Knockout screen of the flagellar proteome of Leishmania mexicana

Description

Motile cilia (eukaryotic flagella) are thin cellular appendages which cells use for movement, to generate fluid flows, to adhere to surfaces and to sense their environment. They are structurally complex, consisting of >600 different proteins, including a highly structured microtubule-based axoneme studded with dynein motor proteins and accessory structures required for the generation and modulation of flagellar waveforms. How each of these proteins and structures contributes to motility and cellular behaviours is not fully understood. In this project we performed a systematic study of loss-of-function phenotypes for flagella proteins in the protist Leishmania. Leishmania has a single motile flagellum and it is a tractable model organisms with well-developed in vitro tools and scalable methods for generating CRISPR-Cas9 assisted mutant libraries. We sed CRISPR-Cas9 to targeted 629 proteome-validated flagellar proteins for gene deletion (replacing target genes with drug-selectable marker cassettes), producing an arrayed library of 473 confirmed knockout mutants. 53 lines were recovered where a copy of the target gene remained detectable ('incomplete knockouts') and for 89 target genes, no viable transfectants were recovered. For all mutant cell lines, we assessed their growth rates, recorded their morphology and measured their swim speed and directionality. These data provide motility profiles for deletion mutants for 75% of the known flagellum proteins in Leishmania.

Primary Contact

Glünz, Eva Miriam

Institute of Cell Biology

Principal Investigator

Gluenz, Eva

Institute of Cell Biology

Investigators