AFERGIN TAGC 2020 poster.pdf (4.24 MB)


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posted on 20.04.2020, 23:45 by Aleksandra Fergin, Gabriel Boesch, Evelyn Lattmann, Charlotte A. Lambert, Alex Hajnal

Poster 1391B for TAGC online 2020. File includes PDF of the poster (AFERGIN_TAGC 2020 poster.pdf)


The sumoylation pathway is involved in a variety of processes in C. elegans including gonadal and vulval fate specification, cell cycle progression, maintenance of chromosome structure, and chromosome segregation. Since many transcription factors crucial for normal development are modified by SUMO, studying the exact role of this posttranslational modification with respect to specific targets is a challenging question. Therefore, we have developed the tools to block sumoylation in a tissue-specific and temporally controlled manner to study how the loss of sumoylation affects vulval development and the AC invasion. For this purpose, we are using the auxin-inducible tissue-specific protein degradation system to down-regulate degron-tagged components of the sumoylation pathway (such as the E3 ligase GEI-17 and SMO-1) in the AC or vulval precursor cells (VPCs).


The auxin-inducible (AID) degradation system is using a modified Arabidopsis thaliana TIR1 (substrate recognition component of Skp1-Cullin-F-box E3 ubiquitin ligase complex), which leads to depletion of the degron-tagged proteins in response to phytohormone auxin.

We applied AID to down-regulate degron-tagged components of the sumoylation pathway (such as the E3 ligase GEI-17 and SMO-1) in the AC or VPCs. We designed the tissue-specific TIR1 construct containing a trans-splicing domain SL2 inserted between TIR1 and mCherry. We crossed them with the degron-tagged SMO-1 and GEI-17 (generated by CRISPR/Cas9 mediated genome editing approach) and performed auxin treatment experiments.


Validation of auxin-inducible degradation system

We observed that upon 1mM auxin treatment, the degradation of GEI-17 in somatic tissues resulted in the approximately 70% of the P0 animals showing a protruding vulva phenotype (Pvl - characteristic of SUMO pathway deletion mutants) and sterility.

Degradation of GEI-17 in somatic tissues leads to various phenotypes

Degradation of GEI-17 in somatic cells resulted in many developmental defects during vulva formation - the AC polarity defects and misplacement of the AC, presence of multiple ACs instead of only one, BM breaching defects and BM breaching shift, various morphogenesis defects including toroid formation, and utse formation defects.

Degradation of GEI-17 in the AC results in defective dorsal lumen opening

Degradation of GEI-17 in the AC resulted in defects in the dorsal lumen formation.

Degradation of GEI-17 in the VPCs leads to defects in cell-fate specification

We crossed [egl-17::YFP] (1° cell fate marker) into animals containing degron-tagged GEI-17 and bar-1p::TIR1::SL2::mCherry to study the role of sumoylation during cell-fate specification. We observed that the expression of EGL-17 is much lower in the animals treated with auxin compared to the control during one-cell, two-cells and four-cells stage. This experiment shows that degradation of GEI-17 in the VPCs results in a disturbance of cell-fate specification.

Degradation of GEI-17 results in toroid formation defects during vulval morphogenesis.

We crossed [ajm-1::GFP] (cell junction marker) into animals containing degron-tagged GEI-17 and TIR1 expressed in somatic tissues to study sumoylation role in toroid formation. 3D reconstruction has revealed that degradation of GEI-17 in somatic tissues leads to diverse toroid formation defects.


We crossed degron-tagged SMO-1 with strains containing a TIR1 driver under somatic, VPC and AC-specific promoters. We treated those animals with 1mM auxin from L2 to L4 stage at 20°C and scored the Pvl phenotype in adults. We observed 100%, or nearly 100% penetrance of the Pvl phenotype in case of degradation of SMO-1 in somatic cells, and 1° and 2° VPCs accordingly. Degradation in 1° VPCs resulted in 25% penetrance of the Pvl phenotype. The AC-specific degradation of SMO-1 caused only 7% of Pvls. This experiment shows that SMO-1 plays a crucial role in secondary VPCs and disruption of its function in this tissue leads to the most severe defects in vulva morphogenesis. Disturbing SMO-1 in the primary VPCs and AC does not contribute in the same extent to the Pvl phenotype.


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