Multiple Requirements for Rab GTPases in the Development ofDrosophilaTracheal Dorsal Branches and Terminal Cells

Abstract

The tracheal epithelium in fruit fly larvae is a popular model for multi- and unicellular migration and morphogenesis. Like all epithelial cells, tracheal cells use Rab GTPases to organize their internal membrane transport, resulting in the specific localization or secretion of proteins on the apical or basal membrane compartments. Some contributions of Rabs to junctional remodelling and governance of tracheal lumen contents are known, but it is reasonable to assume that they play important further roles in morphogenesis. This pertains in particular to terminal tracheal cells, specialized branch-forming cells that drastically reshape both their apical and basal membrane during the larval stages. We performed a loss-of-function screen in the tracheal system, knocking down endogenously tagged alleles of 26 Rabs by targeting the tag via RNAi. This revealed that at least 14 Rabs are required to ensure proper cell fate specification and migration of the dorsal branches, as well as their epithelial fusion with the contralateral dorsal branch. The screen implicated four Rabs in the subcellular morphogenesis of terminal cells themselves. Further tests suggested residual gene function after knockdown, leading us to discuss the limitations of this approach. We conclude that more Rabs than identified here may be important for tracheal morphogenesis, and that the tracheal system offers great opportunities for studying several Rabs that have barely been characterized so far.

Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies

“…Since the identification of Ypt1 (Rab1) in yeast, the Rab GTPase family has been well characterized as an evolutionarily conserved group of proteins involved in the regulation of membrane trafficking in all eukaryotes ( Hutagalung and Novick, 2011 ; Klöpper et al, 2012 ; Lipatova et al, 2015 ; Pfeffer, 2017 ). Rab GTPases have been analyzed in several comparative studies in order to gain a systematic view of membrane trafficking in cells ( Best and Leptin, 2020 ; Chan et al, 2011 ; Dunst et al, 2015 ; Gillingham et al, 2014 ; Gurkan et al, 2005 ; Harris and Littleton, 2011 ; Jin et al, 2012 ; Pfeffer, 1994 ; Stenmark, 2009 ; Zerial and McBride, 2001 ). All comparative studies to date have been based on expression profiling, the expression of GDP- and GTP-locked Rabs or RNAi.…”

“…Over the years, Rab GTPases have repeatedly been analyzed as a gene family to gain insight into membrane trafficking networks ( Best and Leptin, 2020 ; Chan et al, 2011 ; Dunst et al, 2015 ; Gillingham et al, 2014 ; Gurkan et al, 2005 ; Harris and Littleton, 2011 ; Jin et al, 2012 ; Pfeffer, 1994 ; Stenmark, 2009 ; Zerial and McBride, 2001 ). Nonetheless, a complete and comparative null mutant analysis of all family members is currently not available for any multicellular organism.…”

High-precision targeting workflow for volume electron microscopy

“…The Drosophila line used was reported previously (Best and Leptin, 2020) and carries a recombined btl-Gal4 (Shiga et al, 1996) and UAS-DsRed1 (BDSC 6282) element on the third chromosome, driving expression of DsRed in all tracheal cells. The flies were grown on standard cornmeal-agar medium at 25°C.…”

Drosophila Rab39 Attenuates Lysosomal Degradation

“…Rab39 also localizes predominantly to the trans-Golgi, nevertheless, it was claimed to be involved in a variety of cellular mechanisms, e.g., endocytosis, neuritogenesis, oxidative stress response, cannabinoid receptor signaling, Drosophila embryogenesis, Notch signaling, lipid transport, or microtubular organization [ 11 , 31 , 32 , 33 , 34 ].…”