Interplay between Ciliary Ultrastructure and IFT-Train Dynamics Revealed by Single- Molecule Super-resolution Imaging  Felix Oswald, Bram Prevo, Seyda Acar, Erwin J.G. Peterman  Cell Reports  Volume 25, Issue 1, Pages 224-235 (October 2018) DOI: 10.1016/j.celrep.2018.09.019 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Single-Molecule Imaging of IFT-B (OSM-6::eGFP, a Subunit of IFT-Particle Subcomplex B) (A) Time-averaged image of EGFP-tagged IFT-B imaged in phasmid chemosensory cilia of C. elegans (black) overlaid with single-molecule trajectories (white and colored traces; asterisks indicate the start of colored trajectories). Scale bar, 1 μm. The blue rhombus indicates the location of the velocity switch in the blue trajectory (see C). (B–E) Left: time-position (colored) and time-velocity (gray) traces of representative trajectories, highlighted with the same color in (A). Right: sequence of raw images corresponding to time points highlighted with white-filled symbols on the left. Trajectory in DS showing turnaround at (B), retrograde trajectory in PS and TZ (C), anterograde trajectory in TZ and PS (D) and anterograde trajectory in TZ including pauses (E). Scale bar, 500 nm. See also Figure S1. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Resolving Ciliary Ultrastructure by Dynamic Single-Molecule Super-resolution Imaging (A) Representative example of dynamic single-molecule super-resolution imaging on IFT-B: single-molecule localizations (nLoc = 1,785) (yellow circles, bottom) obtained from single-molecule trajectories (nTrajectories = 46) (colored lines, top) are used to obtain a super-resolution image that shows the underlying ciliary structure. Scale bar, 2 μm. (B) Illustration of the ciliary structure on the basis of state-of-the-art EM data (Doroquez et al., 2014) (left, middle) compared with super-resolution images of IFT proteins (right). Left: side view; middle: cross sections. Red highlights the ciliary base, blue the TZ, violet the PS, and gray the DS. Right: super-resolution fluorescence images (side view) of kinesin-II (bottom; nLoc = 493) and OSM-3 (middle [nLoc = 521] and top [nLoc = 833]). Arrows indicate the typical cross-sectional widths in the ciliary subdomains. Scale bar, 0.5 μm. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Single-Molecule Motility Characterization of IFT Components (A–C) Velocity distributions for IFT-B (nCilia = 19, nTrajectories = 202, nDisplacements = 1,157) (A), OSM-3 (nCilia = 38, nTrajectories = 324, nDisplacements = 2,630) (B), and kinesin-II (nCilia = 24, nTrajectories = 120, nDisplacements = 492) (C) in the presence of kinesin-II function. (D and E) Velocity distributions for IFT-B (nCilia = 22, nTrajectories = 143, nDisplacements = 1,632) (D) and OSM-3 (nCilia = 22, nTrajectories = 178, nDisplacements = 881) (E) in the absence of kinesin-II function. (F) Illustrations of kinesin-II, OSM-3, and IFT-B, as well as IFT trains in the presence and absence of kinesin-II function. Velocity distributions in (B) and (C) were fit with single Gaussian functions and those in (A), (D), and (E) with the sum of two Gaussians. Dashed lines indicate the maxima of the Gaussian functions. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Single-Molecule Velocity Histograms Resolved in Ciliary Subdomains (A) Velocity distributions of IFT-B in the presence of kinesin-II function for, from bottom to top, base (nDisplacementsBase = 144), TZ (nDisplacementsTZ = 280), first half of the PS (PS I) (nDisplacementsPSI = 258) (PS I), second half of the PS (PS II) (nDisplacementsPSII = 189), and the DS (nDisplacementsDS = 286). (B) Velocity distributions for IFT-B in absence of kinesin-II function (nDisplacementsBase = 496, nDisplacementsTZ = 337, nDisplacementsPSI = 216, nDisplacementsPSII = 319, nDisplacementsDS = 264). (C) Illustration of the ciliary ultrastructure indicating the subdomains. (D) Table summarizing the results of Gaussian fits of the velocity histograms (mean ± SD). Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 IFT-B Pausing at the Ciliary Base and TZ (A) IFT-B pausing positions with respect to ciliary base (nTotal = 71). Inset (cyan): histogram of pause durations in the TZ (nTZ = 55). (B) As in (A) for IFT-B pauses in the absence of kinesin-II function (nTotal = 101, nTZ = 94). (C) Left: super-resolution image of IFT-B (magenta) overlaid with a representative anterograde single-molecule trajectory (scale bar, 500 nm). Pause localizations highlighted in cyan and anterograde movement in white. Right: time-position trace of the IFT-B molecule visualized on the left. Inset: time-velocity trace of the same trajectory. Pauses are indicated in cyan. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 IFT-B Turnaround Dynamics (A) Cartoon of ciliary ultrastructure with single-molecule turnaround trajectories. (B) Right: position-time and velocity-time traces of single-molecule trajectories highlighted on the left. Top: anterograde-to-retrograde turnarounds; bottom: retrograde-to-anterograde turnarounds (additional example trajectories shown in Figure S1). Left: super-resolution images of IFT-B in magenta, overlaid with turnaround trajectories highlighted in white. Pauses during turnarounds are highlighted in cyan. Scale bar, 500 nm. (C) Position histogram of IFT-B turnarounds. Top: anterograde-to-retrograde turns (na-r = 65); bottom: retrograde-to-anterograde turns (nr-a = 22), both in the presence of kinesin-II function. (D) As in (C) for IFT-B in the absence of kinesin-II function (na-r = 30, nr-a = 13). See also Figure S2. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 Kinesin-II-Dependent Alteration of Ciliary Ultra-structure and IFT-Train Orientation (A) Super-resolution images of base, TZ, and PS of IFT-B (left) (nLocIFTB = 2,917) and TBB-4 (right) (nLocTBB-4 = 1,423) in the presence of kinesin-II function. (B) As in (A), in the absence of kinesin-II function (nLocIFTB = 1,342, nLocTBB-4 = 1,288). Scale bar, 500 nm. (C) Cartoon of data analysis using perpendicular pairwise distances of trajectories. Pairwise distances were measured between the means of perpendicular positions of trajectories in the PS (Experimental Procedures). (D) CPD of pairwise distances of anterograde IFT-B (cyan) (nID = 739, 15 cilia), retrograde IFT-B (magenta) (nID = 417, 15 cilia), and TBB-4 (black) (nID = 1,559, 12 cilia) in the presence of kinesin-II function. Note that in the analysis of the TBB-4 data, only static TBB-4::EGFP was included, in order to select for tubulin incorporated in the axonemal structure, rather than tubulin transported by IFT (Hao et al., 2011). CPDs in dark cyan, dark magenta, and gray correspond to anterograde IFT-B (nID = 244, 11 cilia), retrograde IFT-B (nID = 183, 11 cilia), and TBB-4 (nID = 643, 8 cilia) respectively, in the absence of kinesin-II function. (E) Simulated CPDs obtained from fits to experimental CPDs; color coding as in (D). In the simulations, we reconstituted the nine-fold axonemal geometry and determined pairwise distances between IFT trains randomly placed on MT tracks in the axoneme, mimicking experimental conditions. In this way, by varying the axoneme width in the simulated PS, we generated a CPD library, used to fit the experimental CPDs. See also Figures S3–S5. Cell Reports 2018 25, 224-235DOI: (10.1016/j.celrep.2018.09.019) Copyright © 2018 The Author(s) Terms and Conditions