@article{doherty_aw_warren_hockenberry_whitworth_krohn_howell_diekman_legant_nia_et al._2023, title={Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics}, volume={166}, ISSN={["1878-7568"]}, DOI={10.1016/j.actbio.2023.05.015}, abstractNote={Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.}, journal={ACTA BIOMATERIALIA}, author={Doherty, Elizabeth L. and Aw, Wen Yih and Warren, Emily C. and Hockenberry, Max and Whitworth, Chloe P. and Krohn, Grace and Howell, Stefanie and Diekman, Brian O. and Legant, Wesley R. and Nia, Hadi Tavakoli and et al.}, year={2023}, month={Aug}, pages={346–359} }
 @article{roudot_legant_zou_dean_isogai_welf_david_gerlich_fiolka_betzig_et al._2023, title={u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions}, volume={3}, ISSN={["2667-2375"]}, DOI={10.1016/j.crmeth.2023.100655}, abstractNote={We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.}, number={12}, journal={CELL REPORTS METHODS}, author={Roudot, Philippe and Legant, Wesley R. and Zou, Qiongjing and Dean, Kevin M. and Isogai, Tadamoto and Welf, Erik S. and David, Ana F. and Gerlich, Daniel W. and Fiolka, Reto and Betzig, Eric and et al.}, year={2023}, month={Dec} }
 @article{shi_daugird_legant_2022, title={A quantitative analysis of various patterns applied in lattice light sheet microscopy}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-32341-w}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, author={Shi, Yu and Daugird, Timothy A. and Legant, Wesley R.}, year={2022}, month={Aug} }
 @article{hockenberry_legant_2022, title={Cells in the mechanical spotlight}, volume={121}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2022.09.003}, abstractNote={Brillouin microscopy, a technique that visualizes the transfer of energy from photons of light to phonons of mechanical energy ( 1 Brillouin L. Diffusion de la lumière et des rayons X par un corps transparent homogène. Ann. Phys. (Paris). 1922; 9: 88-122https://doi.org/10.1051/anphys/192209170088 Crossref Google Scholar ), has emerged as a useful tool in the study of biological mechanics. Unlike other approaches to mechanical characterization, such as optical tweezers, atomic force microscopy (AFM), or rheology, Brillouin microscopy does not require physical contact between a probe and the specimen. Instead, the mechanical properties of the specimen are characterized by observing optical frequency shifts in the Brillouin scattered light from the specimen. These frequency shifts arise from energy transfer between the photons of the electromagnetic field and acoustic phonons of vibrational energy within the specimen. The magnitude of this Brillouin shift (νB) is related to the wavelength of incident light (λ), and the density ( ρ ), refractive index ( n ), and longitudinal modulus (Μ) of the material according to Eq. 1, while the linewidth of the Brillouin peak is related to the wavelength, density, refractive index, and viscosity (η) of the specimen according to Eq. 2 ( 2 Prevedel R. Diz-Muñoz A. Antonacci G. et al. Brillouin microscopy: an emerging tool for mechanobiology. Nat. Methods. 2019; 16: 969-977https://doi.org/10.1038/s41592-019-0543-3 Crossref PubMed Scopus (172) Google Scholar ). The wavelength of the incident light is an experimentally controlled parameter; thus, under conditions wherein the specimen density and refractive index are known, or where the relative changes in these values can be controlled or assumed to be constant within the specimen, then the viscoelastic properties of the specimen can be estimated, or relative changes compared, by observing the magnitude of the Brillouin frequency shift and the width of this peak ( 2 Prevedel R. Diz-Muñoz A. Antonacci G. et al. Brillouin microscopy: an emerging tool for mechanobiology. Nat. Methods. 2019; 16: 969-977https://doi.org/10.1038/s41592-019-0543-3 Crossref PubMed Scopus (172) Google Scholar ). M = λ 2 4 ρ n 2 ν B 2 (1) η = λ 2 2 ρ n 2 Δ B (2) Multimodal microscale mechanical mapping of cancer cells in complex microenvironmentsNikolić et al.Biophysical JournalSeptember 3, 2022In BriefThe mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal is to disrupt cancer cells’ ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo two-dimensional (2D) and 3D extracellular matrix environments. Full-Text PDF}, number={19}, journal={BIOPHYSICAL JOURNAL}, author={Hockenberry, Max A. and Legant, Wesley R.}, year={2022}, month={Oct}, pages={3571–3572} }
