Overexpression of Rotini, the Drosophila GOLPH3, affects Wingless signaling by interfering Wntless localization
Date Issued
2015
Date
2015
Author(s)
Huang, Chiao-Hsia
Abstract
Rotini (Rti), the Drosophila Golgi protein GOLPH3, has been shown to regulate the biosynthesis of Heparan Sulfate Proteoglycans (HSPGs) by modulating the retrograde trafficking of glycosyltransferases, Exostosins (EXTs). Loss of function of Rti shifts the distribution of EXTs to the trans-Golgi, while overexpression of Rti results in EXTs mislocalization toward cis-Golgi. Both situations interfere with EXTs activity and result in incomplete HSPG and perturbation of Hh signaling. In addition to Hh, Drosophila Wnt/Wingless (Wg) has been found affected by Rti. Wg is required for a wide range of patterning events, including defining the wing blade and specifying the wing margin. It has been found that Rti overexpression causes the wing disc atrophied and Wg accumulation inside Wg-producing cells while loss-of-function of Rti appears to have no effect on Wg expression. Therefore, we proposed that Rti may involve in modulation of Wg secretion. According to studies in the modulation of Wg secretion, Wntless (Wls) is known to be a dedicated transporter for Wg protein from trans-Golgi to plasma membrane. In Wg-producing cells, Wls proteins accumulate in Golgi which results in strengthen Wls staining. After Wg is released, Wls is endocytosed form the cell surface, and then retromer-mediated retrieval brings it back to the trans-Golgi network for maintenance of continuous Wnt secretion. In Wls mutant clone, Wg proteins accumulate inside Wg-producing cells, which is similar to the Wg mutant phenotype observed in Rti overexpression. Therefore, the aim of the thesis is to investigate whether Rti overexpression interferes with Wls function, thus causing Wg accumulation inside Wg-producing cells. When Rti is overexpressed, the reduction of Wls staining in Wg-producing cells disrupts the partial colocalization of Wls with Golgi marker Galt-GFP. These results suggest that Rti affects the localization of Wls in Wg-producing cells. When use hrsD28 mutant to block lysosomal degradation and observed the Wls staining in Wg-producing cells under different Rti expression dosage, we found that Wls staining was unaffected while Wg staining was increased in hrsD28 mutant clone. This suggests that the strong Wls staining in Wg producing cells is not caused by increase of protein stability. In addition, the reduction of Wls staining in Rti overexpression tissue is suppressed in hrsD28 clone. As our expected, the expression in hrsD28 rti double mutant clone is similar to that in hrsD28 mutant clone. Conclusively, as its role in EXTs regulation, in Wg-producing cells, Rti affects Wls localization by retrograde trafficking.
Subjects
Golgi protein GOLPH3
Wg secretion
retrograde trafficking
Type
thesis
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