title: Investigating the role of Rab GTPases in Wnt secretion in Drosophila melanogaster
creator: Pavlovic, Bojana
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
description: Wnt pathway is one of the main signaling pathways required for development and homeostasis. Wnt proteins must be secreted into the extracellular space in order to trigger the signaling cascade. Regulation of Wnt protein secretion is therefore crucial for Wnt pathway activity. All intracellular membrane trafficking is regulated by Rab GTPases. Very few Rab GTPases, however, have been directly associated with Wnt secretion. I performed a systematic screen by perturbing the 26 Drosophila melanogaster RabGTPases and observing the effects on Wnt secretion in the wing imaginal disc through immunohistochemical analysis. I identified Rab7 as a regulator of Wnt secretion and characterized its role in this process. I found that overexpression of Rab7 induces the accumulation of Drosophila Wnt1 homolog Wingless (Wg) in both secreting and receiving cells, as well as moderate relocalization of Wg from predominantly apical side to the basal side of these cells. The changes in Wg protein localization did not, however, affect the level of secreted Wg in the wing discs or the downstream Wg signaling. By performing colocalization staining, I determined that upon Rab7 overexpression, Wg was localizing to LAMP1- and Arl8-positive lysosomes at the basal side of the wing disc cells. Knockdown of Arl8 in cells with Rab7 overexpression reduced the translocalization of Wg to the basal side of the disc.     In order to study the interaction between Rab GTPases and Wnt secretion in living tissue, I developed novel Drosophila tools, which will be of use to other studies. Firstly, I endogenously tagged the Wg carrier protein Evi/Wls with the fluorescent proteins mScarlet and EGFP. Additionally, I generated a Wg:mScarlet fly line based on the existing Wg:EGFP fly line. Using these tools, I characterized the different fluorescence localization of proteins tagged with EGFP and mScarlet. I confirmed that the fluorescent tags do not affect Wg secretion, but that the difference in signal localization is due to the intrinsic properties of the fluorescent proteins. Secondly, I adapted the LAMA (ligand-modulated antibody fragments) system, an acute protein trap-and-release system previously published in human cell culture, for use in Drosophila. I could show that the system was functional in wing discs and salivary gland cells and that it allowed the trapping of both overexpressed and endogenous GFP-tagged proteins on the mitochondrial membrane and the endoplasmic reticulum (ER). The addition of the release molecule triggered the relocalization of the tagged proteins from the mitochondrial membrane into the cytoplasm. Using the LAMA system, I was able to show that the Wg accumulation induced by Rab7 overexpression appeared within sixty minutes of the Rab7 release.     In summary, my data indicate a role of Rab7 in the translocalization of Wg to the basal side through the late endosomal pathway. Furthermore, the development of experimental tools, especially the adaptation of a protein trap-and-release system for use in Drosophila melanogaster, provide a material contribution to the field.
date: 2024
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/33894/1/Dissertation%20Bojana%20Pavlovic.pdf
identifier: DOI:10.11588/heidok.00033894
identifier: urn:nbn:de:bsz:16-heidok-338946
identifier:   Pavlovic, Bojana  (2024) Investigating the role of Rab GTPases in Wnt secretion in Drosophila melanogaster.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/33894/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng