Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Abstract

Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis within minutes following a temperature shift from 25°C to 37°C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbots displays a strong synergistic interaction with the Syntaxin-1A TS allele syx3-69, recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbots showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbots;syx3-69 double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbots synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbots displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

Efr3a Insufficiency Attenuates the Degeneration of Spiral Ganglion Neurons after Hair Cell Loss

“…The endocytosis mediated by PI 4,5 P is involved in a variety of processes at the cell surface (Kumari et al, 2010; McMahon and Boucrot, 2011), including axonal and neuronal degeneration, collapse, disintegration and death (Weinkove et al, 2008; Kuboyama et al, 2015). In Drosophila , the RBO played a pivotal role in PI 4,5 P-mediated signaling in photoreceptors, and in bulk endocytosis and macropinocytosis in neuronal and non-neuronal cells (Huang et al, 2004, 2006; Vijayakrishnan et al, 2009). In mammalian cells, Efr3 is also important in the control of G-protein-coupled receptor (GPCR)-mediated signaling by affecting the phosphorylation of GPCR (Bojjireddy et al, 2015).…”

Identifying cellular pathways modulated by Drosophila palmitoyl-protein thioesterase 1 function

“…This assay is particularly useful since it can be used to compare endocytosis rates between freshly isolated mutant cells and wildtype controls. Many proteins involved in endocytosis show alterations in fluorescent tracer uptake when examined with this system (Chang et al ., 2002; Satoh et al ., 2005; Vijayakrishnan et al ., 2009). In order to observe endocytosis, we dissected live garland cells from 3 rd instar larvae and monitored endocytosis over a period of 40 minutes by incubating the cells in cell culture medium containing Texas Red-conjugated Avidin (TR-Avidin) and Lysotracker green.…”

“…This approach allowed us to more precisely analyze vesicle uptake in Garland cells (Koenig and Ikeda, 1990; Chang et al ., 2002; Vijayakrishnan et al ., 2009). Freshly dissected Garland cells from wildtype and Ppt1 A179T mutant larvae were incubated with HRP for 5 minutes at room temperature, fixed, and processed for electron microscopy.…”

“…Our results demonstrated a decreased rate of TR-Avidin and HRP tracer uptake in Ppt1 mutant cells that is consistent with a role for Ppt1 in the modulation of early endocytic processes. Drosophila genes that have been shown to play a major role in endocytosis, such as Rab11, Dynamin, Rolling Blackout (RBO), and Hsc70–4, all demonstrate a complete block in the uptake of tracer (fluorescent avidin or HRP) in mutant cells (Koenig and Ikeda, 1990; Satoh et al ., 2005; Chang et al ., 2002; Vijayakrishnan et al ., 2009). A reduction, but not complete cessation of uptake in Ppt1 mutant cells suggests that Ppt1 is not required for endocytosis but rather that its function is necessary for robust endocytosis to occur.…”

“…While labyrinthine channels are absent in Rab11 and Hsc70–4 cells, cells that lack Dynamin or Rolling Blackout (EFRS integral membrane lipase) function show ultrastructural phenotypes similar to Ppt1 (Koenig and Ikeda, 1990; Vijayakrishnan et al ., 2009). Work on both Dynamin and RBO indicate that the enlarged and vacuolated channels are due to a block in vesicle formation during endocytosis (Koenig and Ikeda, 1990; Vijayakrishnan et al ., 2009). Our garland cell experiments and the genetic interaction with Dynamin in the adult eye are consistent with the loss of Ppt1 function impacting endocytosis at the point of vesicle formation.…”