THE ROLE OF PROTEIN SUMOYLATION DURING VULVAL MORPHOGENESIS AND ANCHOR CELL INVASION ALEKSANDRA FERGIN, Gabriel Boesch, Evelyn Lattmann, Charlotte A. Lambert, Alex Hajnal FINDINGS Protruding vulva phenotype [%] [degron::FLAG::SMO-1] SUMOYLATION plays crucial role in the VPCs in securing proper vulva formation. Application of auxin-inducible degradation (AID) system in a tissue-specific manner TIR1 Cul1 E2 Skp1 Rbx1 Target protein degron auxin Ub Ub Ub Ub Ub Ub Tissue-specific promoter TIR1 mCherry SL2 degron GEI-17 GFP FLAG LoxP GEI-17 degron SMO-1 FLAG LoxP Generation of tissue-specific degradation drivers cdh-3p::TIR1::SL2-mCherry egl-17p::TIR1::SL2::mCherry bar-1p::TIR1::SL2::mCherry Generation of degron-tagged target proteins Endogenously integrated by CRISPR/Cas9 control 1mM auxin eft-3p::TIR1::mRuby; GFP::FLAG::degron::GEI-17 control 1mM auxin 0 20 40 60 80 100 ✱✱✱✱ Validation of auxin-inducible degradation system Degradation of GEI-17 in somatic tissues leads to abnormal vulva phenotype [%] egl-17p::TIR1::SL2::mCherry; GFP::FLAG::degron::GEI-17 control 1mM auxin Example of tissue-specific degradation of GEI-17 in primary VPCs Degradation of GEI-17 in somatic tissues leads to various phenotypes Degradation of GEI-17 in the AC results in defective dorsal lumen opening control 1mM auxin 0 2 4 6 8 10 ✱✱✱✱ control 1mM auxin 0 20 40 60 80 ✱✱✱✱ AC shift [o] BM shift ratio control 1mM auxin 0 20 40 60 80 100 ✱✱ BM breaching defect [%] control 1mM auxin 0 20 40 60 80 100 ✱✱✱✱ control 1mM auxin 0 20 40 60 80 100 ✱✱✱✱ Morphogenesis defect [%] AC fusion defect [%] control 1mM auxin 0 20 40 60 80 100 ✱✱✱✱ AC polarity defect [%] control 1mM auxin 0 2 4 6 8 ✱✱✱ Dorsal lumen diameter [µm] Degradation of GEI-17 in the VPCs leads to defects in cell-fate specification bar-1p::TIR1::SL2::mCherry; degron::FLAG::GEI-17; egl-17::YFP P6.p P6.px P6.pxx 1 mM auxin control egl-17::YFP L4.1 L4.1 L4.4 eft-3p::TIR1::mRuby; GFP::FLAG::degron::GEI-17 AC polarity defect multiple AC misplaced AC BM breaching defect AC BM 1mM auxin P6.p control P6.p 1mM auxin P6.px control P6.px 1mM auxin P6.pxx control P6.pxx 1mM auxin 0 50 100 150 200 250 ✱✱ ✱✱✱ ✱✱✱✱ Mean intensity of in P6.p-P6.pxx METHODS Integrated by mos1-mediated Single Copy Insertion (mosSCI) RESULTS Degradation of GEI-17 results in toroid formation defects during vulval morphogenesis control auxin 0.0 0.2 0.4 0.6 0.8 1.0 ✱✱✱✱ Diameter ratio VulB/VulA AC BM INTRODUCTION INTRODUCTION To study how the loss of protein sumoylation affects vulval development and AC invasion, we developed tools to block sumoylation in a tissue-specific and temporally controlled manner. For this purpose, we have been using the auxin-inducible tissue-specific protein degradation system to down- regulate a degron-tagged SUMO E3 ligase (GEI-17) or a degron-tagged SUMO (SMO-1) in different tissues of the worm. We generated transgenic animals expressing a modified Arabidopsis thaliana TIR1, an E3 ubiquitin ligase specific for the degron tag, under the VPC and the AC specific promoters. Tissue-specific degradation of sumoylation pathway components during the AC invasion OR AC BM VPCs 1mM auxin control cdh-3p::TIR1::SL2::mCherry; GFP::FLAG::degron::GEI-17 BM eft-3p::TIR1::mRuby; degron::FLAG::GEI-17; ajm::GFP 1 mM auxin control ajm::GFP eft-3p::TIR1::mRuby; degron::FLAG::GEI-17; ajm::GFP Toroid fusion defect [%] control auxin 0 20 40 60 80 100 ✱✱✱ VulD fusion defect [%] control auxin 0 20 40 60 80 100 ✱✱ no TIR1 eft-3p::TIR1::mRuby bar-1p::TIR1::SL2::mCherry egl-17p::TIR1::SL2::mCherry cdh-3p::TIR1::SL2::mCherry 0 20 40 60 80 100 control 1mM auxin ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱ degron FLAG LoxP Pelisch et al. Mol Cell. 2017 Jan 5;65(1):66-77. P6.p P5.p P7.p