Mutation and/or inactivation of actin regulatory genes relevant for infection using the CRISPR/Cas system in tissue cultured cells

The Arp2/3 complex is a key regulator of the actin cytoskeleton that nucleates branched actin filament networks. However, the nucleation core activity of Arp2/3 is weak by itself and needs to be activated by nucleation promoting factors (NPFs) from the Wiskott-Aldrich Syndrome family. Several NPFs have been identified and can be divided into the two subclasses type I NPFs as well as type II NPFs. Type I NPFs include WASH, WHAMM, and JMY, among others. Importantly, bacterial pathogens can manipulate processes involved in actin rearrangements to survive within host cells and even enforce their uptake. Of particular interest for this study was the connection between actin assemblies on endomembranes and the intracellular lifestyle of pathogenic invaders. Here, I addressed the inactivation of WASH, WHAMM, and JMY that are at play in endomembrane traffic by using the CRISPR/Cas9 technology. Upon somatic gene knockout of these actin regulators, I examined a broad set of cellular and infection processes in murine fibroblasts and human cancer cells. To this end, I have generated several independent clones of NIH/3T3 fibroblasts lacking functional genes of WASH, WHAMM, and JMY. All cell lines were probed for the absence of the respective proteins by western blotting and targeted loci of clones lacking protein expression were genomically sequenced. The cellular phenotypes of obtained knockout cell lines have been characterized with special emphasis on ER- and Golgi morphology as well as on autophagy. WHAMM knockout significantly alters NIH/3T3 morphology, in particular WHAMM-deficient cells exhibited enlarged cell size and nuclear area and displayed higher granularity. In addition, knockout of WHAMM significantly affects integrity of the Golgi apparatus. Microarray analyses revealed a deregulation of genes encoding for enzymes involved in cholesterol biosynthesis as well as of genes being crucial for the adhesion pattern in WHAMM KO clones. Notably, examination of autophagy uncovers significantly higher numbers of LC3-, p62-, and LAMP1-positive vesicles as well as displayed enlarged omegasomes, autophagosomes, and lysosomes in knockout NIH/3T3 cells. Lastly, it could be shown that ablation of WHAMM decreases the infection rate of VSV and HSV-1. Current experiments are designed to unravel potential functions of WHAMM and its relatives in autophagy in more detail and to explore potential membrane alterations by both super-resolution and electron microscopy.