The Secretory Pathways Laboratory studies organelle biogenesis and dynamics during macroautophagy (here called autophagy) in mammalian cells. Autophagy is lysosome-mediated self-degradation process induced in response to a variety of signals including starvation, infection, neurodegenerative disease and cancer. Induction of autophagy leads to the formation of double-membrane vesicles, autophagosomes, (AVi) which envelop cytoplasmic proteins and organelles delivering them to the endosomal/lysosomal (E/LY) system by vesicular fusion (Figure 1). Subsequent degradation of the sequestered proteins and lipids provides the cell with amino acids and other molecules to resume protein and lipid synthesis. Starvation-induced autophagy is a tightly regulated survival response terminated by replenishment of nutrients, however unregulated autophagy can cause cell death, and thus is also called Type II cell death.
The autophagic response is regulated by the number of autophagosomes formed, and their rate of consumption by lysosomal degradation. The laboratory has been investigating both processes to understand the molecular mechanisms underlying the regulation of autophagy. We aim to achieve an understanding of these mechanisms allowing us to extend our findings to address the role of autophagy in human disease, in particular cancer.
Autophagosome formation
Amino acid starvation inactivates TOR (target of Rapamycin), a key growth promoting kinase and a negative regulator of autophagy. In yeast, the Atg1 kinase is downstream of TOR, and is proposed to control early events in autophagy. Ed Chan identified the Unc-51-like kinase 1 (ULK1) in a siRNA kinome screen as the mammalian homologue of Atg1 (Chan et al, J Biol Chem 2007; 282: 25464-25474), and showed that depletion of ULK1, but not the closely related ULK2, inhibited starvation induced autophagy. To further understand the role of ULK1 in autophagy, Ed, in collaboration with Andrea Longatti and Nicole McKnight, uncovered an intra-molecular regulation of the ULK1/2 kinases through autophosphorylation, and modulated by a novel ULK1/2 substrate, KIAA0652, the putative orthologue of yeast Atg13. Mammalian Atg13 interacts with, and is a substrate of ULK1/2, and has a starvation sensitive membrane association. Future work on ULK1/2 and mAtg13 aims to understand the regulation of this complex, and the amino acid signalling pathways controlling the expansion of the isolation membranes (IM) and nascent AVis. Furthermore, Nicole McKnight, with the LRI High Throughput Screening Unit headed by Mike Howell, is conducting a siRNA genome screen for novel regulators of autophagy.
Figure 1. The autophagy pathway in mammalian cells. Autophagy is induced on isolation membranes (IMs) that serve both as signalling platforms and membrane source/acceptor compartments. The source of the IM is unknown. During expansion, the IM grows sequestering cytosolic components (Note; for simplicity the sequestered cytosolic components are not illustrated). After closure of the expanded IM, the immature autophagosome (AVi) fuses with the endosomal compartments (E) and lysosomes (LY), becoming a degradative autophagosome AVd. Degradation of the sequestered cytosolic components occurs in the AVd as it matures to an autolysosome. In the final stage, the AVd is shown in cross-section to aid visualisation of the membrane fusion event(s).
ULK1 is required for mammalian Atg9 trafficking to the autophagosome (Young et al, JCS 2006; 119:3888-900), and mAtg9 is the only transmembrane protein so far thought to be on IMs. mAtg9 cycles between the TGN and endosomes under normal conditions, and after starvation mAtg9 redistributes to autophagosomes. To understand how mAtg9 trafficking is controlled, Andrea Orsi investigates the cytoplasmic domains of mAtg9 to uncover trafficking signals and effectors, and together with Jemma Webber is exploring further the role of ULK1 in mAtg9 trafficking.
Jemma has recently identified a new mAtg9 partner, p38IP (p38α MAPK interacting protein), which is required for starvation-induced mAtg9 trafficking and autophagosome formation. Jemma's hypothesis is that p38MAPK, a negative regulator of basal and starvation induced autophagy, and acts through mAtg9 and p38IP, and provides a mechanistic link between the MAPK signalling pathway and the control of autophagy through mAtg9 trafficking via p38IP.
Formation of the IM requires the Class III Phosphatidylinositol-3-kinase (PtdIns-3-K). The production of PtdIns-3-phosphate is essential for the formation and expansion of the autophagosome. Beclin1 is an essential subunit of the PtdIns-3-K complex, and is required for autophagy. It is not known how PtdIns-3-P production is localised to the IM, and how the Class III kinase is regulated. Harold Jefferies is studying the composition and subcellular localisation of Beclin1-containing complexes to understand more about this issue. Furthermore, a family of PtdIns-3-P, and PtdIns 3,5-P2 binding proteins, the WIPI family, are required for autophagosome formation although what their function is remains unknown. Hannah Polson (in a collaboration with Dr M Claque, University of Liverpool) has been studying this family to determine which WIPI proteins are recruited to the IM, and how. Additional information will be gained by Hannah through the identification of novel WIPI effectors.
Autophagosome maturation and fusion
In the final stages of autophagy, autophagosome fuses with endosomes and lysosomes and become degradative (AVds). How this fusion occurs is not known, although it is likely based on membrane fusion paradigms developed from the well studied endosome-late endosome fusion. The laboratory has a long-standing interest in membrane fusion events in the secretory pathway, and Grant Otto has continued this interest with his work on the SNARE protein Syntaxin 6. Grant has characterised a novel Syntaxin 6-binding protein, KIAA0701,which he showed binds and regulates Syntaxin 6 function the in endocytic recycling pathway. Grant¿s work has potentially uncovered a novel regulation of SNAREs and membrane traffic.
Finally, we aim to understand more about autophagosomeendosome fusion. Minoo Razi has shown that early endosomes fuse with autophagosomes by preventing early endosome function by siRNA depletion of the coat protein complex COPI. Loss of COPI. causes an accumulation of non-degradative autophagosomes, and an inhibition of autophagy. However, it is not clear if fusion with early endosomes is a pre-requisite for fusion of autophagosomes with late endosomes. Thus, to investigate the molecular requirements for autophagosome fusion with endosomes, Joëlle Morvan and Harold Jefferies have exploited have exploited an in vitro fusion assay that reconstitutes AVi-E/LY fusion. They found this fusion event has several unique properties, the most significant being a lack of dependence on ATP, and suggest that the fusion between the autophagosome and the endosome is an unusual type of fusion. It remains to be understood if the SNARE fusion machinery is catalysing this fusion.
For a list of refereed research papers, see Publications (in navigation on left).
