Honors & Awards

  • Mona M. Burgess Graduate Fellow, Bio-X SIGF, Stanford University (2018 Sept - 2021)
  • CMAD Fellow, The Center for Molecular Analysis and Design, Stanford University (2016 July - 2018 June)

Education & Certifications

  • B.Sc, University of Michigan, Biochemistry, summa cum laude (2014)

Lab Affiliations

All Publications

  • Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. Proceedings of the National Academy of Sciences of the United States of America Dahlberg, P. D., Saurabh, S., Sartor, A. M., Wang, J., Mitchell, P. G., Chiu, W., Shapiro, L., Moerner, W. E. 2020


    Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of 30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

    View details for DOI 10.1073/pnas.2001849117

    View details for PubMedID 32513734

  • Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation. Proceedings of the National Academy of Sciences of the United States of America Zhou, X., Wang, J., Herrmann, J., Moerner, W. E., Shapiro, L. 2019


    The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

    View details for DOI 10.1073/pnas.1906119116

    View details for PubMedID 31315982

  • Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging. Journal of the American Chemical Society Dahlberg, P. D., Sartor, A. M., Wang, J., Saurabh, S., Shapiro, L., Moerner, W. E. 2018; 140 (39): 12310–13


    Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.

    View details for PubMedID 30222332

  • Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bayas, C. A., Wang, J., Lee, M. K., Schrader, J. M., Shapiro, L., Moerner, W. E. 2018; 115 (16): E3712–E3721


    We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

    View details for PubMedID 29610352

  • Mg2+ Shifts Ligand-Mediated Folding of a Riboswitch from Induced-Fit to Conformational Selection JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Suddala, K. C., Wang, J., Hou, Q., Water, N. G. 2015; 137 (44): 14075-14083


    Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg(2+) and three ligands of distinct affinities. We show that, in the absence of Mg(2+), both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 μM Mg(2+) generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg(2+)-mediated RNA folding.

    View details for DOI 10.1021/jacs.5b09740

    View details for Web of Science ID 000364727600019

    View details for PubMedID 26471732

  • Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014 BIOPOLYMERS Bartke, R. M., Cameron, E. L., Cristie-David, A. S., Custer, T. C., Denies, M. S., Daher, M., Dhakal, S., Ghosh, S., Heinicke, L. A., Hoff, J. D., Hou, Q., Kahlscheuer, M. L., Karslake, J., Krieger, A. G., Li, J., Li, X., Lund, P. E., Vo, N. N., Park, J., Pitchiaya, S., Rai, V., Smith, D. J., Suddala, K. C., Wang, J., Widom, J. R., Walter, N. G. 2015; 103 (5): 296-302


    Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

    View details for DOI 10.1002/bip.22603

    View details for Web of Science ID 000350306700005

    View details for PubMedID 25546606

  • Recent Advances in Radical SAM Enzymology: New Structures and Mechanisms ACS CHEMICAL BIOLOGY Wang, J., Woldring, R. P., Roman-Melendez, G. D., McClain, A. M., Alzua, B. R., Marsh, E. N. 2014; 9 (9): 1929-1938


    The radical S-adenosylmethionine (SAM) superfamily of enzymes catalyzes an amazingly diverse variety of reactions ranging from simple hydrogen abstraction to complicated multistep rearrangements and insertions. The reactions they catalyze are important for a broad range of biological functions, including cofactor and natural product biosynthesis, DNA repair, and tRNA modification. Generally conserved features of the radical SAM superfamily include a CX3CX2C motif that binds an [Fe4S4] cluster essential for the reductive cleavage of SAM. Here, we review recent advances in our understanding of the structure and mechanisms of these enzymes that, in some cases, have overturned widely accepted mechanisms.

    View details for DOI 10.1021/cb5004674

    View details for Web of Science ID 000342121200003

    View details for PubMedID 25009947