Loss- and gain-of-function analysis of the lipid raft proteins Reggie/Flotillin in Drosophila: They are posttranslationally regulated, and misexpression interferes with wing and eye development

Abstract

Reggie/Flotillin proteins are upregulated after optic nerve dissection and evolutionary highly conserved components of lipid rafts. Whereas many biochemical and cell culture studies suggest an involvement in the assembly of multiprotein complexes at cell contact sites, not much is known about their biological in vivo functions. We therefore set out to study the expression pattern and the effects of loss- and gain-of-function in the Drosophila melanogaster model system. We found that in flies these proteins are mainly expressed in axons at the root of fiber tracts, in places where strong fasciculation is required, e.g. at the neck of the peduncle of the mushroom bodies and in the optic chiasms. Despite their evolutionary conservation which implies fundamental and important functions, a P-element-induced null mutant (KG00210) of reggie1/flotillin2 (reggie1/flo2) in D. melanogaster shows no apparent phenotypic defects. This was even more surprising as we show that in this reggie1/flo2 null mutant the paralogous Reggie2/Flo1 protein is unstable and degraded, while the transcript is still present. The requirement of Reggie1/Flo2 for Reggie2/Flo1 stabilization is confirmed by misexpression experiments. Reggie2/Flo1 can only be misexpressed when Reggie1/Flo2 is provided as well. Conversely, Reggie1/Flo2 immunoreactivity can be detected, when its transgene is misexpressed alone. Using appropriate Gal4 driver lines, misexpression of Reggie1/Flo2 alone or together with Reggie2/Flo1 in the eye imaginal disc results in a specific and severe mislocalization of cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) (while DE-Cadherin is unaffected) and in differentiation defects pointing to impaired signaling. In the wing imaginal disc, global overexpression of Reggie/Flotillin proteins leads to a significant extension of the Wingless signal and severely disrupts normal wing development. Our data support the notion that Reggie/Flotillin proteins are implicated in signaling processes at cellular contact sites.

Function of Flotillins in Receptor Tyrosine Kinase Signaling and Endocytosis: Role of Tyrosine Phosphorylation and Oligomerization

“…A P-element-induced null mutant for flotillin-2 was viable, fertile and with no apparent deficits in behavior or overall activity. In this flotillin-2 null mutant, the expression of flotillin-1 was undetectable even though the mRNA transcript was present (83). Conversely, overexpression of flotillin-1 and flotillin-2 proteins either alone or together in the eye imaginal discs lead to mislocalization of adhesion molecules of the immunoglobulin superfamily (IgCAMs; please also refer to the chapter "Functions of flotillins in cell migration and adhesion").…”

“…Hoehne et al carried out initial studies of loss-and gain-of function of flotillins in wild type and mutant Drosophila melanogaster to investigate their role during morphogenesis and organogenesis (83). According to this study, flotillin proteins are highly expressed in embryonic neuronal tissues, and during later developmental stages flotillins showed an enhanced expression pattern in axon fascicles (83). Additionally, flotillin specific antibodies labeled in particular the Drosophila mushroom body, which is a paired structure of insect central brain responsible for higher-order sensory integration and learning.…”

Eukaryotic operon-like transcription of functionally related genes in Drosophila

“…6D). flotillin is an integral membrane protein and Cdk5 (cyclin-dependent kinase 5) is a soluble protein (17,18). Although they are not known to participate in a common biochemical pathway, they both contribute to neuronal signal transduction, and both have been implicated in the pathogenesis of Alzheimer's disease in humans (19,20).…”

Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type II cells

“…Among F-2 shRNAs, F-2 shRNA (C) was the most effective in silencing flotillin-2 expression. This shRNA also reduced flotillin-1 expression ( Figure 1A and 1B), probably because the stability of flotillin-1 is dependent on flotillin-2 [8]. Using F-1 shRNA (A) and F-2 shRNA (C), we determined the effect of flotillin-1 and -2 silencing on surfactant secretion.…”