Alex Choi

All Publications

• Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion. bioRxiv : the preprint server for biology Unger, B. A., Wu, C. Y., Choi, A. A., He, C., Xu, K. 2024

  Abstract

  As with most intermediate filament systems, the hierarchical self-assembly of vimentin into nonpolar filaments requires no nucleators or energy input. Utilizing a set of live-cell, single-molecule, and super-resolution microscopy tools, here we show that in mammalian cells, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing differently charged constructs, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges (-18 per vimentin protein) along the filament are less screened or otherwise intensified, and stabilized when the charges are better screened or otherwise reduced. Generalizing this model to other intermediate filaments, we further show that whereas the negatively charged GFAP cytoskeleton is similarly subject to fast disassembly under hypotonic stress, the cytokeratin, as a copolymer of negatively and positively charged subunits, does not exhibit this behavior. Thus, in cells containing both vimentin and keratin cytoskeletons, hypotonic stress disassembles the former but not the latter. Together, our results both provide new handles for modulating cell behavior and call for new attention to the effects of net charges in intracellular protein interactions.

  View details for DOI 10.1101/2024.07.08.602555

  View details for PubMedID 39026705

  View details for PubMedCentralID PMC11257561

• Single-Molecule Diffusivity Quantification Unveils Ubiquitous Net Charge-Driven Protein-Protein Interaction JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Choi, A. A., Xu, K. 2024; 146 (15): 10973-10978

  Abstract

  Recent microscopy and nuclear magnetic resonance (NMR) studies have noticed substantial suppression of intracellular diffusion for positively charged proteins, suggesting an overlooked role of electrostatic attraction in nonspecific protein interactions in a predominantly negatively charged intracellular environment. Utilizing single-molecule detection and statistics, here, we quantify in aqueous solutions how protein diffusion, in the limit of low diffuser concentration to avoid aggregate/coacervate formation, is modulated by differently charged interactor proteins over wide concentration ranges. We thus report substantially suppressed diffusion when oppositely charged interactors are added at parts per million levels, yet unvaried diffusivities when same-charge interactors are added beyond 1%. The electrostatic attraction-driven suppression of diffusion is sensitive to the protein net charge states, as probed by varying the solution pH and ionic strength or chemically modifying the proteins and is robust across different diffuser-interactor pairs. By converting the measured diffusivities to diffuser diameters, we further show that in the limit of excess interactors, a positively charged diffuser molecule effectively drags along just one monolayer of negatively charged interactors, where further interactions stop. We thus unveil ubiquitous, net charge-driven protein-protein interactions and shed new light on the mechanism of charge-based diffusion suppression in living cells.

  View details for DOI 10.1021/jacs.4c02475

  View details for Web of Science ID 001200580500001

  View details for PubMedID 38576203

  View details for PubMedCentralID PMC11023747

• A composite electrodynamic mechanism to reconcile spatiotemporally resolved exciton transport in quantum dot superlattices. Science advances Yuan, R., Roberts, T. D., Brinn, R. M., Choi, A. A., Park, H. H., Yan, C., Ondry, J. C., Khorasani, S., Masiello, D. J., Xu, K., Alivisatos, A. P., Ginsberg, N. S. 2023; 9 (42): eadh2410

  Abstract

  Quantum dot (QD) solids are promising optoelectronic materials; further advancing their device functionality requires understanding their energy transport mechanisms. The commonly invoked near-field Förster resonance energy transfer (FRET) theory often underestimates the exciton hopping rate in QD solids, yet no consensus exists on the underlying cause. In response, we use time-resolved ultrafast stimulated emission depletion (STED) microscopy, an ultrafast transformation of STED to spatiotemporally resolve exciton diffusion in tellurium-doped cadmium selenide-core/cadmium sulfide-shell QD superlattices. We measure the concomitant time-resolved exciton energy decay due to excitons sampling a heterogeneous energetic landscape within the superlattice. The heterogeneity is quantified by single-particle emission spectroscopy. This powerful multimodal set of observables provides sufficient constraints on a kinetic Monte Carlo simulation of exciton transport to elucidate a composite transport mechanism that includes both near-field FRET and previously neglected far-field emission/reabsorption contributions. Uncovering this mechanism offers a much-needed unified framework in which to characterize transport in QD solids and additional principles for device design.

