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. 2022 Aug 5;221(9):e202112097. doi: 10.1083/jcb.202112097

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Figure 5. — PLP is essential for centriole motility and interacts with KHC. (A) 10-min time projections of centriole movement (colored tracks) in PCs following knockdown of PLP with tub-GAL4. Scale bars: 5 µm. (B and C) Quantification of mean squared displacement (B) and average velocity (C) following PLP knockdown (control average velocity = 128.8 nm/s ± 51.48, n = 49; PLPRNAi²⁹⁶ = 40.19 nm/s ±12.04, n = 54; PLPRNAi⁶⁴⁵ = 36.6 nm/s ±12.48, n = 112, data = mean ± SD, ANOVA: P = <0.0001, Dunnett’s pairwise comparison P = 0.0001 between Ctrl and RNAi conditions, ****). (D) Diagram showing the three independent interactions (black lines) found through yeast two-hybrid screening of PLP and KHC subfragments (Fig. S4). Blue = predicted coiled coils. Yellow = KHC motor domain. The PACT domain of PLP is located in the final C-terminal fragment (purple). (E) Mitochondrial recruitment assay validating interactions found through Y2H. Prey fragments (gray/cyan) accumulate in nucleus (magenta dashed line). In the presence of a co-transfected bait fragments (red, arrowhead) that is targeted to the mitochondria via a Tom20-RFP tag, a positive interacting fragment (gray/cyan) is also recruited to the mitochondria. (F) Examples showing mNG::KHC (green) localization in control or plp⁻ PCs. Yellow arrowheads indicated centriolar localization determined by Asterless staining (magenta). Blue = nucleus. (G) Quantification of mNG::KHC localization determined by the mean intensity at the centriole divided by mean cytoplasmic intensity. Mean ± SD: Control = 1.078 ± 0.05, n = 116 centrioles; plp²¹⁷²/Df = 1.05 ± 0.06, n = 69 centrioles, unpaired t test: P = 0.003, **. (H) Examples showing endogenous PLP (green) localization in control or KHCRNAi expressing PCs. Yellow arrowheads indicated centriolar localization determined by Asterless staining (magenta). Blue = nucleus. (I) Quantification of PLP localization determined by the mean intensity at the centriole divided by mean cytoplasmic intensity. Mean ± SD: Control = 2.8 ± 0.5, n = 44 centrioles; KHCRNAi = 2.9 ± 0.8, n = 71 centrioles, unpaired t test: P = 0.3, ns.