual role-that of an ENM undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the exceptional optical properties exhibited by noncovalent hybrids of single-stranded DNA and SWCNTs (DNA-SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA-SWCNTs were observed to scale with increasing time after internalization. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with submicron spatial resolution. This approach uncovered correlations between DNA-SWCNT concentration, dielectric modulation, and irreversible aggregation within single intracellular vesicles. An immunofluorescence assay was designed to directly observe the DNA-SWCNTs in labeled endosomal vesicles, revealing a distinct relationship between the physical state of organelle-bound DNA-SWCNTs and the dynamic luminal conditions during endosomal maturation processes. Finally, we trained a machine learning algorithm to predict endosome type using the Raman spectra of the vesicle-bound DNA-SWCNTs, enabling major components in the endocytic pathway to be simultaneously visualized using a single intracellular reporter.

Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.

Transistor-like Ultra-pH-Sensitive Polymeric Nanoparticles.

Electronic transistors have revolutionized the fields of microelectronics, computers, and mobile devices. Their ability to digitize electronic signals allows high fidelity data transfer as well as formation of logic gates. Inspired by electronic transistors, transistor-like organic materials have been under intensive investigation to amplify biological signals in a broad range of applications such as biosensing, diagnostic imaging, and therapeutic delivery. This Account highlights the inception and implementation of a "proton transistor" nanoparticle that can digitize acidotic pH signals in biological systems. Similar to electronic transistors, the ultra-pH-sensitive (UPS) nanoparticles derive their binary threshold response from phase separation phenomena. Hydrophobic micellization drives nanophase separation from unimers to aggregated polymeric micelles, which is responsible for the all-or-nothing proton distribution between the micelle and unimer states. Depending on the assembly status, conjugated fluorophores are quenched (micelle state) or freely fluoresce (solution unimer state) allowing robust detection of the phase transition behavior across a narrow pH range. Based on this mechanistic insight, we created a UPS nanoparticle library encompassing a broad physiological pH range from 4.0 to 7.4. For biological applications, we engineered a barcode-like nanosensor capable of digitizing multiple pH signals at a single organelle resolution in live cells. The barcode system allowed easy identification of mutant Kirsten rat sarcoma viral oncogene (KRAS), a common mutation involved in tumorigenesis, which leads to rapid cellular proliferation, as the protein driver for accelerated organelle acidification and lysosome catabolism in a broad set of isogenic as well as heterogeneous cancer cell lines. Adoption of the technology to an ON-OFF/Always-ON design allowed the quantification of proton flux across the membranes of endocytic organelles. For medical applications, we demonstrate the ability to achieve binary detection of solid cancers with clear tumor margin delineation by near-infrared fluorescence imaging. Image-guided resection of head/neck and breast tumors resulted in significantly improved long-term survival over white light or tumor debulking surgeries in tumor-bearing mice, catapulting the clinical evaluation of the UPS nanosensor in cancer patients. This Account serves as the first comprehensive summary of the molecular mechanism and biological applications of the digital pH threshold sensors. Building on the concept of cooperative phase transition behavior, we hope this Account will promote the rational design and development of additional transistor-like chemical sensors to digitize analog biological signals.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Digitization of Endocytic pH by Hybrid Ultra-pH-Sensitive Nanoprobes at Single-Organelle Resolution.

A multispectral hybrid nanotransistor consisting of modular fluorescent block copolymers with discrete and sharp pH transitions in one nanoparticluate system is presented. This nanotransistor probe allows digitized reporting of dynamic maturation at single-organelle resolution over time. This nanotechnology platform offers a powerful tool in fundamental studies of organelle biology, cell signaling in diseases characterized by pH dysregulation in the endosomes or lysosomes.

A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles.

Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells. Deployment in cells allows quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. Together, these biological applications indicate the robustness and adaptability of this nanotechnology-enabled ‘detection and perturbation' strategy.

Optical microwell arrays for large-scale studies of single mitochondria metabolic responses.

Most of the methods dedicated to the monitoring of metabolic responses from isolated mitochondria are based on whole-population analyses. They rarely offer an individual resolution though fluorescence microscopy allows it, as demonstrated by numerous studies on single mitochondria activities in cells. Herein, we report on the preparation and use of microwell arrays for the entrapment and fluorescence microscopy of single isolated mitochondria. Highly dense arrays of 3 μm mean diameter wells were obtained by the chemical etching of optical fiber bundles (850 μm whole diameter). They were manipulated by a micro-positioner and placed in a chamber made of a biocompatible elastomer (polydimethylsiloxane or PDMS) and a glass coverslip, on the platform of an inverted microscope. The stable entrapment of individual mitochondria (extracted from Saccharomyces cerevisiae yeast strains, inter alia, expressing a green fluorescent protein) within the microwells was obtained by pretreating the optical bundles with an oxygen plasma and dipping the hydrophilic surface of the array in a concentrated solution of mitochondria. Based on the measurement of variations of the intrinsic NADH fluorescence of each mitochondrion in the array, their metabolic status was analyzed at different energetic respiratory stages: under resting state, following the addition of an energetic substrate to stimulate respiration (ethanol herein) and the addition of a respiratory inhibitor (antimycin A). Statistical analyses of mean variations of mitochondrial NADH in the population were subsequently achieved with a single organelle resolution.

Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo.

Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with single-organelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function. This approach revealed that axonal mitochondria undergo spontaneous 'contractions' that are accompanied by reversible redox changes. These contractions are amplified by neuronal activity and acute or chronic neuronal insults. Multiparametric imaging reveals that contractions constitute respiratory chain-dependent episodes of depolarization coinciding with matrix alkalinization, followed by uncoupling. In contrast, permanent mitochondrial damage after spinal cord injury depends on calcium influx and mitochondrial permeability transition. Thus, our approach allows us to identify heterogeneity among physiological and pathological redox signals, correlate such signals to functional and structural organelle dynamics and dissect the underlying mechanisms.

Mitochondrial morphological features are associated with fission and fusion events.

Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes.

Optical microwell array for large scale studies of single mitochondria metabolic responses

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-μm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29% and 35% under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach

Many essential cellular processes are affected by transmembrane H+ gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.