Cells of our body need to deal with similar logistical challenges as we do. They need, for instance, transport cargo from one place to another with high fidelity and efficiency within a very short period of time. Moreover, cells need to degrade unwanted or damaged cellular material to prevent accumulation of molecular waste. This, however, imposes another challenge to cells: they need to identify cytoplasmic trash and degrade it in a highly selective manner. All this needs to be achieve in the crowded cytoplasmic environment harbouring a high density of membrane bound organelles such as mitochondria, endoplasmic reticulum, golgi, or lysosomes. The latter are degradative organelles that contain an amanda of lytic enzymes that degrade all material that is delivered to these recycling stations.
How do cells recycle cytoplasmic material?
Cell have evolved a dedicated pathway which selects cargo and transports it to lysosomes for degradation. This pathway has been termed “autophagy” which means self digestion. In autophagy, a crescent shaped membrane is formed which encloses cargo entirely to give rise to an autophagosome that transports its content to lysosomes. Deficits in selective degradation of protein aggregates is closely related to the onset of a number of neurodegenerative diseases such as Alzheimer’s, Parkinson’s or Huntington’s diseases.
Our research therefore aims to reveal molecular mechanisms involved in autophagy, focussing on its relation to the onset of neurodegenerative diseases.To address these questions, we are using a combination of cell biology, biochemistry, high resolution microscopy, electron tomography and genome editing tools.
Upon cytotoxic stress or starvation, however, cells loose specificity and their waste bags start to capture cytoplasm randomly. This process is particularly important during the development of cancer. We could show that a protein shell at the surface of autophagic membranes function as scaffold to support autophagosome maturation. Our hypothesis is that this protein shell functions like a trash bin that physically support the intrinsically dynamic and flexible autophagic membrane.
Autophagy in the test tube
Using in vitro reconstitution techniques is particularly challenging if not only proteins, but also membranes are involved. We established a wide variety of different model membrane systems, including giant, large, and small unilamellar vesicles and supported lipid bilayers, to recapitulate the biogenesis of autophagosomes in the test tube. Fluorescent labeled autophagy related proteins are added to these membranes to reveal their molecular function. We used this technique to reconstitute the autophagic scaffold on membranes and investigated it by fluorescent techniques such as fluorescent recovery after photobleaching.
The movie shows such an experiment with a giant unilamellar vesicles (membrane, red) at which the protein shell (green) was reconstituted. FRAP (fluorescence recovery after photobleaching) revealed that the proteins which make up the shell are immobile, whereas the lipids of the membrane diffuse freely in and out of the bleached area. This example demonstrates the power of our combinatorial approach in discovering fundamental molecular mechanisms in autophagy.