Ashwin Balaji

All Publications

• Structural remodeling of target-SNARE protein complexes by NSF enables synaptic transmission. Nature communications White, K. I., Khan, Y. A., Qiu, K., Balaji, A., Couoh-Cardel, S., Esquivies, L., Pfuetzner, R. A., Diao, J., Brunger, A. T. 2025; 16 (1): 8371

  Abstract

  Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

  View details for DOI 10.1038/s41467-025-62764-0

  View details for PubMedID 40993127

  View details for PubMedCentralID 6378885

• Efficient Double Helix Detection with Steerable Filters. bioRxiv : the preprint server for biology Barentine, A. E., Balaji, A., Moerner, W. E. 2025

  Abstract

  We present an efficient detection scheme for localization of Double Helix point-spread functions for 3D single-molecule localization microscopy or tracking. Using steerable filters, we extract both 2D position and lobe orientation (axial position) estimates using just 7 convolutions, orders of magnitude less than used in deep learning based approaches. For a complete SMLM analysis pipeline, we pair this detection with a fitter using an optimally parameterized double Gaussian model, and implement both as a plugin for the open source PYthon Microscopy Environment (PYME).

  View details for DOI 10.1101/2025.08.14.670427

  View details for PubMedID 40894605

  View details for PubMedCentralID PMC12393288

• A Super-Resolution Spatial Atlas of SARS-CoV-2 Infection in Human Cells. bioRxiv : the preprint server for biology Andronov, L., Han, M., Balaji, A., Zhu, Y., Qi, L. S., Moerner, W. E. 2025

  Abstract

  The spatial organization of viral and host components dictates the course of infection, yet the nanoscale architecture of the SARS-CoV-2 life cycle remains largely uncharted. Here, we present a comprehensive super-resolution Atlas of SARS-CoV-2 infection, systematically mapping the localization of nearly all viral proteins and RNAs in human cells. This resource reveals that the viral main protease, nsp5, localizes to the interior of double-membrane vesicles (DMVs), challenging existing models and suggesting that polyprotein processing is a terminal step in replication organelle maturation. We identify previously undescribed features of the infection landscape, including thin dsRNA "connectors" that physically link DMVs, and large, membrane-less dsRNA granules decorated with replicase components, reminiscent of viroplasms. Finally, we show that the antiviral drug nirmatrelvir induces the formation of persistent, multi-layered bodies of uncleaved polyproteins. This spatial Atlas provides a foundational resource for understanding coronavirus biology and offers crucial insights into viral replication, assembly, and antiviral mechanisms.

  View details for DOI 10.1101/2025.08.15.670620

  View details for PubMedID 40894656

  View details for PubMedCentralID PMC12393340

• High-resolution dynamic imaging of chromatin DNA communication using Oligo-LiveFISH. Cell Zhu, Y., Balaji, A., Han, M., Andronov, L., Roy, A. R., Wei, Z., Chen, C., Miles, L., Cai, S., Gu, Z., Tse, A., Yu, B. C., Uenaka, T., Lin, X., Spakowitz, A. J., Moerner, W. E., Qi, L. S. 2025

  Abstract

  Three-dimensional (3D) genome dynamics are crucial for cellular functions and disease. However, real-time, live-cell DNA visualization remains challenging, as existing methods are often confined to repetitive regions, suffer from low resolution, or require complex genome engineering. Here, we present Oligo-LiveFISH, a high-resolution, reagent-based platform for dynamically tracking non-repetitive genomic loci in diverse cell types, including primary cells. Oligo-LiveFISH utilizes fluorescent guide RNA (gRNA) oligo pools generated by computational design, in vitro transcription, and chemical labeling, delivered as ribonucleoproteins. Utilizing machine learning, we characterized the impact of gRNA design and chromatin features on imaging efficiency. Multi-color Oligo-LiveFISH achieved 20-nm spatial resolution and 50-ms temporal resolution in 3D, capturing real-time enhancer and promoter dynamics. Our measurements and dynamic modeling revealed two distinct modes of chromatin communication, and active transcription slows enhancer-promoter dynamics at endogenous genes like FOS. Oligo-LiveFISH offers a versatile platform for studying 3D genome dynamics and their links to cellular processes and disease.

  View details for DOI 10.1016/j.cell.2025.03.032

  View details for PubMedID 40239646

• Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates. Nature communications Aris, K. D., Cofsky, J. C., Shi, H., Al-Sayyad, N., Ivanov, I. E., Balaji, A., Doudna, J. A., Bryant, Z. 2025; 16 (1): 2939

  Abstract

  The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5bp initial RNA-DNA hybridization and a ~17bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

  View details for DOI 10.1038/s41467-025-57703-y

  View details for PubMedID 40133266

• Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission. bioRxiv : the preprint server for biology White, K. I., Khan, Y. A., Qiu, K., Balaji, A., Couoh-Cardel, S., Esquivies, L., Pfuetzner, R. A., Diao, J., Brunger, A. T. 2024

  Abstract

  Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, alpha-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

  View details for DOI 10.1101/2024.10.11.617886

  View details for PubMedID 39416070

• Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles. Nature communications Andronov, L., Han, M., Zhu, Y., Balaji, A., Roy, A. R., Barentine, A. E., Patel, P., Garhyan, J., Qi, L. S., Moerner, W. E. 2024; 15 (1): 4644

  Abstract

  The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

  View details for DOI 10.1038/s41467-024-48991-x

  View details for PubMedID 38821943

  View details for PubMedCentralID 7951565

• Revealing the 3D nanoscale organization of MyosinH in the apical complex of toxoplasma gondii through single-molecule localization microscopy with the double-helix point spread function Balaji, A., Zarko, L., Dahlberg, P. D., Boothroyd, J. C., Moerner, W. E. CELL PRESS. 2024: 30A-31A

  View details for Web of Science ID 001194120700146

• Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles. bioRxiv : the preprint server for biology Andronov, L., Han, M., Zhu, Y., Roy, A. R., Barentine, A. E., Garhyan, J., Qi, L. S., Moerner, W. E. 2023

  Abstract

  The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

  View details for DOI 10.1101/2023.11.07.566110

  View details for PubMedID 37986994

• Stabilizing a centrifugal force microscope to study DNA folding Orlowski, A. A., Huq, M., Balaji, A., Carter, A. R. CELL PRESS. 2023: 548A

  View details for Web of Science ID 000989629703040

• Stabilizing a centrifugal force microscope to study DNA folding. Biophysical journal Orlowski, A. A., Huq, M., Balaji, A., Carter, A. R. 2023; 122 (3S1): 548a

  View details for DOI 10.1016/j.bpj.2022.11.2898

  View details for PubMedID 36784841

• Characterizing the distribution of myosin H in the apical complex of conoid protruded and conoid retracted Toxoplasma gondii Balaji, A., Dahlberg, P. D., Segev-Zarko, L., Sun, S., Chiu, W., Boothroyd, J., Moerner, W. E. CELL PRESS. 2022: 409A

  View details for Web of Science ID 000759523002535

• Single-molecule analysis of DNA interrogation by Cas9 and Cas12a on supercoiled DNA Aris, K. D. P., Cofsky, J., Wright, A. V., Ivanov, I. E., Balaji, A., Doudna, J. A., Bryant, Z. CELL PRESS. 2022: 288A

  View details for Web of Science ID 000759523001663

• Construction of a centrifugal force microscope Golden, G. N., Carter, A., Balaji, A. CELL PRESS. 2022: 417A-418A

  View details for Web of Science ID 000759523002577