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Sirota, S. S.

Publications and source records attributed to Sirota, S. S..

2 recordsLinked to original sources

Transcriptional regulation and repressive condensates modulate a proliferative-invasive cellular switch in vivo

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of nhr-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

developmental biology↗

Expanding the Caenorhabditis elegans auxin-inducible degron system toolkit with internal expression and degradation controls and improved modular constructs for CRISPR/Cas9-mediated genome editing

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell-types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying an equimolar co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. We have also constructed a set of plasmids to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These templates can be produced through frequently used cloning systems (Gibson assembly or SapTrap) or through ribonucleoprotein complex-mediated insertion of PCR-derived, linear repair templates. We have generated a set of sgRNA plasmids carrying modifications shown to boost editing efficiency, targeting standardized transgene insertion sites on chromosomes I and II. Together these reagents should complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID* tagging of factors of interest. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for facile assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

genetics↗