During autophagy, unwanted or dangerous cellular material is captured in vesicles called autophagosomes that deliver the cargo to lysosomes, the waste disposal and recycling centers of the cell. The precursors of autophagosomes are phagophores, small patches of membrane that nucleate the autophagic machinery and engulf the cargo as they grow.
A key protein for phagophore growth and cargo binding is called LC3. LC3 is conjugated to phospholipids on the autophagosomal membrane in a reaction dictated by an enzyme complex (E3). This complex is recruited to the phagophore by sensor proteins (WIPI2) which themselves depend on the activity of a lipid kinase (PI3KC3-C1). The kinase generates a key autophagic signal in the form of another phospholipid (PI3P) that recruits WIPI2 to the pre-autophagosomal membrane. The researchers reconstituted these key steps of membrane targeting and activation in vitro and unravelled how the different “cogwheels” of the autophagy machinery interlock to efficiently generate autophagosomes.
Intriguingly, they showed that the recruitment of PI3KC3-C1 and WIPI2 is mutually enhanced in a positive feedback loop. The more of the kinase was present the more WIPI2 was recruited and vice versa. WIPI2 was also observed to activate the E3 complex at a site other than the active site, a phenomenon known as allosteric activation. “These two effects help explain how the autophagy machine rapidly recruits LC3 and generates the autophagosomal membrane on a timescale similar to that in cells”, says Dorotea Fracchiolla, first author of the study and Postdoc in the Martens lab.
The results are part of a bigger effort between the Martens lab and the Hurley (UC Berkeley), Hummer (MPI, Frankfurt) and Yoshimori (Osaka) labs to reconstitute each step of mammalian autophagosome biogenesis in the lab from start to finish. In live cells, many different processes happen at the same time, making it difficult to observe certain mechanisms in detail. The scientists used purified, recombinant proteins together with giant unilamellar vesicles (GUVs) to characterise individual steps. By systematically adding each component (lipids and proteins) they could study their specific roles in the process, a key advantage over in vivo methods. The research was funded by the Human Frontiers Science Programme (HFSP).
Science Sketch made by Dorotea Fracchiolla:
https://www.youtube.com/watch?v=soWx_tuJm_g
Publication:
Dorotea Fracchiolla, Chunmei Chang, James H. Hurley, Sascha Martens: A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy. J Cell Biol (2020) 219 (7): e201912098.
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Early evolution of animal genome regulation
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Dissecting how gene regulatory domains reconfigure in evolution and development
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Sister chromatid cohesion is asymmetric
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How HUSH protects your genome from reverse genetic flow
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Single-cell driven enhancer modelling and design
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Cell stress and the evolution of novel cell types
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New insights in gene regulation and chromatin folding by enhancers and cohesin
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Designing and building a minimal divisome
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Genomic Analysis of Osteosarcoma: Making Sense of a Complex Genome to Advance Translational Research and Targeted Therapy
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Imaging of cell division across scales
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Characterisation of Vpr1, a novel protein involved in DSB repair
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m5C-modifications of ribosomal RNA modulate healthy lifespan and development
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Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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Visualization of Macromolecular Assemblies in Meiosis Using Cryo-Electron Tomography
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Title to be announced
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Control of meiotic crossover placement in Drosophila
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Title to be announced
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Identification of novel ciliogenesis genes using a phylogenetic profiling approach
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Title to be announced
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Comprehensive and quantitative characterization of aneuploidy phenotypes
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Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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Monday Seminar (Internal)
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Title to be announced
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Title to be announced
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Monday Seminar (Internal)
Speaker: ATTRILL Sarah (Dolan), UIJTTEWAAL Esther (Elling), SCIBISZ Grzegorz (Karagöz)Institute: Open only to IMP/IMBA/GMI/Max Perutz Labs
Monday Seminar (Internal)
Speaker: DOLESCHALL Balint (Knoblich), FENG Sonjie (Bücker), BOURGUET Pierre (Berger)Institute: Open only to IMP/IMBA/GMI/Max Perutz Labs
Title to be announced
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Monday Seminar (Internal)
Speaker: HORENKAMP Simone (Tanaka), CASEY Chloe (Dolan), KAPRAL Thomas (Zagrovic)Institute: Open only to IMP/IMBA/GMI/Max Perutz Labs
Monday Seminar (Internal)
Speaker: KOBAKHIDZE George (Dong), ANGELOPOULOU Eleni (Ellis), PACHANO Tomas (Stark)Institute: Open only to IMP/IMBA/GMI/Max Perutz Labs
Title to be announced
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Monday Seminar (Internal)
Speaker: RAMIREZ America (Köhler), VÖLKL Isabella (Bücker), HOANG Oi Pui (Technau)Institute: Open only to IMP/IMBA/GMI/Max Perutz Labs
Title to be announced
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Title to be announced
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Title to be announced
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Title to be announced
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