Tension induced autophagosome formation: substrates and mechanisms

• Applicants: Dr. Bernd Hoffmann; Professor Dr. Pitter Huesgen
• Subject Area: Cell Biology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 388932620

Mechanosensory proteins play an outstanding role for the detection of mechanical signals, especially strain, that enable adaptation to dynamic changes in environmental parameters both on the single cell level and in tissues. Mechanosensoric proteins usually convert this strain into a chemical signal cascade by opening their tertiary structure to facilitate protein binding or phosphorylation. Subsequently, their refolding typically occurs. However, their fate remains largely unclear in the case of faulty refolding, as shown for high strain amplitudes or cyclic strain. We have demonstrated a clear activation of autophagosomal processes under cyclic strain. The number of autophagosomes formed correlates in time with the amount of stress applied to the cells by stretching. Reorientation of the cells as a cell response for stress reduction causes a simultaneous, closely coupled reduction of formed autophagosomes. Blocking autophagosomal degradation reduces the reorientation behavior. Autophagosome formation is further associated with the number of mechanosensitive structures, particularly stress fibers and cell-matrix adhesions. Mechanosensitivity, -response and autophagosome formation seem to be largely influenced by BAG3 without affecting the total amount of known mechanosensitive proteins much. Based on these investigations we will generate a detailed correlation map between strength of mechanosensitive protein complex formation, mechanosensitive response and autophagy. Studies will be performed for both, cellular and tissue-specific mechanosensitive complexes. Targeted activation and inactivation of complex formation and autophagy complete the analyses. Proteins degraded by stress-induced autophagy will be characterized by autophagosome purification under different stress conditions and subsequent mass spectrometric and immunoblot analyses. In addition, BAG3- and HSPB interactomes in differently stressed myotubes will be analysed to characterize the underlying autophagy induction machinery. Changes in the exchange kinetics of mechanosensitive proteins are investigated by FRAP under conditions of maximum autophagosome formation. The effect of blocked autophagy, constitutively active mutants of the CASA machinery (e.g. BAG3-WAWA) and BAG3-phosphomutants on exchange kinetics of these proteins and cellular mechanoresponse will be investigated. Analyses will be extended to total muscle analysis (rat soleus muscle) using an already developed tissue stretcher. In addition to the verification of known and newly identified connections between stress application and autophagy at the cell culture level, the tissue will allow us to determine to what extent electrically induced contraction (negative strain) and stretching (positive strain) differ with respect to their influence on mechanosensitive proteins and autophagy.

• DFG Programme: Research Units
• Subproject of: FOR 2743: Mechanical Stress Protection
• Co-Investigator: Professor Dr. Rudolf Merkel