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Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

Fig 3

Identification of motility phenotypes.

All deletion cell lines generated in this study and the ΔPF16 mutant [23] were analysed for defects in swimming speed or directionality (the ratio of velocity to speed). (A) Mean swimming speeds. Speeds were measured three times and the mean of all three replicates (●) and the individual replicates (○) are shown. Error bars represent the standard deviation. Red dotted lines indicate two standard deviations above and below the parental cell line (Cas9 T7) mean swimming speed. Cas9 T7 cells killed with 1% formaldehyde (Cas9 T7 fixed) were used as a completely immotile control. *** p<0.0005, ** p<0.005, * p<0.05 (Student’s t-test compared to the parental cell line). SBL1, SBL2 and SBL3 are barcoded parental cell lines used in pooled assays. For a sub-set of mutants, an addback copy of the deleted gene was introduced and swimming speeds restored toward the wild-type. (B) The swimming speeds of all knockout mutants (○), as in (A), plotted against mean directionality. Error bars represent the standard deviation of the three replicates. Four main mutant phenotype clusters are apparent: Paralysed (including mutants lacking a long flagellum), uncoordinated (which move slowly, but with greatly reduced directionality), slow (which move slowly, but with reduced directionality and speed) and fast (which move faster, with a tendency for higher directionality). (C) Speed and directionality of all knockout mutants in (B) with deletion of IFT components, mutants with a tendency for curling of the flagellum (S9 Fig) or disrupted PFR structure (Fig 5) highlighted. (D) Speed and directionality of knockout mutants in (B) with those passaged though sand flies (Fig 6) highlighted.

Fig 3