TY - GEN T1 - Characterizing Dynein’s Role in P-cell Nuclear Migration using an Auxin-Induced Degradation System AU - Ho, Jamie AU - Venecia A. Valdez, AU - Ma, Linda AU - Starr, Daniel A. DO - 10.17912/W2W96J UR - https://www.micropublication.org/journals/biology/w2w96j/ AB - Here, we tested the hypothesis that the unc-83/unc-84 pathway works through dynein to move P-cell nuclei using the auxin-inducible degradation system (AID) to knock down dynein specifically during P-cell nuclear migration (Zhang et al., 2015). The Arabidopsis thaliana TIR1 gene was expressed downstream of the C. elegans P-cell specific hlh-3 promoter (Bone et al., 2016) in the strain UD550 (oxIs12[unc-47::GFP]; ycEx253[phlh-3::TIR-1::mRuby; odr-1::rfp]). Next, UD550 was crossed to a strain with dhc-1(ie28[dhc-1::degron::GFP]) (Zhang et al., 2015) to make UD551 (dhc-1(ie28[dhc-1::degron::GFP]) I; oxIs12[unc-47::GFP] X; ycEx253[phlh-3::TIR-1::mRuby; odr-1::rfp]). UD550 and UD551 animals were synchronized to mid L1 as previously described (Bone et al., 2016) and exposed to 1mM auxin for five hours at 25°C. P-cell nuclear migration defects were quantified by counting GABA neurons marked with UNC-47::GFP by fluorescence microscopy in L4 animals after the auxin treatment. Wild-type animals have 19 GABA neurons and missing GABA neurons indicate that P-cell nuclear migration failed (Chang et al., 2013; Bone et al., 2016). As a negative control, UD550 animals, which lack the dhc-1 degron tag, had no P-cell nuclear migration defects when exposed to auxin. This result is similar to UD551 animals that were not exposed to auxin (Figure 1). In support of our hypothesis, larvae exposed to auxin that expressed both the TIR1 gene in P cells and the dhc-1 degron tag (UD551) had an average of 12.3 GABA neurons compared to sibling larvae not exposed to auxin that had an average of 18.1 GABA neurons (Figure 1). Thus, the dynein-degraded larvae had an average of 5.8 missing GABA neurons (p<0.0001), indicative of a severe P-cell nuclear migration defect. These results further strengthen our model that dynein plays a major role in generating forces to move nuclei in P-cells through constricted spaces. Finally, the phlh-3::TIR1 line will be a valuable reagent to knock down other essential proteins to determine their roles during P-cell nuclear migration. PY - 2018 PB - Micropublication:biology ER -