Generation of Phosphatase knockout strain of Tetrahymena thermophila

Abstract

Dynamin family of proteins are a group of large GTPases which remodel their target membranes to cause membrane fission, fusion or tubulation.1,2 These proteins also perform other important cellular processes like endocytosis endoplasmic reticulum tubulation, cell plate formation and antiviral activity, etc. by localizing at their site of action. These proteins have properties like self-assembly and membrane binding, 3 which together determine the localization of these proteins and are regulated by various post-translational modifications (PTM). There are several examples of PTM which affect the function of dynamins. For example Drp1 undergoes phosphorylation which stimulates its fission activity. Phosphorylation is done by kinase and dephosphorylation is carried out by phosphatase.4 In our lab we study Dynamin related proteins 6 (Drp6), belonging to the dynamin superfamily, which is responsible for macronucleus (MAC) expansion in Tetrahymena thermophila.1 Previously in our lab it was reported that phosphorylation in a single serine residue in the GTPase domain enhances membrane fusion function as well as enhanced nuclear localization of Drp6.9 As phosphorylation affects the function of Drps, we hypothesized that dephosphorylation might have a role in Drp6 functioning. So we generate a Tetrahymena strain of phosphatase KO and we found that phosphatase is not required for normal growth of Tetrahymena but it is required for maintaining endoplasmic reticulum morphology in Tetrahymena.