Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen‐age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior–posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis–diffusion–degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen‐age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): study protocol for a two-factorial randomized controlled multi-arm trial with cluster sampling

Background: To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. Methods: This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals' self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups (individuals vs. all household members) and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Discussion: Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures. Trial registration: ClinicalTrials.gov DRKS00023271. Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien

Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast [Dataset]

Data accompanying the paper "Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast" by Buchmuller and Herbst et al, 2019, Nat Communications. This contains raw NGS data for all genotyping analysis in the publication as well as the source code of the castR design software (v1.0)

Protein quality control at the inner nuclear membrane.

International audienceThe nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM

Genome-wide C-SWAT library for high-throughput yeast genome tagging

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.</p