A study of Fez1 and Fez2. Establishment of Fez1 and Fez2 knock out cells lines. Localization of Fez1 and Fez2

Abstract

Autophagy is an essential cellular process that is important to maintain homeostasis by degrading proteins, lipids and organelles during critical times like cellular or environmental stress conditions. Autophagy plays a critical role in human pathologies, since defective autophagy can lead to diseases like cancer, neurodegenerative- and inflammatory diseases. Fez1 (Fasciculation and elongation protein zeta 1) is a protein that has been reported to inhibit autophagy. In the first part of this study an attempt to establish stable knock out cell lines of Fez1, its homologue Fez2 and double Fez1-Fez2 by using CRISPR/Cas9 technology was performed. The knock out strategy was applied on two different human cell lines, Hek293 Flp-In and the neuronal cell line SH-SY5Y, and more than 200 putative clones were screened. Unfortunately, none of them displayed complete knock out of Fez1 or Fez2. This may indicate that Fez1 and Fez2 are essential for cell survival or cell growth in these cell lines.

In the second part of this study, confocal imaging was applied to study the localization of Fez1 and Fez2, and if they colocalized with proteins involved in autophagy and intracellular transport. Interestingly, upon over-expression both Fez1 and Fez2 colocalized with proteins involved in autophagosome initiation and maturation like WIPI2 and the Atg8 proteins. However, they did not colocalize with syntaxin17, which is involved in the fusion step between autophagosomes and lysosomes. Both proteins also colocalized with tubulin and the transport protein KIF5B. This may suggest a role for Fez1 and Fez2 in the transport of early autophagosomes. The colocalization with tubulin, specifically γ tubulin, was verified at the endogenous level by immunostaining.