Biology subjects

Sepers, J. J.

Publications and source records attributed to Sepers, J. J..

3 recordsLinked to original sources

bioRxiv2022

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

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.

molecular biology↗

bioRxiv2022

Systemic coagulopathy drives host lethality in a new Drosophila tumor model

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvoraks characterization of tumors as wounds that never heal 1. Some of these responses lead to profound defects in blood clotting, such as Disseminated Intravascular Coagulopathy (DIC), which correlate with poor prognoses 2-4. Here, we demonstrate that a new tumor model in Drosophila provokes phenotypes that recapitulate coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by malignant growth exhaustion of host clotting components. Interestingly, clinical studies have suggested that lethality in patients with high serum levels of clotting components can be independent of thrombotic events 5,6. We show that rescuing coagulopathy improves survival of tumor-bearing flies, despite the fact that flies have an open circulatory system. Our work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality, as well as exploring other conserved mechanisms of host responses to chronic wounds.

developmental biology↗

bioRxiv2021

ERM-1 phosphorylation and NRFL-1 redundantly control lumen formation in the C. elegans intestine

Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defect in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.

cell biology↗