Light-microscopy methods in C. elegans research

Ever since Caenorhabditis elegans was introduced as a model system it has been tightly linked to microscopy, which has led to significant advances in understanding biology over the last decades. Developing new technologies therefore is an essential part in the endeavor to gain further mechanistic insights into developmental biology. This review will discuss state-of-the-art developments in quantitative light microscopy in the context of C. elegans research as well as the impact these technologies have on the field. We will highlight future developments that currently promise to revolutionize biological research by combining sequencing-based single-cell technologies with high-resolution quantitative imaging

Constraining Stellar Rotation at the Zero-Age Main Sequence with TESS

The zero-age main sequence (ZAMS) is a critical phase for stellar angular momentum evolution, as stars transition from contraction-dominated spin-up to magnetic wind-dominated spin-down. We present the first robust observational constraints on rotation for FGK stars at $\approx40$ Myr. We have analyzed TESS light curves for 1410 members of five young open clusters with ages between 25-55 Myr: IC 2391, IC 2602, NGC 2451A, NGC 2547, and Collinder 135. In total, we measure 868 rotation periods, including 96 new, high-quality periods for stars around 1 ${M_{\odot}}$. This is an increase of ten times the existing literature sample at the ZAMS. We then use the $\tau^2$ method to compare our data to models for stellar angular momentum evolution. Although the ages derived from these rotation models do not match isochronal ages, we show these observations can clearly discriminate between different models for stellar wind torques. Finally, $\tau^2$ fits indicate that magnetic braking and/or internal angular momentum transport significantly impact rotational evolution even on the pre-main sequence.Comment: 20 pages, 11 figures, 4 tables; ApJ Accepte

mRNA stability and m(6)A are major determinants of subcellular mRNA localization in neurons

For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here, we assess the role of mRNA stability in localization by combining the isolation of the soma and neurites of mouse primary cortical and mESC-derived neurons, SLAM-seq, m(6)A-RIP-seq, the perturbation of mRNA destabilization mechanisms, and the analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m(6)A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons

RS-FISH: Precise, interactive, fast, and scalable FISH spot detection

Fluorescent in-situ hybridization (FISH)-based methods are powerful tools to study molecular processes with subcellular resolution, relying on accurate identification and localization of diffraction-limited spots in microscopy images. We developed the Radial Symmetry-FISH (RS-FISH) software that accurately, robustly, and quickly detects single-molecule spots in two and three dimensions, making it applicable to several key assays, including single-molecule FISH (smFISH), spatial transcriptomics, and spatial genomics. RS-FISH allows interactive parameter tuning and scales to large sets of images as well as tera-byte sized image volumes such as entire brain scans using straight-forward distributed processing on workstations, clusters, and in the cloud

Poster CS20.5 - Weakened magnetic braking supported by asteroseismic rotation

Studies using asteroseismic ages and rotation rates from star-spot rotation have indicated that standard age-rotation relations may break down roughly half-way through the main sequence lifetime, a phenomenon referred to as weakened magnetic braking. While rotation rates from spots can be difficult to determine for older, less active stars, rotational splitting of asteroseismic oscillation frequencies can provide rotation rates for both active and quiescent stars, and so can confirm whether this effect really takes place on the main sequence. In this talk, I’ll show how we obtained asteroseismic rotation rates of 91 main sequence stars showing high signal-to-noise modes of oscillation. Using these new rotation rates, along with effective temperatures, metallicities and seismic masses and ages, we built a hierarchical Bayesian mixture model that showed that our new ensemble more closely agreed with weakened magnetic braking, over a standard rotational evolution scenario

RS-FISH: precise, interactive, fast, and scalable FISH spot detection

Fluorescent in-situ hybridization (FISH)-based methods extract spatially resolved genetic and epigenetic information from biological samples by detecting fluorescent spots in microscopy images, an often challenging task. We present Radial Symmetry-FISH (RS-FISH), an accurate, fast, and user-friendly software for spot detection in two- and three-dimensional images. RS-FISH offers interactive parameter tuning and readily scales to large datasets and image volumes of cleared or expanded samples using distributed processing on workstations, clusters, or the cloud. RS-FISH maintains high detection accuracy and low localization error across a wide range of signal-to-noise ratios, a key feature for single-molecule FISH, spatial transcriptomics, or spatial genomics applications

Massively parallel identification of mRNA localization elements in primary cortical neurons

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called ‘zipcodes’. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3′ untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons—cell bodies and neurites—with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes

The H4K20 demethylase DPY-21 regulates the dynamics of condensin DC binding

Condensin is a multi-subunit SMC complex that binds to and compacts chromosomes. Here we addressed the regulation of condensin binding dynamics using C. elegans condensin DC, which represses X chromosomes in hermaphrodites for dosage compensation. We established fluorescence recovery after photobleaching (FRAP) using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes its binding. Next, we performed FRAP in the background of several chromatin modifier mutants that cause varying degrees of X-chromosome derepression. The greatest effect was in a null mutant of the H4K20me2 demethylase DPY-21, where the mobile fraction of condensin DC reduced from ∼30% to 10%. In contrast, a catalytic mutant of dpy-21 did not regulate condensin DC mobility. Hi-C data in the dpy-21 null mutant showed little change compared to wild type, uncoupling Hi-C measured long-range DNA contacts from transcriptional repression of the X chromosomes. Together, our results indicate that DPY-21 has a non-catalytic role in regulating the dynamics of condensin DC binding, which is important for transcription repression

The H4K20 demethylase DPY-21 regulates the dynamics of condensin DC binding

Condensin is a multi-subunit SMC complex that binds to and compacts chromosomes. Unlike cohesin, in vivo regulators of condensin binding dynamics remain unclear. Here we addressed this question using C. elegans condensin DC, which specifically binds to and represses transcription of both X chromosomes in hermaphrodites for dosage compensation. Mutants of several chromatin modifiers that regulate H4K20me and H4K16ac cause varying degrees of X chromosome derepression. We used fluorescence recovery after photobleaching (FRAP) to analyze how these modifiers regulate condensin DC binding dynamics in vivo. We established FRAP using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes its binding. The greatest effect on condensin DC dynamics was in a null mutant of the H4K20me2 demethylase DPY-21, where the mobile fraction of the complex reduced from ∼30% to 10%. In contrast, a catalytic mutant of dpy-21 did not regulate condensin DC mobility. Separation of catalytic and non-catalytic activity is also supported by Hi-C data in the dpy-21 null mutant. Together, our results indicate that DPY-21 has a non-catalytic role in regulating the dynamics of condensin DC binding, which is important for transcription repression

Implementation of the Random Forest Method for the Imaging Atmospheric Cherenkov Telescope MAGIC

The paper describes an application of the tree classification method Random Forest (RF), as used in the analysis of data from the ground-based gamma telescope MAGIC. In such telescopes, cosmic gamma-rays are observed and have to be discriminated against a dominating background of hadronic cosmic-ray particles. We describe the application of RF for this gamma/hadron separation. The RF method often shows superior performance in comparison with traditional semi-empirical techniques. Critical issues of the method and its implementation are discussed. An application of the RF method for estimation of a continuous parameter from related variables, rather than discrete classes, is also discussed.Comment: 16 pages, 8 figure