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Research & Publications

*Chen J-H F., *Mirvis M, *Ekman A, Vanslembrouck B, Le Gros M, Larabell C, & Marshall WF. “Automated Segmentation of Soft X-Ray Tomography: Native Cellular Structure with Sub-Micron Resolution at High Throughput for Whole-Cell Quantitative Imaging in Yeast.” bioRxiv, November 1, 2024. https://doi.org/10.1101/2024.10.31.621371. Soft X-ray tomography (SXT) is an invaluable tool for quantitatively analyzing cellular structures at sub-optical isotropic resolution. However, it has traditionally depended on manual segmentation, limiting its scalability for large datasets. Here, we leverage a deep learning-based auto-segmentation pipeline to segment and label cellular structures in hundreds of cells across three Saccharomyces cerevisiae strains. This task-based pipeline employs manual iterative refinement to improve segmentation accuracy for key structures, including the cell body, nucleus, vacuole, and lipid droplets, enabling high-throughput and precise phenotypic analysis. Using this approach, we quantitatively compared the 3D whole-cell morphometric characteristics of wild-type, VPH1-GFP, and vac14 strains, uncovering detailed strain-specific cell and organelle size and shape variations. We show the utility of SXT data for precise 3D curvature analysis of entire organelles and cells and detection of fine morphological features using surface meshes. Our approach facilitates comparative analyses with high spatial precision and statistical throughput, uncovering subtle morphological features at the single cell and population level. This workflow significantly enhances our ability to characterize cell anatomy and supports scalable studies on the mesoscale, with applications in investigating cellular architecture, organelle biology, and genetic research across diverse biological contexts.

Perlaza K, Mirvis M, Ishikawa H, & Marshall WF. “The Short Flagella 1 (SHF1) Gene in Chlamydomonas Encodes a Crescerin TOG-Domain Protein Required for Late Stages of Flagellar Growth.” Molecular Biology of the Cell 33, no. 2 (February 2022): ar12. https://doi.org/10.1091/mbc.E21-09-0472. Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagellar tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

Mirvis M, Siemers KA, Nelson WJ, & Stearns TP. “Primary Cilium Loss in Mammalian Cells Occurs Predominantly by Whole-Cilium Shedding.” PLOS Biology 17, no. 7 (July 17, 2019): e3000381. https://doi.org/10.1371/journal.pbio.3000381. The primary cilium isacentral signaling hub incell proliferation and differentiation and is built and disassembled every cell cycle inmany animal cells. Disassembly iscritically important, as misregulation ordelay ofcilia loss leads tocell cycle defects. The physical means by which cilia are lost are poorly understood but are thought toinvolve resorption ofciliary components into the cell body. To investigate cilium loss inmammalian cells, we used live-cell imaging tocomprehensive lycharacterize individual events. The predominant mode ofcilium loss was rapid deciliation, inwhich the membrane and axoneme ofthe cilium was shed from the cell. Gradual resorption was also observed, as well as events inwhich aperiod ofgradual resorption was followed by rapid deciliation. Deciliation resulted inintact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels ofkatanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently ofcalcium. Together, these results suggest that mammalian ciliary loss involves atunable decision between deciliation and resorption.

Mirvis, M, Stearns TP, & Nelson WJ. “Cilium Structure, Assembly, and Disassembly Regulated by the Cytoskeleton.” Biochemical Journal 475, no. 14 (July 31, 2018): 2329–53. https://doi.org/10.1042/BCJ20170453. The cilium, once considered a vestigial structure, is a conserved, microtubule-based organelle critical for transducing extracellular chemical and mechanical signals that control cell polarity, differentiation, and proliferation. The cilium undergoes cycles of assembly and disassembly that are controlled by complex inter-relationships with the cytoskeleton. Microtubules form the core of the cilium, the axoneme, and are regulated by post-translational modifications, associated proteins, and microtubule dynamics. Although actin and septin cytoskeletons are not major components of the axoneme, they also regulate cilium organization and assembly state. Here, we discuss recent advances on how these different cytoskeletal systems­ affect cilium function, structure, and organization.

Bowen, AM, Musalgaonkar S, Moomau CA, Gulay SP, Mirvis M, & Dinman JD. “Ribosomal Protein uS19 Mutants Reveal Its Role in Coordinating Ribosome Structure and Function.” Translation 3, no. 2 (July 3, 2015): e1117703. https://doi.org/10.1080/21690731.2015.1117703. Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges.

Jegalian AG, Eberle FC, Pack SD, Mirvis M, Raffeld M, Pittaluga S, & Jaffe ES. “Follicular Lymphoma in Situ: Clinical Implications and Comparisons with Partial Involvement by Follicular Lymphoma.” Blood 118, no. 11 (September 15, 2011): 2976–84. https://doi.org/10.1182/blood-2011-05-355255. Follicular lymphoma in situ (FLIS) was first described nearly a decade ago, but its clinical significance remains uncertain. We reevaluated our original series and more recently diagnosed cases to develop criteria for the distinction of FLIS from partial involvement by follicular lymphoma (PFL). A total of 34 cases of FLIS were identified, most often as an incidental finding in a reactive lymph node. Six of 34 patients had prior or concurrent FL, and 5 of 34 had FLIS composite with another lymphoma. Of patients with negative staging at diagnosis and available follow-up (21 patients), only one (5%) developed FL (follow-up: median, 41 months; range, 10-118 months). Follow-up was not available in 2 cases. Fluorescence in situ hybridization for BCL2 gene rearrangement was positive in all 17 cases tested. PFL patients were more likely to develop FL, diagnosed in 9 of 17 (53%) who were untreated. Six patients with PFL were treated with local radiation therapy (4) or rituximab (2) and remained with no evidence of disease. FLIS can be reliably distinguished from PFL and has a very low rate of progression to clinically significant FL. FLIS may represent the tissue counterpart of circulating t(14;18)-positive B cells.

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