Visualisation of native Atg9 complexes using in situ cryo-EM

Abstract text (incl. figure legends and references)

Autophagy is a conserved process that allows for recycling of cellular components by sequestration into a double-membrane-bounded compartment which fuses with the lysosome for degradation. Among the core machinery that monitors the steps towards autophagy is Atg9, the only multi-spanning membrane protein. Atg9 forms a complex with Atg2 and Atg18 and is essential for extending the isolation membrane. So-called Atg9 vesicles, stored in the cytoplasm, provide a lipid reservoir for the growing isolation membrane. Recently a scramblase activity has been identified in both the human and yeast Atg9 [1,2]. In order to elucidate the exact mechanism of the Atg9-Atg2-Atg18 complex, I observed native Atg9 complexes and surrounding membrane environment in S. cerevisiae using cryo-confocal microscopy followed by focussed ion beam (FIB) milling and in situ tomography. Thus far, I established the reproducible preparation of FIB lamellae in yeast. I have screened a range of knock-out mutants, tested different fluorophores and growth conditions. These optimizations enriched Atg9-containing punctae around lysosomal membranes and will improve the success of localisation for down-stream FIB-milling and any associated image analysis such as subtomogram averaging. With functional mutants of the complex components in situ we will be able to address mechanistic questions of isolation membrane elongation.

References:

[1] Maeda, S. et al. Nat Struct Mol Biol 27, 1194-1201 (2020).

[2] Matoba, K., et al. Nat Struct Mol Biol 27, 1185-1193 (2020).