A global genetic interaction network by single-cell imaging and machine learning

Heigwer, Florian; Scheeder, Christian; Bageritz, Josephine; Yousefian, Schayan; Rauscher, Benedikt; Laufer, Christina; Beneyto-Calabuig, Sergi; Funk, Maja C; Peters, Vera Maria; Boulougouri, Maria; Bilanovic, Jana; Miersch, Thilo; Schmitt, Barbara; Blass, Claudia; Port, Fillip; Boutros, Michael

doi:10.1016/j.cels.2023.03.003

Cited by 7 publications

(3 citation statements)

References 91 publications

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“…In conclusion, we suggest that a stably coupled, interconnected system of endogenous oscillators and clocks oscillating at defined frequency bands defines dynamic homeostatic setpoints of physiology and behavior. This systems-based concept comprising different time scales and different levels of complexity differs from the currently dominating hierarchical concepts of chronobiology (e.g., (Harvey et al, 2020;Rosbash, 2021;Patton and Hastings, 2023)) but appears to find increasing support (Hall, 2005;Stengl et al, 2015;Stengl and Arendt, 2016;Rojas et al, 2019;Jaumouillé et al, 2021;Stengl and Schröder, 2021;Yang et al, 2022;Heigwer et al, 2023). Furthermore, contrasting the current hypothesis (Hughes et al, 2009;Zhu et al, 2017;Ballance and Zhu, 2021) we propose an equally important role for plasma membrane associated PTFL clocks as compared to TTFL clocks associated with nuclear gene regulatory networks.…”

Section: Discussion: Our Novel Hypothesis Final Conclusion and Outlookcontrasting

confidence: 65%

Contribution of membrane-associated oscillators to biological timing at different timescales

Stengl¹,

Schneider²

2023

Preprint

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Environmental rhythms have selected for endogenous clocks that predict regular environmental changes like the daily light-dark cycle. Endogenous clocks are based on positive feedforward and negative feedback loops that generate rhythms even under constant conditions. Autonomous coupling of oscillators is the basis of self-organized timing that determines the repetitive sequence of events of physiology and behavior while mechanisms of homeostatic plasticity serve to maintain and stabilize physiological and behavioral homeostasis. The best studied biological clocks are circadian clock neurons of insects and mammals. Their molecular clockwork in the nucleus consists of transcriptional and translational feedback loops (TTFLs). This clockwork generates ~24 h rhythms in clock gene expression entrained to the light-dark cycle. It is assumed to drive all circadian oscillations in clock neurons such as rhythms in membrane potential, second messenger levels, and neurotransmitter release, thereby controlling circadian rhythms in physiology and behavior. It is not discerned yet whether the membrane-bound rhythms are mere outputs of the TTFL clockwork or whether instead they are driven by an autonomous posttranscriptional feedback loop (PTFL) clockwork in the plasma membrane. Furthermore, next to circadian rhythms the plasma membrane generates rhythms on much faster or slower timescales. It is not understood whether and how these multiscale rhythms are linked. Based on our work in peripheral and central insect multiscale clock neurons, we hypothesize that the excitable plasma membrane constitutes an adaptive endogenous PTFL clockwork at multiple timescales. Multiscale membrane-associated rhythms are suggested to be coupled to, but not driven by, the TTFL clockwork in the nucleus. Furthermore, we suggest that negative feedback loops via protein kinases and phosphatases on the one hand provide for mechanisms of homeostatic plasticity that maintain physiological homeostasis, keeping the period of cellular clocks stable. On the other hand, they also serve as coupling factors and links between cellular oscillators and clocks at multiple time scales controlling the temporal sequence and/or synchronization of physiological and behavioral processes. Our novel hypothesis is up for challenge in future experiments in different species.

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Section: Discussion: Our Novel Hypothesis Final Conclusion and Outlookcontrasting

confidence: 65%

Contribution of membrane-associated oscillators to biological timing at different timescales

Stengl¹,

Schneider²

2023

Preprint

View full text Add to dashboard Cite

show abstract

“…We suggest that a stably coupled, interconnected system of endogenous oscillators and clocks oscillating at defined frequency bands defines dynamic homeostatic setpoints of physiology and behavior. This systems-based concept comprising different time scales and different levels of complexity differs from the currently dominating hierarchical concepts of chronobiology (e.g., Harvey et al, 2020 ; Rosbash, 2021 ; Patton and Hastings, 2023 ) but appears to find increasing support ( Hall, 2005 ; Stengl et al, 2015 ; Stengl and Arendt, 2016 ; Rojas et al, 2019 ; Jaumouillé et al, 2021 ; Stengl and Schröder, 2021 ; Yang et al, 2022 ; Heigwer et al, 2023 ). Furthermore, contrasting the current hypothesis ( Hughes et al, 2009 ; Zhu et al, 2017 ; Ballance and Zhu, 2021 ) we propose an equally important role for plasma membrane associated PTFL clocks as compared to TTFL clocks associated with nuclear gene regulatory networks.…”

Section: Discussion: Our Novel Hypothesis Final Conclusion and Outlookmentioning

confidence: 78%

Contribution of membrane-associated oscillators to biological timing at different timescales

Stengl,

Schneider

2024

Front. Physiol.

