Chemical-genetic control or autophagy – Autophagy

Autophagy, which occurs in general or organelle-specific variants, plays a fundamental role in the maintenance of cytoplasmic homeostasis, both at baseline and in response to multiple stressors. Deranged autophagy is involved in neurodegeneration, progeroid syndromes, oncogenesis and cardiovascular diseases. Autophagy is usually studied upon exposure of cells or organisms to nutrient deprivation or pharmacological agents, which however have multiple additional effects on cellular physiology, do not induce autophagy in all cells simultaneously, and fail to induce truly organelle-specific (as opposed to general) autophagy, rendering difficult data interpretation. Here, we propose to construct an entirely new system for the experimental induction and inhibition of autophagy.
For this, we will create chemical biological tools (Workpackage 1) consisting in inducible genetic systems to stimulate organelle-specific autophagy. Cells will be stably transfected with tamoxifen-inducible Cre (Cre-ERT2) plus, downstream of a Lox-Stop-Lox cassette, a polycistronic vector expressing ATG5 plus LC3 N-terminally fused to a streptavidin binding peptide (SBP) tag. In addition, cells will be transduced with streptavidin directed to the surface of distinct organelles, namely centrosomes (by fusion with centrin-2), endoplasmic reticulum (CD74/Ii), mitochondria (TOM20), nuclear membrane (KASH domain), peroxisomes (PEX3), plasma membrane (KRAS) or ribosomes (RPL11). In this system, organelle specific autophagy can be controlled at two levels, namely (i) by adding tamoxifen to express ATG5 and the SBP-LC3 fusion protein, and (ii) by adding and removing biotin and a reversible biotin mimetic (AliS-1), which out-competes the interaction between SBP-LC3 and organelle-targeted streptavidin, hence inhibiting and inducing organelle-specific autophagy, respectively. Optogenetic tools for the induction of autophagy will be developed as well (Workpackage 2), as a backup of the chemical biological tool box.

This experimental system will then be used to study the kinetics and biochemistry of autophagy of distinct organelles (Workpackage 3) in vitro, in cultured human cells and in vivo, in cells transplanted into animals, as well as in transgenic mice. Purification of autophagosomes and lysosomes (facilitated by expression of transgenic HA-tagged LC3 and LAMP2, allowing to immuno-purify HA-positive particles) followed by mass spectrometric analysis of degradation products will furnish fundamental knowledge on the autophagic process and its impact on cellular metabolism. Local biotinylation approaches will also be developed to avoid subcellular fractionation.

The technology that we will develop will allow us to generate cells that lack specific organelles (such as centrosomes, mitochondria, peroxisomes…) and hence study (Workpackage 4) the cell biological consequences of a unique type of “organellar knockout” (or partial depletion of organelles: “organellar knockdown”). This type of approach should yield basic insights into the contribution of organelles to cellular organization, signaling processes and metabolic pathways. It will also allow to characterize the mechanisms through which autophagy of particular organelles improves cellular fitness (in vitro), organismal health (in vivo) or, alternatively, may cause cell death.

Given the cardinal importance of autophagy in health and disease, these insights will contribute to the “Challenge No. 4: Life, health and wellbeing”, as defined In the ANR call. Indeed, fundamental research in cell stress and death pathways may yield important insights for the discovery of diagnostic biomarkers and novel therapeutic procedures.