Autophagy is a catabolic process essential for cell homeostasis, at the core of which is the formation of double-membrane organelles called autophagosomes. Atg9 is the only known transmembrane protein required for autophagy and is proposed to deliver membrane to the preautophagosome structures and autophagosomes. We show here that mammalian Atg9 (mAtg9) is required for the formation of DFCP1-positive autophagosome precursors called phagophores. mAtg9 is recruited to phagophores independent of early autophagy proteins, such as ULK1 and WIPI2, but does not become a stable component of the autophagosome membrane. In fact, mAtg9-positive structures interact dynamically with phagophores and autophagosomes without being incorporated into them. The membrane compartment enriched in mAtg9 displays a unique sedimentation profile, which is unaltered upon starvation-induced autophagy. Correlative light electron microscopy reveals that mAtg9 is present on tubular–vesicular membranes emanating from vacuolar structures. We show that mAtg9 resides in a unique endosomal-like compartment and on endosomes, including recycling endosomes, where it interacts with the transferrin receptor. We propose that mAtg9 trafficking through multiple organelles, including recycling endosomes, is essential for the initiation and progression of autophagy; however, rather than acting as a structural component of the autophagosome, it is required for the expansion of the autophagosome precursor.

Groundbreaking advances in volume electron microscopy and specimen preparation are enabling the 3-dimensional visualisation of specimens with unprecedented detail, and driving a gratifying resurgence of interest in the ultrastructural examination of cellular systems. Serial section techniques, previously the domain of specialists, are becoming increasingly automated with the development of systems such as the automatic tape-collecting ultramicrotome, and serial blockface and focused ion beam scanning electron microscopes. These changes are rapidly broadening the scope of biomedical studies to which volume electron microscopy techniques can be applied beyond the brain. Further innovations in microscope design are also in the pipeline, which have the potential to enhance the speed and quality of data collection. The recent introduction of integrated light and electron microscopy systems will revolutionise correlative light and volume electron microscopy studies, by enabling the sequential collection of data from light and electron imaging modalities without intermediate specimen manipulation. In doing so, the acquisition of comprehensive functional information and direct correlation with ultrastructural details within a 3-dimensional reference space will become routine. The prospects for volume electron microscopy are therefore bright, and the stage is set for a challenging and exciting future.

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.