 @article{moore_fogerson_tulu_yu_cox_sican_li_legant_weigel_crawford_et al._2022, title={Superresolution microscopy reveals actomyosin dynamics in medioapical arrays}, volume={33}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.E21-11-0537}, abstractNote={ Superresolution microscopy produced images of individual actin and myosin filaments and their three-dimensional distribution in medioapical arrays of cells in developing embryos. Arrays are part of a modified cell cortex that condenses and relaxes with isotropic and anisotropic contraction and expansion-they are recruited to ameliorate cell bulging. }, number={11}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Moore, Regan P. and Fogerson, Stephanie M. and Tulu, U. Serdar and Yu, Jason W. and Cox, Amanda H. and Sican, Melissa A. and Li, Dong and Legant, Wesley R. and Weigel, Aubrey V and Crawford, Janice M. and et al.}, year={2022}, month={Sep} }
 @article{moore_ellen c. o'shaughnessy_shi_nogueira_heath_hahn_legant_2021, title={A multi-functional microfluidic device compatible with widefield and light sheet microscopy}, ISSN={["1473-0189"]}, DOI={10.1039/d1lc00600b}, abstractNote={FEP-based microfluidics enable diverse applications in light sheet microscopy.}, journal={LAB ON A CHIP}, author={Moore, Regan P. and Ellen C. O'Shaughnessy and Shi, Yu and Nogueira, Ana T. and Heath, Katelyn M. and Hahn, Klaus M. and Legant, Wesley R.}, year={2021}, month={Nov} }
 @article{speiser_mueller_hoess_matti_obara_legant_kreshuk_macke_ries_turaga_2021, title={Deep learning enables fast and dense single-molecule localization with high accuracy}, ISSN={["1548-7105"]}, DOI={10.1038/s41592-021-01236-x}, abstractNote={Single-molecule localization microscopy (SMLM) has had remarkable success in imaging cellular structures with nanometer resolution, but standard analysis algorithms require sparse emitters, which limits imaging speed and labeling density. Here, we overcome this major limitation using deep learning. We developed DECODE (deep context dependent), a computational tool that can localize single emitters at high density in three dimensions with highest accuracy for a large range of imaging modalities and conditions. In a public software benchmark competition, it outperformed all other fitters on 12 out of 12 datasets when comparing both detection accuracy and localization error, often by a substantial margin. DECODE allowed us to acquire fast dynamic live-cell SMLM data with reduced light exposure and to image microtubules at ultra-high labeling density. Packaged for simple installation and use, DECODE will enable many laboratories to reduce imaging times and increase localization density in SMLM.}, journal={NATURE METHODS}, author={Speiser, Artur and Mueller, Lucas-Raphael and Hoess, Philipp and Matti, Ulf and Obara, Christopher J. and Legant, Wesley R. and Kreshuk, Anna and Macke, Jakob H. and Ries, Jonas and Turaga, Srinivas C.}, year={2021}, month={Sep} }
 @article{ahn_davis_daugird_zhao_quiroga_uryu_li_storey_tsai_keeley_et al._2021, title={Phase separation drives aberrant chromatin looping and cancer development}, ISSN={["1476-4687"]}, DOI={10.1038/s41586-021-03662-5}, abstractNote={The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.}, journal={NATURE}, author={Ahn, Jeong Hyun and Davis, Eric S. and Daugird, Timothy A. and Zhao, Shuai and Quiroga, Ivana Yoseli and Uryu, Hidetaka and Li, Jie and Storey, Aaron J. and Tsai, Yi-Hsuan and Keeley, Daniel P. and et al.}, year={2021}, month={Jun} }
 @article{pamula_carlini_forth_verma_suresh_legant_khodjakoy_betzig_kapoor_2019, title={High-resolution imaging reveals how the spindle midzone impacts chromosome movement}, volume={218}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201904169}, abstractNote={In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.}, number={8}, journal={JOURNAL OF CELL BIOLOGY}, author={Pamula, Melissa C. and Carlini, Lina and Forth, Scott and Verma, Priyanka and Suresh, Subbulakshmi and Legant, Wesley R. and Khodjakoy, Alexey and Betzig, Eric and Kapoor, Tarun M.}, year={2019}, month={Aug}, pages={2529–2544} }
 @article{moore_legant_2018, title={Improving probes for super-resolution}, volume={15}, ISSN={["1548-7105"]}, DOI={10.1038/s41592-018-0120-1}, abstractNote={Chemically modified DNA aptamers enable quantitative super-resolution imaging.}, number={9}, journal={NATURE METHODS}, author={Moore, Regan P. and Legant, Wesley R.}, year={2018}, month={Sep}, pages={659–660} }