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Environmental rhythms such as the daily light-dark cycle selected for endogenous clocks. These clocks predict regular environmental changes and provide the basis for well-timed adaptive homeostasis in physiology and behavior of organisms. Endogenous clocks are oscillators that are based on positive feedforward and negative feedback loops. They generate stable rhythms even under constant conditions. Since even weak interactions between oscillators allow for autonomous synchronization, coupling/synchronization of oscillators provides the basis of self-organized physiological timing. Amongst the most thoroughly researched clocks are the endogenous circadian clock neurons in mammals and insects. They comprise nuclear clockworks of transcriptional/translational feedback loops (TTFL) that generate ∼24 h rhythms in clock gene expression entrained to the environmental day-night cycle. It is generally assumed that this TTFL clockwork drives all circadian oscillations within and between clock cells, being the basis of any circadian rhythm in physiology and behavior of organisms. Instead of the current gene-based hierarchical clock model we provide here a systems view of timing. We suggest that a coupled system of autonomous TTFL and posttranslational feedback loop (PTFL) oscillators/clocks that run at multiple timescales governs adaptive, dynamic homeostasis of physiology and behavior. We focus on mammalian and insect neurons as endogenous oscillators at multiple timescales. We suggest that neuronal plasma membrane-associated signalosomes constitute specific autonomous PTFL clocks that generate localized but interlinked oscillations of membrane potential and intracellular messengers with specific endogenous frequencies. In each clock neuron multiscale interactions of TTFL and PTFL oscillators/clocks form a temporally structured oscillatory network with a common complex frequency-band comprising superimposed multiscale oscillations. Coupling between oscillator/clock neurons provides the next level of complexity of an oscillatory network. This systemic dynamic network of molecular and cellular oscillators/clocks is suggested to form the basis of any physiological homeostasis that cycles through dynamic homeostatic setpoints with a characteristic frequency-band as hallmark. We propose that mechanisms of homeostatic plasticity maintain the stability of these dynamic setpoints, whereas Hebbian plasticity enables switching between setpoints via coupling factors, like biogenic amines and/or neuropeptides. They reprogram the network to a new common frequency, a new dynamic setpoint. Our novel hypothesis is up for experimental challenge.

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“…In addition, this high-content screen allows for further interpretation directly from screening data. Indeed, using dimensionality reduction and unsupervised clustering 24,25,27 , we identify groups of genes that similarly affect STING trafficking when knocked out and we show that this dataset is useful for assigning functional roles to previously uncharacterized genes. We focused on two groups of genes regulating STING degradation at distinct stages and found that the HOPS complex is required for signaling shutdown at the endolysosome.…”

Section: Introductionmentioning

confidence: 98%

Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening

Gentili,

Carlson,

Liu

et al. 2024

Preprint

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STING is an innate immune sensor that traffics across many cellular compartments to carry out its function of detecting cyclic di-nucleotides and triggering defense processes. Mutations in factors that regulate this process are often linked to STING-dependent human inflammatory disorders. To systematically identify factors involved in STING trafficking, we performed a genome-wide optical pooled screen and examined the impact of genetic perturbations on intracellular STING localization. Based on subcellular imaging of STING protein and trafficking markers in 45 million cells perturbed with sgRNAs, we defined 464 clusters of gene perturbations with similar cellular phenotypes. A higher-dimensional focused optical pooled screen on 262 perturbed genes which assayed 11 imaging channels identified 73 finer phenotypic clusters. In a cluster containing USE1, a protein that mediates Golgi to ER transport, we found a gene of unknown function, C19orf25. Consistent with the known role of USE1, loss of C19orf25 enhanced STING signaling. Other clusters contained subunits of the HOPS, GARP and RIC1-RGP1 complexes. We show that HOPS deficiency delayed STING degradation and consequently increased signaling. Similarly, GARP/RIC1-RGP1 loss increased STING signaling by delaying STING exit from the Golgi. Our findings demonstrate that genome-wide genotype-phenotype maps based on high-content cell imaging outperform other screening approaches, and provide a community resource for mining for factors that impact STING trafficking as well as other cellular processes observable in our dataset.

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A global genetic interaction network by single-cell imaging and machine learning

Cited by 7 publications

References 91 publications

Contribution of membrane-associated oscillators to biological timing at different timescales

Contribution of membrane-associated oscillators to biological timing at different timescales

Contribution of membrane-associated oscillators to biological timing at different timescales

Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening

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