Optogenetic control of Nodal signaling patterns

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Апрель 12, 2024

Abstract A crucial step in early embryogenesis is the establishment of spatial patterns signaling activity. Tools to perturb morphogen signals with high resolution space and time can help reveal how embryonic cells decode these make appropriate fate decisions. Here, we present new optogenetic reagents an experimental pipeline for creating designer Nodal live zebrafish embryos. receptors were fused light-sensitive heterodimerizing pair Cry2/CIB1N, Type II receptor was sequestered cytosol. The improved optoNodal2 eliminate dark activity improve response kinetics, without sacrificing dynamic range. We adapted ultra-widefield microscopy platform parallel light patterning up 36 embryos demonstrated precise control over downstream gene expression. Patterned activation drove precisely controlled internalization endodermal precursors. Further, used patterned illumination generate synthetic mutants, rescuing several characteristic developmental defects. This study establishes toolkit systematic exploration

Язык: Английский

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Light-directed evolution of dynamic, multi-state, and computational protein functionalities

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 2, 2024

Abstract Directed evolution is a powerful method in biological engineering. Current approaches draw on time-invariant selection mechanisms, ideal for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to continuously evolve dynamic, multi-state, computational functionalities, e.g., on-off kinetics, state-specific activity, stimulus-responsiveness, switching and logic capabilities. These require pressure all of the states protein interest (POI) transitions between them. We realized that optogenetics cell cycle oscillations could be leveraged novel directed paradigm (‘optovolution’) germane this need: designed signaling cascade budding yeast where optogenetic input switches POI off (0) (1) states. In turn, controls Cdk1 cyclin, which re-engineered system essential one stage but poisonous another. Thus, cyclin must oscillate (1-0-1-0…) proliferation. system, can act efficiently dynamics, transient states, input-output relations every cycle. Further, controlling pacemaker, light, directs tunes pressures. Optovolution vivo, continuous, self-selecting, efficient. first evolved two systems, relay 0/1 output: obtained 25 new variants widely used LOV transcription factor El222 were stronger, less leaky, green-and red-responsive. The latter was conjectured impossible domains needed multiplexing lowering phototoxicity. Evolving PhyB-Pif3 we discovered loss YOR1 makes supplementing expensive unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, demonstrate generality by destabilized rtTA factor, performs an AND operation transcriptional doxycycline input. coveted, difficult-to-change functionalities evolvable.

Язык: Английский

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In vivomeasurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 7, 2025

A lack of tools for detecting receptor activity in vivo has limited our ability to fully explore receptor-level control developmental patterning. Here, we extend a new class biosensors tyrosine kinase (RTK) activity, the pYtag system, visualize endogenous RTK Drosophila . We build three RTKs that function across stages and tissues. By characterizing Torso::pYtag during terminal patterning early embryo, find Torso differs from downstream ERK two surprising ways: is narrowly restricted poles but produces broader gradient ERK, decreases over time while sustained. This decrease driven by pathway-dependent negative feedback. Our results suggest an updated model where narrow domain tuned amplitude feedback, locally activates signaling effectors which diffuse through syncytial embryo form gradient. Altogether, this work highlights usefulness pYtags investigating regulation

Язык: Английский

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Temporal changes in PTTH/Egf signaling and ERK target gene expressions during chilling in Bombyx mori eggs

Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology, Год журнала: 2025, Номер 307, С. 111884 - 111884

Опубликована: Июнь 2, 2025

Язык: Английский

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