Regulation of Expression of Autophagy Genes by Atg8a-Interacting Partners Sequoia, YL-1, and Sir2 in Drosophila.

Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.

Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update.

Visualization of single neurons and glia, as well as neural lineages within their complex environment is a pivotal step towards uncovering the mechanisms that control neural circuit development and function. This chapter provides detailed technical information on how to use Drosophila variants of the mouse Brainbow-2 system, called Flybow, for stochastic labeling of individual cells or lineages with different fluorescent proteins in one sample. We describe the genetic strategies and the heat shock regime required for induction of recombination events. Furthermore, we explain how Flybow and the mosaic analysis with a repressible cell marker (MARCM) approach can be combined to generate wild-type or homozygous mutant clones that are positively labeled in multiple colors. This is followed by a detailed protocol as to how to prepare samples for imaging. Finally, we provide specifications to facilitate multichannel image acquisition using confocal microscopy.