563 Publications

Microtubule reorganization during female meiosis in C. elegans

Ina Lantzsch, S. Fürthauer

Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic Caenorhabditis elegans spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.

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June 11, 2021

Metallic Microswimmers Driven up the Wall by Gravity

Q. Brosseau, F. Balboa Usabiaga, E. Lushi, Y. Wu, L. Ristroph, M. D. Ward, M. Shelley, J. Zhang

Experiments on autophoretic bimetallic nanorods propelling within a fuel of hydrogen peroxide show that tail-heavy swimmers preferentially orient upwards and ascend along inclined planes. We show that such gravitaxis is strongly facilitated by interactions with solid boundaries, allowing even ultraheavy microswimmers to climb nearly vertical surfaces. Theory and simulations show that the buoyancy or gravitational torque that tends to align the rods is reinforced by a fore-aft drag asymmetry induced by hydrodynamic interactions with the wall.

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June 11, 2021

Temporal integration of inductive cues on the way to gastrulation

Sarah McFann, Sayantan Dutta, Jared E. Toettcher, S. Shvartsman

In early development, cells commit to a single germ fate despite receiving multiple, conflicting inductive cues. Here, we examine how cells in the Drosophila embryo integrate promesodermal and proendodermal signals. We find that proendoderm signals repress transcriptional determinants of mesodermal cell movements during a critical time window in the early embryo. Based on precise optogenetic perturbations, live imaging, and computational modeling, our work provides a framework for quantitative understanding of combinatorial control of gastrulation dynamics. All study data are included in the article and/or supporting information.

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Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites

Parisa Hosseinzadeh, Paris R. Watson, Timothy W. Craven, V. Mulligan, et al.

Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain..

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Axisymmetric membranes with edges under external force: buckling, minimal surfaces, and tethers

L. Jia, Steven Pei, Robert A. Pelcovits, Thomas R. Powers

We use theory and numerical computation to determine the shape of an axisymmetric fluid membrane with a resistance to bending and constant area. The membrane connects two rings in the classic geometry that produces a catenoidal shape in a soap film. In our problem, we find infinitely many branches of solutions for the shape and external force as functions of the separation of the rings, analogous to the infinite family of eigenmodes for the Euler buckling of a slender rod. Special attention is paid to the catenoid, which emerges as the shape of maximal allowable separation when the area is less than a critical area equal to the planar area enclosed by the two rings. A perturbation theory argument directly relates the tension of catenoidal membranes to the stability of catenoidal soap films in this regime. When the membrane area is larger than the critical area, we find additional cylindrical tether solutions to the shape equations at large ring separation, and that arbitrarily large ring separations are possible. These results apply for the case of vanishing Gaussian curvature modulus; when the Gaussian curvature modulus is nonzero and the area is below the critical area, the force and the membrane tension diverge as the ring separation approaches its maximum value. We also examine the stability of our shapes and analytically show that catenoidal membranes have markedly different stability properties than their soap film counterparts.

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June 2, 2021

Single nucleus multi-omics regulatory atlas of the murine pituitary

F Ruf-Zamojski, Z. Zhang, M Zamojski, O. Troyanskaya, S Sealfon, et al.

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.

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Single-cell gene regulatory network inference at scale: The Inferelator 3.0

C. Skok Gibbs, A. Watters, N. Carriero, R. Bonneau, et al.

Gene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. In this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator 3.0 reliably learns informative networks from the model organisms Bacillus subtilis and Saccharomyces cerevisiae. We demonstrate its capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression data set with paired single-cell chromatin accessibility data.

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A stable and accurate scheme for solving the Stefan problem coupled with natural convection using the Immersed Boundary Smooth Extension method

S. Ju, H. Chu, M. Shelley, J. Zhang

In cellular vortical flows, short but flexible filaments can show simple random walks through their stretch-coil interactions with flow stagnation points. Here, we study the dynamics of semi-rigid filaments long enough to broadly sample the vortical field. Using simulation, we find a surprising variety of long-time transport behavior -- random walks, ballistic transport, and trapping -- depending upon the filament's relative length and effective flexibility. Moreover, we find that filaments execute Lévy walks whose diffusion exponents generally decrease with increasing filament length, until transitioning to Brownian walks. Lyapunov exponents likewise increase with length. Even completely rigid filaments, whose dynamics is finite-dimensional, show a surprising variety of transport states and chaos. Fast filament dispersal is related to an underlying geometry of "conveyor belts". Evidence for these various transport states are found in experiments using arrays of counter-rotating rollers, immersed in a fluid and transporting a flexible ribbon.

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A hydraulic instability drives the cell death decision in the nematode germline

N. T. Chartier, A. Mukherjee, Sebastian Fürthauer, et al.

Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow—typically at the expense of others that undergo cell death. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.

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SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma

J. Choe, P. Watchmaker, M. Simic , O. Troyanskaya, et al.

Two major hurdles in chimeric antigen receptor (CAR) T cell therapy for solid tumors are ensuring specificity to tumor cells without affecting healthy cells and avoiding tumor escape due to antigen loss. To address these challenges, Hyrenius-Wittsten et al. and Choe et al. developed synthetic notch (synNotch)–CAR T cells targeting solid tumor antigens and used them to treat mouse models of mesothelioma, ovarian cancer, and glioblastoma. In both studies, the authors demonstrated that synNotch-CAR T cells were better at controlling tumors than traditional CAR T cells and did not result in toxicity or damage to healthy tissue. These results suggest that synNotch-CAR T cells may be an effective treatment strategy for solid tumors.

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