BLOCKING ENDOCYTOSIS IN DROSOPHILA'S CIRCADIAN PACEMAKER NEURONS INTERFERES WITH THE ENDOGENOUS CLOCK IN A PDF-DEPENDENT WAY

Abstract

The neuropeptide pigment-dispersing factor (PDF) plays an essential role in the circadian clock of the fruit fly Drosophila melanogaster, but many details of PDF signaling in the clock network are still unknown. We tried to interfere with PDF signaling by blocking the GTPase Shibire in PDF neurons. Shibire is an ortholog of the mammalian Dynamins and is essential for endocytosis of clathrin-coated vesicles at the plasma membrane. Such endocytosis is used for neurotransmitter reuptake by presynaptic neurons, which is a prerequisite of synaptic vesicle recycling, and receptor-mediated endocytosis in the postsynaptic neuron, which leads to signal termination. By blocking Shibire function via overexpression of a dominant negative mutant form of Shibire in PDF neurons, we slowed down the behavioral rhythm by 3 h. This effect was absent in PDF receptor null mutants, indicating that we interfered with PDF receptor-mediated endocytosis. Because we obtained similar behavioral phenotypes by increasing the PDF level in regions close to PDF neurons, we conclude that blocking Shibire did prolong PDF signaling in the neurons that respond to PDF. Obviously, terminating the PDF signaling via receptor-mediated endocytosis is a crucial step in determining the period of behavioral rhythms. (Author correspondence: [email protected])

Key Words:

• Drosophila
• Dynamin
• Shibire
• Pigment-dispersing factor
• Neuropeptide signaling

ACKNOWLEDGMENTS

The authors thank Toshihiro Kitamoto for the uas-shi^ts1 line, Jae Park for the Pdf-gal4 line, Heiner Dircksen for anti-PDH, Alois Hofbauer and Orie Shafer for comments on the manuscript, and Angelika Kühn for excellent technical assistance. This work was supported by the DFG (grant Fo207-10 to C.H.-F.) and EUCLOCK.

The authors declare no conflicts of interest.