Research Group Graef

Autophagy, lipid metabolism, and mitochondria – a complex relationship

The de novo formation of autophagosomes requires substantial membrane sources and rearrangements, which are poorly described as yet. In our previous work, we found using proteomic, cytological and functional analyses, that the formation of autophagosomes is spatially, physically and functionally linked to specialized regions of the endoplasmic reticulum (ER), so-called ER exit sites (ERES), which play a role in the biogenesis of COPII vesicles. Our data show that ERES are core autophagy components required for the assembly of the autophagy machinery during the early stages of autophagosome formation and for the expansion of phagophores, likely by providing a proximal membrane source. Significantly, our experiments in mammalian cells revealed that ERES-associated autophagosome biogenesis is a conserved feature of autophagy (Graef et al., 2013).

Our recent work revealed that lipid droplets (LD), dynamic organelles dedicated to neutral lipid storage, while dispensable as lipid sources for autophagosomal biogenesis, are required for intact autophagy regulation by buffering excess fatty acids and maintaining the phospholipid composition of membranes. In particular, absence of LD results in chronic stress and morphological alterations in the endoplasmic reticulum (ER), and severely compromises autophagosome biogenesis (Velazquez et al., 2016; Velazquez and Graef, 2016). We are exploring the mechanistic underpinning of these phenotypes and, in addition, examining the involvement of central lipid metabolism pathways and their potential roles in the regulation of autophagy.

In our previous work, we found that mitochondrial dysfunction compromises the capacity of cells to regulate autophagy in dependence of metabolic conditions (Graef and Nunnari, 2011). Hence, in contrast to the general view that mitochondrial dysfunction induces selective quality control autophagy targeting mitochondria, so-called mitophagy, our data indicate the interrelationship of mitochondria and the autophagy machinery is more complex, and we are currently dissecting these multifaceted relationships.

Genetic-metabolic profiling of autophagy

Given the plasticity and complexity of the known core and auxiliary autophagy machinery (>200 factors), comprehensive understanding of autophagy regulation requires system-wide approaches amenable to high throughput analysis. We have developed a fluorescence-based reporter system, which simultaneously quantifies two key aspects of autophagy, autophagy flux and gene induction, by automated flow cytometry. We are in the process of generating autophagy response profiles (ARP) for each gene in the yeast genome against a variety of autophagy-inducing/metabolic conditions. Using ARP-based cluster analysis, we will assemble a genetic-metabolic map of autophagy regulation.

Genetic-metabolic profiling of mitotic ageing

Mitotic ageing (MA) describes the limited capacity of cells to divide. A lack of appropriate tools has hampered system-wide analysis of MA. We have engineered a synthetic genetic programme in yeast cells, the daughter extinction programme (DEP), which specifically prevents daughter cell division after asymmetric separation from mother cells. Consequently, DEP growth rates in culture solely depend on mother cell division and reflect mitotic ageing rates (MAR) over time. Using fully automated, high throughput growth measurements and a newly developed bioinformatics pipeline, we have determined genome-wide MAR at single gene resolution and assembled functional networks. We are dissecting the mechanisms of novel gene clusters accelerating or slowing MA.