Chemically induced dislocation and mutation of essential cytoskeletal proteins: A case study focusing on Coronin-A

Abstract

Actin is one of the most highly conserved proteins—the actin cytoskeleton or actin-like structures are found in all kingdoms of life. In this work, Coronin-A, a central player involved in actin dynamics, is considered as an example to compare the effect of four mutation strategies on its function inDictyostelium discoideum. Two distinct genetic knockout methods (selection-linked integration and classical gene disruption), RNAi knockdown, and sudden loss-of-function generated by chemically induced dislocation (CID) are compared and the cell lines are characterized according to actin dependent processes: appearance, development, and motility. These phenotypic features are affected to different degrees in the mutant cell lines, providing a basis to discuss the strengths and weaknesses of each approach. This comparative study highlights the advantages of the newly introduced selection-linked integration method to knock out genes over commonly used gene disruption, and for expressing a CID-based knock-sideways system under the native promotor of the protein of interest.