FLIM measurement of OSM-3 conformation in C. elegans sensory cilia. (A) Schematic diagram designed for FLIM-FRET assay. Lifetime of GFP was measured and analyzed. (B) Diagram illustrating the conformational change between autoinhibited and active OSM-3 with different fluorescence labeling. (C) Schematic of the C. elegans amphid neurons. The dashed boxes are enlarged on the right, representing cilia and soma, respectively. Each cilium contains a transition zone (t.z.), a middle segment (m.s.), and a distal segment (d.s.). (D) Representative FLIM images of OSM-3 with different fluorescence labeling. OSM-3::GFP, OSM-3::GFP::mScarlet, and mScarlet::OSM-3::GFP are expressed under the control of Pdyf-1 in osm-3 null mutant animals. Fluorescence lifetimes of GFP are shown with a scale of pseudo color ranging from 2.50 to 3.00 nanoseconds (ns). Scale bar, 5 μm. (E) Statistics of lifetimes of GFP measured from different regions of amphid neurons in worms of indicated genotypes. Data are mean ± SD. Comparisons were performed between OSM-3::GFP and OSM-3::GFP::mScarlet or mScarlet::OSM-3::GFP. ***P < 0.001 by one-way ANOVA using BH method to adjust P values. (F) Diagram illustrating conformational changes of homodimeric OSM-3 kinesins in different regions of C. elegans sensory cilia suggested by FLIM-FRET.