Biology subjects

Mittal, J.

Publications and source records attributed to Mittal, J..

52 records · Page 3Linked to original sources

bioRxiv2023

Expanding the molecular language of protein liquid-liquid phase separation

Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a "molecular grammar" for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

biophysics↗

bioRxiv2023

Role of Strong Localized vs. Weak Distributed Interactions in Disordered Protein Phase Separation

Interaction strength and localization are critical parameters controlling the single-chain and condensed-state properties of intrinsically disordered proteins (IDPs). Here, we decipher these relationships using coarse-grained heteropolymers comprised of hydrophobic (H) and polar (P) monomers as model IDPs. We systematically vary the fraction of P monomers XP and employ two distinct particle-based models that include either strong localized attractions between only H-H pairs (HP model) or weak distributed attractions between both H-H and H-P pairs (HP+ model). To compare different sequences and models, we first carefully tune the attraction strength for all sequences to match the single-chain radius of gyration. Interestingly, we find that this procedure produces similar conformational ensembles, nonbonded potential energies, and chain-level dynamics for single chains of almost all sequences in both models, with some deviations for the HP model at large XP. However, we observe a surprisingly rich phase behavior for the sequences in both models that deviates from the expectation that similarity at the single-chain level will translate to similar phase-separation propensity. Coexistence between dilute and dense phases is only observed up to a model-dependent XP despite the presence of favorable interchain interactions, which we quantify using the second virial coefficient. Instead, the limited number of attractive sites (H monomers) leads to the self-assembly of finite-sized clusters of different sizes depending on XP. Our findings strongly suggest that models with distributed interactions favor the formation of liquid-like condensates over a much larger range of sequence compositions compared to models with localized interactions.

biophysics↗

bioRxiv2022

Aliphatic residues contribute significantly to the phase separation of TDP-43 C-terminal domain

TAR DNA binding protein 43 (TDP-43) is involved in key processes in RNA metabolism such as splicing, stability and transcription. TDP-43 dysfunction is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone and harbors the majority (~90%) of all ALS-related mutations. Recent studies have established that TDP-43 CTD can undergo liquid-liquid phase separation (LLPS) in isolation and is important for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (IDR1/2) have also been found to play a critical role in PS and aggregation. However, TDP-43 CTD has a distinct sequence composition compared with other phase separating proteins, including many aliphatic residues. These residues have been suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution to phase separation has been relatively ignored. Here, we utilized a multiscale simulation and experimental approach to assess the residue-level determinants of TDP-43 CTD phase separation. Single chain and condensed phase simulations performed at the atomistic and coarse-grained level respectively, identified the importance of aromatic residues (previously established) while also suggesting an essential role for aliphatic methionine residues in LLPS. In vitro experiments confirmed the role of phenylalanine, methionine, and leucine (but not alanine) residues in driving the phase separation of CTD, which have not been previously considered essential for describing the molecular grammar of PS. Finally, NMR experiments also showed that phenylalanine residues in the disordered flanking regions and methionine residues both within and outside the CR contribute important contacts to CTD interactions. Broadly, our work highlights the importance of non-alanine aliphatic residues such as methionine and leucine, and potentially valine and isoleucine, in determining the LLPS propensity, expanding the molecular grammar of protein phase separation to include critical contributions from aliphatic residues.

biophysics↗

bioRxiv2022

Effect of Charge Distribution on the Dynamics of Polyampholytic Disordered Proteins

The stability and physiological function of many biomolecular coacervates depend on the structure and dynamics of intrinsically disordered proteins (IDPs) that typically contain a significant fraction of charged residues. Although the effect of relative arrangement of charged residues on IDP conformation is a well-studied problem, the associated changes in dynamics are far less understood. In this work, we systematically interrogate the effects of charge distribution on the chain-level and segmental dynamics of polyampholytic IDPs in dilute solutions. We study a coarse-grained model polyampholyte consisting of an equal fraction of two oppositely charged residues (glutamic acid and lysine) that undergoes a transition from an ideal chain-like conformation for uniformly charge-patterned sequences to a semi-compact conformation for highly charge-segregated sequences. Changes in the chain-level dynamics with increasing charge segregation correlate with changes in conformation. The chain-level and segmental dynamics conform to simple homopolymer models for uniformly charge-patterned sequences but deviate with increasing charge segregation, both in the presence and absence of hydrodynamic interactions. We discuss the significance of these findings, obtained for a model polyampholyte, in the context of a charge-rich intrinsically disordered region of the naturally occurring protein LAF-1. Our findings have important implications for understanding the effects of charge patterning on the dynamics of polyampholytic IDPs in dilute conditions using polymer scaling theories.

biophysics↗

bioRxiv2022

Molecular interactions underlying the phase separation of HP1: Role of phosphorylation, ligand and nucleic acid binding

Heterochromatin protein 1 (HP1) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1 functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1 can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1 and that it involves interactions between the positively charged hinge region of HP1 and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1 under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1 binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1 can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1 dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1 where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1 and that can contribute to heterochromatin regulation and function.