  View details for DOI 10.1126/sciadv.adh2410

  View details for PubMedID 37862422

  View details for PubMedCentralID PMC10588942

• Size-Dependent Suppression of Molecular Diffusivity in Expandable Hydrogels: A Single-Molecule Study. The journal of physical chemistry. B Park, H. H., Choi, A. A., Xu, K. 2023; 127 (14): 3333-3339

  Abstract

  By repurposing the recently popularized expansion microscopy to control the meshwork size of hydrogels, we examine the size-dependent suppression of molecular diffusivity in the resultant tuned hydrogel nanomatrices over a wide range of polymer fractions of ∼0.14-7 wt %. With our recently developed single-molecule displacement/diffusivity mapping (SMdM) microscopy methods, we thus show that with a fixed meshwork size, larger molecules exhibit more impeded diffusion and that, for the same molecule, diffusion is progressively more suppressed as the meshwork size is reduced; this effect is more prominent for the larger molecules. Moreover, we show that the meshwork-induced obstruction of diffusion is uncoupled from the suppression of diffusion due to increased solution viscosities. Thus, the two mechanisms, respectively, being diffuser-size-dependent and independent, may separately scale down molecular diffusivity to produce the final diffusion slowdown in complex systems like the cell.

  View details for DOI 10.1021/acs.jpcb.3c00761

  View details for PubMedID 37011131

• Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes. Journal of the American Chemical Society Choi, A. A., Xiang, L., Li, W., Xu, K. 2023

  Abstract

  While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400 μs separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 μm2/s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ∼60-70% of that in vitro, up to ∼250 μm2/s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments.

  View details for DOI 10.1021/jacs.3c00597

  View details for PubMedID 37027457

  View details for PubMedCentralID PMC10558625

• Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes. Journal of the American Chemical Society Aparin, I. O., Yan, R., Pelletier, R., Choi, A. A., Danylchuk, D. I., Xu, K., Klymchenko, A. S. 2022; 144 (39): 18043-18053

  Abstract

  Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.

  View details for DOI 10.1021/jacs.2c07542

  View details for PubMedID 36153973

• Displacement Statistics of Unhindered Single Molecules Show no Enhanced Diffusion in Enzymatic Reactions. Journal of the American Chemical Society Choi, A. A., Park, H. H., Chen, K., Yan, R., Li, W., Xu, K. 2022; 144 (11): 4839-4844

  Abstract

  Recent studies have sparked debate over whether catalytic reactions enhance the diffusion coefficients D of enzymes. Through high statistics of the transient (600 μs) displacements of unhindered single molecules freely diffusing in common buffers, we here quantify D for four enzymes under catalytic turnovers. We thus formulate how ∼ ±1% precisions may be achieved for D, and show no changes in diffusivity for catalase, urease, aldolase, and alkaline phosphatase under the application of wide concentration ranges of substrates. Our single-molecule approach thus overcomes potential limitations and artifacts underscored by recent studies to show no enhanced diffusion in enzymatic reactions.

  View details for DOI 10.1021/jacs.1c12328

  View details for PubMedID 35258969

  View details for PubMedCentralID PMC8975259

• Systematic Analysis of Fatty Acids in Human Cells with a Multiplexed Isobaric Tag (TMT)-Based Method. Journal of proteome research Sun, F., Choi, A. A., Wu, R. 2018; 17 (4): 1606-1614

  Abstract

  Fatty acids (FAs) are essential components in cells and are involved in many cellular activities. Abnormal FA metabolism has been reported to be related to human diseases such as cancer and cardiovascular diseases. Identification and quantification of FAs provide insights into their functions in biological systems, but it is very challenging to analyze them due to their structures and properties. In this work, we developed a novel method by integrating FAs tagged with stable isotope labeled aminoxy tandem mass tags (aminoxyTMTs) and mass spectrometric analysis in the positive mode. On the basis of their structures, the aminoxyTMT reagents reacted with the carboxylic acid group of the FAs, resulting in an amine group with high proton affinity covalently attached to the analytes. This enabled the analysis of FAs under the positive electrospray ionization-mass spectrometry (ESI-MS) mode, which is normally more popular and sensitive compared to the negative mode. More importantly, the multiplexed TMT tags allowed us to quantify FAs from several samples simultaneously, which increased the experimental throughput and quantification accuracy. FAs extracted from three types of breast cells, i.e., MCF 10A (normal), MCF7 (minimally invasive) and MDA-MB-231 (highly invasive) cells, were labeled with the six-plexed aminoxyTMTs and quantified by LC-MS/MS. The results demonstrated that the abundances of some FAs, such as C22:5 and C20:3, were markedly increased in MCF7 and MDA-MB-231 cancer cells compared to normal MCF 10A cells. For the first time, aminoxyTMT reagents were exploited to label FAs for their identification and quantification in complex biological samples in the positive MS mode. The current method enabled us to confidently identify FAs and to accurately quantify them from several samples simultaneously. Because this method does not have sample restrictions, it can be extensively applied for biological and biomedical research.

  View details for DOI 10.1021/acs.jproteome.7b00896

  View details for PubMedID 29521506

  View details for PubMedCentralID PMC5937127