Control of clathrin-mediated endocytosis by NIMA family kinases
Fig 6
Clathrin localization is altered in NEKL-depleted adults.
(A) Model of auxin-inducible degradation of NEKL kinases containing the auxin inducible degron (NEKL-2::AID and NEKL-3::AID). The addition of auxin triggers ubiquitination of NEKL::AID proteins followed by proteolysis. (B) Western blot showing complete loss of the full-length NEKL-2::AID and NEKL-3::AID proteins after auxin treatment. (C) Bar plot showing the percentage of molting defects among NEKL-2::AID and NEKL-3::AID strains in the presence and absence of auxin. p-Values were determined using Fischer’s exact test; ****p < 0.0001. (D–K) CRISPR-tagged GFP::CHC-1 localization was analyzed in NEKL-2::AID and NEKL-3::AID strains in the presence and absence of auxin (20 h) in day-2 adults. (D,G) Representative confocal images of day-2 adults expressing GFP::CHC-1 within the apical region of the hyp7 epidermal syncytium. Background subtraction was performed using the same parameters for all images; minimum and maximum pixel values were kept consistent for all images. Inverted fluorescence was used to aid clarity. Bar size in D = 5 μm (for D and G). (E,F,H,I) Mean GFP::CHC-1 intensities (E,H) and the percentage of GFP-positive pixels above threshold (F,I) were determined for individual adults. (J,K) Summary comparison of data from panels E,F,H,I and S2 Fig (B,C). (E,F,H–K) The group mean and 95% confidence interval (error bars) are shown. p-Values were determined using two-tailed Mann-Whitney tests; ****p < 0.0001, ***p < 0.001, **p < 0.01. Raw data are available in S1 File.