biophysics↗

bioRxiv2022

Zn-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

The toxic gain of function of Cu/Zn superoxide dismutase (SOD1) associated with the neurodegenerative disease - Amyotrophic lateral sclerosis (ALS), is believed to occur via misfolding and/or aggregation. SOD1 is also associated with stress granules (SGs) which are a type of membraneless organelle believed to form via liquid-liquid phase separation (LLPS) of several proteins containing low-complexity, disordered regions. Using a combination of experiments and computer simulations, we report here that structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The phase-separated droplets give rise to aggregates which eventually form toxic amyloids upon prolonged incubation. The addition of exogenous Zn to immature, metal-free SOD1 and the severe ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. In contrast, the Zn-induced inhibition of LLPS and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In conclusion, our work establishes a role for Zn-dependent modulation of SOD1 disorder and LLPS as a precursor phenomenon which may lead to the formation of toxic amyloids associated with ALS. Significance StatementThe formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. SOD1 is a metalloenzyme which is stabilized by the metal co-factor - Zn. In this work, we utilize experimental and simulation techniques to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.

biophysics↗

bioRxiv2021

Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

biophysics↗

bioRxiv2020

Molecular details of protein condensates probed by microsecond-long atomistic simulations

The formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS), and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Molecular simulations can reveal the specific amino acid interactions driving LLPS, which is hard to obtain from experiment. Coarse-grained simulations have been used to directly observe the sequence determinants of phase separation but have limited spatial resolution, while all-atom simulations have yet to be applied to LLPS due to the challenges of large system sizes and long time scales relevant to phase separation. We present a novel multiscale computational framework by obtaining initial molecular configurations of a condensed protein-rich phase from equilibrium coarse-grained simulations, and back mapping to an all-atom representation. Using the specialized Anton 2 supercomputer, we resolve microscopic structural and dynamical details of protein condensates through microsecond-scale all-atom explicit-solvent simulations. We have studied two IDPs which phase separate in vitro: the low complexity domain of FUS and the N-terminal disordered domain of LAF-1. Using this approach, we explain the partitioning of ions between phases with low and high protein density, demonstrate that the proteins are remarkably dynamic within the condensed phase, identify the key residue-residue interaction modes stabilizing the dense phase, all while showing good agreement with experimental observations. Our approach is generally applicable to all-atom studies of other single and multi-component systems of proteins and nucleic acids involved in the formation of membraneless organelles.

biophysics↗

bioRxiv2020

A predictive coarse-grained model for position-specific effects of post-translational modifications on disordered protein phase separation

Biomolecules undergo liquid-liquid phase separation (LLPS) resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed charge atomistic forcefields, we parameterize the size and atomistic hydropathy of the coarse-grained modified amino acid beads, and hence the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the proteins single chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of FUS and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution. Statement of SignificancePost-translational modifications are important regulators of liquid-liquid phase separation (LLPS) which drives the formation of biomolecular condensates. Theoretical methods can be used to characterize the biophysical properties of intrinsically disordered proteins (IDPs). Our recent framework for molecular simulations using a C-centered coarse-grained model can predict the effect of various perturbations such as mutations (Dignon et al. PloS Comput. Biol, 2018) and temperature (Dignon et al, ACS Cent. Sci., 2019) on LLPS. Here, we expand this framework to incorporate modified residues like phosphothreonine, phosphoserine and acetylysine. This model will prove useful for simulating the phase separation of post-translationally modified IDPs and predicting how position-specific modifications can control phase behavior across the large family of proteins known to be phosphorylated and acetylated.

biophysics↗

bioRxiv2020

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration

mRNA transport in neurons is a ubiquitous process but has been often overlooked as a contributor to disease. Mutations of transport granule protein hnRNPA2 cause hereditary proteinopathy of neurons, myocytes, and bone. Here, we examine transport granule component specificity, assembly/disassembly, and the link to neurodegeneration. hnRNPA2 transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet they each partition specifically into hnRNPA2 liquid phases. hnRNPA2 tyrosine phosphorylation dissociates granule interactions by reducing hnRNPA2 phase separation and preventing partitioning of hnRNPF and ch-TOG; tyrosine phosphorylation also decreases aggregation of hnRNPA2 disease mutants. A C. elegans model of hnRNPA2 D290V-associated neurodegeneration exhibits TDP-43 ortholog-dependent glutamatergic neurodegeneration. Expression of the tyrosine kinase that phosphorylates hnRNPA2 reduces glutamatergic neurodegeneration. The evidence for specific partitioning of granule components as well as disruption of these interactions and reduction of neurodegeneration by tyrosine phosphorylation suggest transport granule biology has a role in the pathogenesis of neurodegeneration.

biochemistry↗

bioRxiv2020

Hydropathy patterning complements charge patterning to describe conformational preferences of disordered proteins

