Function and regulation of podin proteins under mechanical stress in muscle

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Podin proteins (SYNPO, SYNPO2, SYNPO2L) are multi-adapter proteins involved in multiple cellular activities related to mechanical stress: by binding to filamin, alpha-actinin and actin a subset of podin proteins contributes to actin cytoskeleton architecture, while others are involved in filamin proteostasis via chaperone-assisted selective autophagy (CASA) and signaling under mechanical stress. In the first funding period we established cell type-specific podin isoform expression patterns and identified important novel podin protein interactions: (1) a SYNPO2 PDZ domain interaction with YAP/TAZ transcription factors, (2) binding of SYNPO2 and SYNPO2L to the calcineurin inhibitor calsarcin, (3) phosphorylation-regulated LC3-binding and (4) a phosphoinositide recognition motif in SYNPO2. Bimolecular fluorescence complementation showed that a SYNPO2 isoform-specific interaction directs BAG3 to myofibrillar Z-discs or contraction-induced lesions. Electrical pulse stimulation (EPS) was exploited to study differential protein expression, changes in the phosphoproteome and effects on protein dynamics in response to mechanical stress. Here, the PDZ domain-containing SYNPO2b variant showed slower dynamics in FRAP assays than the PDZ-less SYNPO2 variants and mechanical stress had an impact on the short but not the long SYNPO2 isoforms. By contrast, the recovery of SYNPO2b is strongly depending on protein synthesis. Our data imply that lesions are a degradation-independent compartment stabilising local myofibrillar damage induced by mechanical stress. The role of podins in this process is further emphasised by the fulminant increase in lesion formation in SYNPO2 knockdown cells. Phosphoproteome analyses revealed complex contraction-induced phosphorylation patterns in myotubes, and for some of these we showed an impact on protein-protein interactions. The overall importance of the podin protein family is further highlighted by the identification of mutations in the SYNPO2 gene in nephrotic syndrome patients. A major focus of the planned work program will be to confine distinct and overlapping functions of the podin proteins. In particular, we will establish how contraction-induced (de)phosphorylations regulate protein interactions of podin proteins and what their respective functional impact is. Biochemical analyses of protein complexes will be complemented with light microscopical analyses including FRAP and BiFC in conjunction with EPS and podin protein knockdown cells. Of particular interest will further be to elucidate the role of podin proteins regarding the newly dicovered links to phosphoinositide signaling and calcineurin signaling, as well as the direct link to YAP/TAZ signaling. Our collaborations in this research unit will enable us to unravel the key functions of podin proteins for the regulation of protein homeostasis and signalling under conditions of mechanical stress in muscle.