A study of Fez1 and Fez2: Localization and knock-out

Abstract

Autophagy is a fundamental cellular process where cell components get digested in autolysosomes and are recycled. Dysregulation of autophagy is involved in major diseases like cancer, neurodegeneration, inflammation and ischemia. In this thesis we have worked with fasciculation and elongation zeta (Fez) proteins, which are reported to inhibit autophagy. There are at least two mammalian Fez proteins, Fez1 and Fez2. Fez1 has three light chain three interaction regions (LIRs). Fez1 can use these to interact with LIR docking sites (LDS) on the autophagy Atg8 proteins. Using the Flp-In system, ten Hek293 cell lines were established. These cell lines have tetracycline inducible expression of EGFP-Fez1 mutants, and one cell line has inducible expression of EGFP-Fez2. Seven of the cell lines expressing EGFP-Fez1 are mutated in the LIR motifs. The other two express a phosphorylation mimicking mutant of Fez1 (S58E) and the un-phosphorylated form Fez1 (S58A). Fez1 binds kinesin-1. Phosphorylation of Fez1 S58 regulates the kinesin-1 binding. The second LIR is close to Fez1 S58 and phosphorylation of S58 may also regulate Atg8 interaction. The second LIR of Fez1 is recently proposed to bind to LDS in a reverse direction. As far as we know, this reverse binding is novel. The Expression and localization of Fez1 mutants and Fez2 was characterized by confocal microscopy. Immunofluorescent staining of endogenous Gabarap in the Flp-In cell lines suggest that either the reverse LIR2 is important or Fez1 Gabarap co-localization in a perinuclear dot is independent of all three Fez1 LIRs. A nuclear localization signal (NLS) is predicted in Fez1. Here the Fez1 NLS was tested experimentally. The NLS was cloned into a plasmid in front of EGFP-gal and localization imaged by confocal microscopy. Our data indicate that the NLS is functional. Furthermore, various EGFP-Fez1 deletions constructs were made. Their localization was studied by confocal microscopy. The results indicate that Fez1 has a second NLS and also a nuclear export sequence (NES), both in the Fez1 2-130 region. Fez1 is expressed in the brain while Fez2 is ubiquitously expressed. They are both hub proteins with many interaction partners. There is little research on Fez2. The EGFP-Fez2 cell line established here shows that Fez2 is mainly cytoplasmic, with strong enrichment in a perinuclear dot. Interestingly, immunofluorescent staining of Gabarap showed that Gabarap co-localizes with Fez2 in this dot. The Fez1 LIR2 is not conserved in Fez2. An attempt to establish Hek293 Flp-In cell lines with the Fez1 and Fez2 genes knocked out was performed using the CRISPR/Cas9 technology. One potential Fez1 and one potential Fez2 knock out cell line was obtained. These cell lines will hopefully be useful in future research of Fez1 and Fez2.