Understanding the conformational ensemble of an intrinsically disordered protein (IDP) is of great interest due to its relevance to critical intracellular functions and diseases. It is now well established that the polymer scaling behavior can provide a great deal of information about the conformational properties as well as liquid-liquid phase separation of an IDP. It is, therefore, extremely desirable to be able to predict an IDPs scaling behavior from the protein sequence itself. The work in this direction so far has focused on highly charged proteins and how charge patterning can perturb their structural properties. As naturally occurring IDPs are composed of a significant fraction of uncharged amino acids, the rules based on charge content and patterning are only partially helpful in solving the problem. Here, we propose a new order parameter, sequence hydropathy decoration (SHD), which can provide a near quantitative understanding of scaling and structural properties of IDPs devoid of charged residues. We combine this with a charge patterning parameter, sequence charge decoration (SCD), to obtain a general equation, parameterized from extensive coarse-grained simulation data, for predicting protein dimensions from the sequence. We finally test this equation against available experimental data and find a semi-quantitative match in predicting the scaling behavior. We also provide guidance on how to extend this approach to experimental data, which should be feasible in the near future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/919498v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1042309org.highwire.dtl.DTLVardef@984e73org.highwire.dtl.DTLVardef@1467afeorg.highwire.dtl.DTLVardef@34f8ec_HPS_FORMAT_FIGEXP M_FIG Graphical TOC Entry C_FIG

biophysics↗

bioRxiv2020

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bio-inspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation. Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the proteins propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

biophysics↗

bioRxiv2019

TDP-43 α-helical structure tunes liquid-liquid phase separation and function

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. Present in several MLOs, TDP-43 undergoes LLPS and is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some disease variants of TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes in a dimeric TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a mini-gene assay. Therefore, TDP-43 helical region serves as a short but uniquely tunable module that shows promise as for controlling assembly and function in cellular and synthetic biology applications of LLPS.

biochemistry↗

bioRxiv2018

Computational modeling highlights disordered Formin Homology 1 domain’s role in profilin-actin transfer

Formins accelerate actin polymerization, assumed to occur through flexible FH1 domain mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of mouse mDia1, mDia2; budding yeast Bni1, Bnr1; fission yeast Cdc12, For3, and Fus1 FH1s. We find FH1 has flexible regions between high propensity polyproline helix regions. A coarse-grained model retaining sequence-specificity, assuming rigid polyproline segments, describes their size. Multiple profilins and profilin-actin complexes can simultaneously bind, expanding mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show the leading FH1 can better transfer profilin or profilin-actin, having decreasing probability with increasing distance from FH2.

biophysics↗

bioRxiv2017

Sequence determinants of protein phase behavior from a coarse-grained model

Membraneless organelles important to intracellular compartmentalization have recently been shown to comprise assemblies of proteins which undergo liquid-liquid phase separation (LLPS). However, many proteins involved in this phase separation are at least partially disordered. The molecular mechanism and the sequence determinants of this process are challenging to determine experimentally owing to the disordered nature of the assemblies, motivating the use of theoretical and simulation methods. This work advances a computational framework for conducting simulations of LLPS with residue-level detail, and allows for the determination of phase diagrams and coexistence densities of proteins in the two phases. The model includes a short-range contact potential as well as a simplified treatment of electrostatic energy. Interaction parameters are optimized against experimentally determined radius of gyration data for multiple unfolded or intrinsically disordered proteins (IDPs). These models are applied to two systems which undergo LLPS: the low complexity domain of the RNA-binding protein FUS and the DEAD-box helicase protein LAF-1. We develop a novel simulation method to determine thermodynamic phase diagrams as a function of the total protein concentration and temperature. We show that the model is capable of capturing qualitative changes in the phase diagram due to phosphomimetic mutations of FUS and to the presence or absence of the large folded domain in LAF-1. We also explore the effects of chain-length, or multivalency, on the phase diagram, and obtain results consistent with Flory-Huggins theory for polymers. Most importantly, the methodology presented here is flexible so that it can be easily extended to other pair potentials, be used with other enhanced sampling methods, and may incorporate additional features for biological systems of interest.\n\nAuthor summaryLiquid liquid phase separation (LLPS) of low-complexity protein sequences has emerged as an important research topic due to its relevance to membraneless organelles and intracellular compartmentalization. However a molecular level understanding of LLPS cannot be easily obtained by experimental methods due to difficulty of determining structural properties of phase separated protein assemblies, and of choosing appropriate mutations. Here we advance a coarse-grained computational framework for accessing the long time scale phase separation process and for obtaining molecular details of LLPS, in conjunction with state of the art enhanced sampling methods. We are able to capture qualitatively the changes of phase diagram due to specific mutations, inclusion of a folded domain, and to variation of chain length. The model is flexible and can be used with different knowledge-based potential energy functions, as we demonstrate. We expect a wide application of the presented framework for advancing our understanding of the formation of liquid-like protein assemblies.

biophysics↗

bioRxiv2017

A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux

Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, which responds to lipid accumulation via modulation of the nanotubes optical bandgap. The engineered nanomaterial, composed of short-single stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation, or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis (DIPL), was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned sub-cellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers new capabilities for drug development processes and the investigation of lipid-linked diseases.

bioengineering↗