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. 2015 Feb 12;11(2):e1004974. doi: 10.1371/journal.pgen.1004974

Figure 5. NUP153 and RAN are required for circadian rhythms and interact with TIM.

Figure 5

(A) Knockdown of Nup153 disrupts rest:activity rhythms. Representative activity records of individual pdf-Gal4/+; dicer/+ (left) and pdf-Gal4/nup153 RNAi; dicer/+ (right) flies are shown. (B) PDF expression in nup153 knockdown flies was detected in l-LNvs, but not in the s-LNvs. In nup153 knockdown flies (right), the s-LNv dorsal tract was impaired as indicated by the white arrow. Low, but cytoplasmic expression of PER (green) was detected in l-LNvs at ZT1. In control flies (left), PER (green) was nuclear in both s- and l-LNvs at ZT1. (C) S2 cells were transfected with pIZ-V5 or pIZ-tim-V5 constructs. After 60 hours, cell lysates were immunoprecipitated with an anti-V5 antibody and western blots were probed with an anti-NUP153 antibody (left). GST pulldown assays were also conducted to test for an interaction between the FG repeat of NUP153 (GST-FG) and TIM (right). Cell extracts from S2 cells transfected with pIZ-tim-V5 were incubated with purified recombinant GST-FG or a GST control. Proteins pulled down by GST were analyzed with an anti-V5 antibody (that recognizes TIM-V5). Similar results were obtained in three independent experiments. (D) Expression of a dominant negative form of Ran (RanDN) in PDF+ cells during adulthoods leads to arrhythmia in DD. RanDN was expressed under control of an RU486-inducible Pdf-GS driver. Flies were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) GST pulldown experiments detect interactions between TIM expressed in S2 cells and purified GST-RAN fusion proteins. RAN was tested in GDP-bound (RanGDP) and GTP-bound (RanGTP) forms. GST alone served as a control. The bound protein was analyzed with an anti-V5 antibody. In five independent experiments we have confirmed that TIM shows slight preference (1.64 ± 0.18) for Ran GTP over Ran GDP. (F) Expression of RanDN in central clock cells leads to severe morphological defects (white arrow) on the 3rd day after RU486 treatment. On the 1st day after RU486 treatment, morphological effects were not visible and PER (green) was cytoplasmic in both l-LNvs and s-LNvs at ZT1.