RNAi-Mediated Reverse Genetic Screen IdentifiedDrosophilaChaperones Regulating Eye and Neuromuscular Junction Morphology

Abstract

Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila. Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development.

Developmental Expression and Functions of the Small Heat Shock Proteins in Drosophila

“…Like Hsp23, Hsp26 also falls into the sHsp group with high CNS expression ( Figure 2 B) and is one of the sHsps with an essential developmental role [ 2 ]; as revealed by pan-neural knockdown, Hsp26 plays a role in neural development and in particular in the proper formation of NMJs. The Hsp26 loss of function results in an adversely affected presynaptic cytoskeleton, and a reduced number of synaptic buttons at NMJs.…”

“…This specific muscular expression prompted us to test for dCryAB functions. It was recently found that ubiquitous RNAi knockdown of l(2)efl / dCryAB could lead to lethality [ 2 ]. Consistent with this, muscle-specific RNAi attenuation of dCryAB leads to severe morphological and ultrastructural defects in body wall muscles ( Figure 5 ).…”

“…There is also growing evidence for stress-independent and tissue-specific expression of several members of the Hsp gene family. Recent work in Drosophila [ 2 ] revealed that several Hsp genes could have vital developmental functions. Among 95 Hsp genes tested, the ubiquitous gene knockdown of 42 of them resulted in F1 lethality, indicating their essential role in fly development.…”

“…They play a crucial role in the maintenance of protein homeostasis, preventing nonspecific aggregation of the substrate protein in an ATP-independent manner [ 3 ]. We will focus on sHsps identified in Drosophila , in which high-throughput developmental gene expression ( ) and several functional studies have already been performed [ 2 , 4 , 5 , 6 , 7 , 8 ]. Importantly, ubiquitous RNAi knockdown of six Drosophila sHsps ( Hsp23 , Hsp26 , Hsp27 , CG4461 , l(2)efl , and CG14207 ) resulted in lethality [ 2 ], suggesting they play vital developmental roles.…”

Small heat shock proteins determine synapse number and neuronal activity during development

“…HSPs are molecular chaperones that represent an intracellular protein quality system to maintain cellular protein homeostasis (proteostasis), preventing aggregation and promoting protein de novo folding or refolding and degradation of misfolded proteins [8]. In addition, HSPs participate in developmental functions in a stress-independent manner [9,10]. In Drosophiladevelopment small Heat Shock proteins (sHsps) have a specific temporal and spatial pattern of expression [10].…”