Pilot Project 9 (2023)

Light-regulated protein degradation of fly Nephrin to monitor slit diaphragm dynamics in Drosophila nephrocytes

Many renal diseases show a severe phenotype of filtration defects in the glomeruli of the kidney. Since podocytes and the slit diaphragms they compose are essential to establish the renal filtration barrier, defects are often caused by impaired or absent podocyte slit diaphragms and the mislocalization of the main slit diaphragm protein component Nephrin.

However, to investigate underlying pathomechanisms of podocyte biology, suitable cell culture models are lacking. Due to the conserved morphological, molecular and functional features of vertebrate podocytes and Drosophila melanogaster nephrocytes, these cells have emerged as model system to study podocyte function and disease (Helmstädter et al., 2017a; Helmstädter et al., 2017b; Hermle et al., 2017). Therefore, Drosophila nephrocytes are a useful tool for investigating the assembly and maintenance of the slit diaphragm. The main components of the Drosophila slit diaphragm are Sticks and Stones (Sns, orthologue of Nephrin in podocytes) and Kin of Irre (Kirre, Neph1 homologue), which are essential for slit diaphragm development and maintenance in nephrocytes (Zhuang et al., 2009).

In the last years, optogenetic approaches find increasing application in biomedical research. By using a generic photosensitive degron module, light-regulated degradation can be transferred to model organisms, enabling the light-regulated manipulation of protein levels, as well as biological processes. Renicke et al. have introduced a light-controlled degron, which allows protein degeneration when exposed to blue light (Renicke et al., 2013) Generation of photosensitive mutants showed blue light dependent growth inhibition when they fused the photosensitive degron (psd) module to a target protein. Optimization of the degron module in following works resulted in rapid degradation when exposed to blue light (Hasenjäger et al., 2019). Fusion of this psd-module to the nephrocyte protein Sns will allow us to investigate slit diaphragm assembly and maintenance and could give new insights into slit diaphragm dynamics.