An engineered, orthogonal auxin analog/<i>At</i>TIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in<i>Caenorhabditis elegans</i>

Hills-Muckey, Kelly; Martinez, Michael A Q; Stec, Natalia; Hebbar, Shilpa; Saldanha, Joanne; Medwig-Kinney, Taylor N.; Moore, Frances E Q; Ivanova, M. V.; Morao, Ana Karina; Ward, Jordan D.; Moss, Eric G.; Ercan, Sevinç; Zinovyeva, Anna Y.; Matus, David Q.; Hammell, Christopher M.

doi:10.1093/genetics/iyab174

Cited by 43 publications

(76 citation statements)

References 47 publications

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“…Adding Auxin further reduced ADL dye filling ( Figure 4—figure supplement 2C IV and Figure 4—figure supplement 2D ) compared to a control strain expressing TIR1 in ADL in the absence of the osm-6::AID allele. This is consistent with recent reports that TIR1 can target proteins for degradation in the absence of Auxin ( Hills-Muckey et al, 2021 ). Taken together these data confirm cell-specific knockdown of OSM-6 and that increased knockdown progressively impairs ADL cilia integrity.…”

Section: Resultssupporting

confidence: 94%

Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans

Valperga

Bono

2022

eLife

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Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. We probe such mechanisms by performing a forward genetic screen for mutants with enhanced O2 perception in Caenorhabditis elegans. Multiple mutants exhibiting increased O2 responsiveness concomitantly show defects in other sensory responses. One mutant, qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+ responses in the ADL pheromone-sensing neurons. At the same time, ADL responsiveness to pre-synaptic input from O2-sensing neurons is heightened in qui-1, and other sensory defective mutants, resulting in enhanced neurosecretion although not increased Ca2+ responses. Expressing qui-1 selectively in ADL rescues both the qui-1 ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling ADL neurons in qui-1 mutants highlights extensive changes in gene expression, notably of many neuropeptide receptors. We show that elevated ADL expression of the conserved neuropeptide receptor NPR-22 is necessary for enhanced ADL neurosecretion in qui-1 mutants, and is sufficient to confer increased ADL neurosecretion in control animals. Sensory loss can thus confer cross-modal plasticity by changing the peptidergic connectome.

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Section: Resultssupporting

confidence: 94%

Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans

Valperga

Bono

2022

eLife

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“…To this end, we inserted an auxin-inducible degron (AID) ( Zhang et al, 2015 ) into the ceh-34 locus using CRISPR/Cas9 genome engineering. Together with a ubiquitously expressed At TIR1 F79G ubiquitin ligase that recognizes the degron ( rps-28 driver; cshIs140 ; Hills-Muckey et al, 2022 ), this approach allows for temporal depletion of CEH-34 protein through addition of an auxin derivative (5-Ph-IAA) to the worm diet ( Figure 7A and B ). Postembryonic 5-Ph-IAA addition at either larval or adult stages resulted in downregulation of four tested markers for the differentiated state of different pharyngeal neuron classes, eat-4/VGluT, unc-17/VAChT, ser-7, and spp-12 ( Figure 7B and C ).…”

Section: Resultsmentioning

confidence: 99%

“… ( A ) Schematic of the AIDv2 system ( Hills-Muckey et al, 2022 ). Skp1, Cul1, Rbx1, and E2 are phylogenetically conserved components of the E3 ligase complex.…”

Section: Resultsmentioning

confidence: 99%

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The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34

Vidal

Gülez

Cao

et al. 2022

eLife

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Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. elegans homolog of the Drosophila Sine oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine oculis homolog, our findings invite speculations about the early evolution of nervous systems.

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“…(5-Ph-IAA) (Hills-Muckey et al, 2022;Negishi et al, 2022). The use of this TIR1 variant greatly reduces leaky degradation, while requiring low micromolar concentrations of 5-Ph-IAA that are less likely to affect the physiology of exposed animals.…”

Section: Introductionmentioning

confidence: 99%

The mIAA7 degron improves auxin-mediated degradation in C. elegans

Sepers

Verstappen

et al. 2022

Preprint

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Auxin-inducible degradation (AID) is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the AID system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in C. elegans, as previously reported in human cells. We tested depletion of a series of proteins with various sub-cellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for five out of six proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for two of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system (C.e.AID2), which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the AID system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

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An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system inCaenorhabditis elegans

Cited by 43 publications

References 47 publications

Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans

Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans

The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34

The mIAA7 degron improves auxin-mediated degradation in C. elegans

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