Andrew Barentine

All Publications

• 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

• Stimulated emission does not radiate in a pure dipole pattern OPTICA Barentine, A. S., Moerner, W. E. 2024; 11 (4): 464-470

  View details for DOI 10.1364/OPTICA.515226

  View details for Web of Science ID 001234696100006

• An integrated platform for high-throughput nanoscopy NATURE BIOTECHNOLOGY Barentine, A. S., Lin, Y., Courvan, E. M., Kidd, P., Liu, M., Balduf, L., Phan, T., Rivera-Molina, F., Grace, M. R., Marin, Z., Lessard, M., Chen, J., Wang, S., Neugebauer, K. M., Bewersdorf, J., Baddeley, D. 2023; 41 (11): 1549-+

  Abstract

  Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions.

  View details for DOI 10.1038/s41587-023-01702-1

  View details for Web of Science ID 000958280000002

  View details for PubMedID 36914886

  View details for PubMedCentralID PMC10497732

• DMA-tudor interaction modules control the specificity of invivo condensates. Cell Courchaine, E. M., Barentine, A. E., Straube, K., Lee, D., Bewersdorf, J., Neugebauer, K. M. 2021; 184 (14): 3612

  Abstract

  Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation invivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.

  View details for DOI 10.1016/j.cell.2021.05.008

  View details for PubMedID 34115980

• 3D super-resolution deep-tissue imaging in living mice. Optica Velasco, M. G., Zhang, M., Antonello, J., Yuan, P., Allgeyer, E. S., May, D., M'Saad, O., Kidd, P., Barentine, A. E., Greco, V., Grutzendler, J., Booth, M. J., Bewersdorf, J. 2021; 8 (4): 442-450

  Abstract

  Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

  View details for DOI 10.1364/OPTICA.416841

  View details for PubMedID 34239948

  View details for PubMedCentralID PMC8243577

• 3D super-resolution deep-tissue imaging in living mice OPTICA Velasco, M. M., Zhang, M., Antonello, J., Yuan, P., Allgeyer, E. S., May, D., M'Saad, O., Kidd, P., Barentine, A. S., Greco, V., Grutzendler, J., Booth, M. J., Bewersdorf, J. 2021; 8 (4): 442-450

  View details for DOI 10.1364/OPTICA.416841

  View details for Web of Science ID 000642200300003