Principal Supervisor: Dr Sovan Sarkar, Institute of Cancer and Genomic Sciences
Co-supervisor: Professor Jon Frampton, Institute of Cancer and Genomic Sciences
PhD project title: Identifying the regulators of vesicle fusion events in autophagy using human stem cells
University of Registration: University of Birmingham
Background: The proteolytic system plays a fundamental role in cellular functioning and survival, deregulation of which leads to various diseases including cancer and neurodegeneration. One central node implicated in diverse human physiological and pathological conditions is autophagy, an intracellular degradation pathway for aggregation-prone proteins and unwanted organelles that would otherwise cause cell death upon accumulation when this process malfunctions. Thus, autophagy is critical for organismal health by maintaining cellular and energy homeostasis1. This process involves multiple vesicle fusion events through the generation of double-membrane vesicles called autophagosomes that are destined to fuse with the lysosomes forming autolysosomes where the autophagic cargo is degraded. This occurs via two avenues: (i) Multi-step route where autophagosomes first fuse with endosomes to form amphisomes, then fuse with lysosomes forming autolysosomes; (ii) Direct route where autophagosomes fuse with lysosomes to form autolysosomes1. Various molecular mediators including SNARE proteins are known to facilitate membrane tethering and autophagic vesicle fusion, especially for the multi-step route1. However, our data point to the existence of yet-to-be-identified molecular players of the direct route of autophagosome maturation2.
Question: This proposal addresses a core issue related to the mechanistic control of autophagy: What are the molecular mediators of direct autophagosome-lysosome fusion? We hypothesize the need to directly capture this vesicle fusion event. This can be achieved by using the cellular model of a lysosomal storage disorder (LSD) where we have previously shown that the multi-step route is blocked2. In this context, stimulation of autophagy restored autophagic flux via the direct route2. Therefore, this disease-based system provides us with a relevant experimental platform to study the basic biology, which can also be relevant for certain neurodegenerative and lysosomal storage disorders associated with defective autophagosome maturation.
Objectives: (1) Identify the molecular mediators of autophagosome-lysosome fusion by genetic screens. (2) Validate their function via CRISPR/Cas9-mediated gene knockout in a human induced pluripotent stem cell (hiPSC) model3 of LSD. (3) Investigate their role in autophagosome maturation in physiologically-relevant, isogenic human cell-types using human embryonic stem cells (hESCs).
Methodology: We have undertaken genetic screens in a LSD cellular platform expressing autophagy reporters. Our preliminary data indicate 2 potential genes regulating direct autophagosome-lysosome fusion. These will be further characterized through molecular, biochemical and cell biology techniques for their ability in membrane tethering and vesicle fusion. Their binding partners and any specific SNARE complex formation will be identified by mass spectrometry. We will then employ hESCs/hiPSCs for differentiating into somatic cell-types with isogenic background. CRISPR/Cas9-mediated gene knockout approaches in the LSD hiPSC model3 will validate the abrogation of autophagosome-lysosome fusion. Similar loss-of-function studies will be undertaken in physiologically-relevant human context using hESC lines expressing autophagy reporters. Functional analysis will be done in these hESCs and hESC-derived isogenic cell-types (such as fibroblasts, neurons, hepatic cells) on autophagosome-lysosome fusion by cell biological and biochemical methods including mathematical approaches to quantify autophagic flux.
Outcome: Understanding the regulators of autophagosome maturation will provide drug targets for improving defective autophagic flux in age-related pathologies like neurodegeneration. Our findings will thus be of basic and biomedical relevance.
- 1Sarkar S. Biochem Soc Trans 41:1103, 2013; 2Sarkar S. et al. Cell Rep 5:1302, 2013; 3Maetzel D., Sarkar S. et al. Stem Cell Rep 2:866, 2014.
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- Culture of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)
- Differentiation of hESCs/hiPSCs into fibroblasts, neural precursors, neurons and hepatic cells
- Genome engineering using CRISPR/Cas9 system for creating gene knockout
- Gene knockdown by siRNA and lentiviral shRNA
- Autophagy assays
- In vitro vesicle fusion assays
- Live-cell and confocal microscopy
- High-content image-based screening and analysis
- Immunoblotting and Southern blot analyses
- Flow cytometry
- Molecular cloning
- Mutagenesis analysis
- Mass spectrometry and LUMIER assay
- Bioinformatics and mathematical approaches
- Statistical analyses
- A range of molecular, biochemical and cell biological techniques.
Contact: Dr Sovan Sarkar, Institute of Cancer and Genomic Sciences