Fig 2: TTC5 loss impairs arborization, axonal outgrowth and microtubule dynamics.(A) Representative images of mScarlet-expressing D12 NT or TTC5 KD i3Neurons (also see Movie S1); scale bar: 100 µm.(B) Arborization metrics: sum of neurite lengths, number of primary branches, and total number of neurites (primary, secondary and tertiary) averaged across 72 timepoints per neuron (Methods). Individual dots represent individual neurons. n = 102, 79 for NT (gray) and TTC5 KD (teal), respectively, from two independent experiments (circle/triangle symbol); error bars, mean +/− SD; ****, p < 0.0001, *, p < 0.05 by Welch’s t-test.(C) Neurospheres of mScarlet- and EB1-EGFP-expressing NT and TTC5 KD neurons (Methods) at D4, D6 and D8 post-differentiation; scale bar: 1 mm.(D) Axonal outgrowth at each timepoint, quantified as the radius of the thresholded signal normalized to the radius of the inner sphere of cell bodies (Methods). Individual dots represents individual neurospheres; n = 31, 32 (D4), 28, 29 (D6), 22, 22 (D8) neurospheres for NT (gray) and TTC5 KD (teal), respectively, across two independent differentiations (circle/triangle symbol); error bars indicate mean +/− SD; ***, p<0.001, ****, p <0.0001 by Welch’s t-test.(E) Representative frame from a time lapse of EB1-EGFP comets in growth cones at the outer edge of a neurosphere (top) with magnification of a single growth cone (bottom); EB1-GFP comets indicated by arrowheads; scale bar: 5 µm.(F) Comet density (top) and velocity (bottom) in growth cones of D5 NT (grey) and TTC5 KD (teal) i3Neurons (Methods, also see Movie S2). Individual dots represent means from individual growth cones from three independent experiments; n = 85 growth cones per condition; error bars and symbol indicate paired means +/− S.E.M. of each independent experiment, *, p < 0.05, **, p < 0.01 by paired t-test.

Fig 3: TTC5 loss results in hypermotility in a glia-guided neuronal migration assay.(A) Representative images of Halo-NLS-expressing NT control and TTC5 KD neurospheres at D6, D9 and D12 post-differentiation showing nuclear distribution over time (maximum intensity projections). Scale bar: 500 µm.(B) Nuclear migration profiles at each timepoint quantified as the averaged intensity of a line extending from the sphere center to the periphery (black dotted line in graphic in rightmost panel) and rotated radially. Each profile depicts the mean (solid line) and S.D. (shadow) across n = 8 neurospheres for both the NT (gray) and TTC5 KD (teal) condition, respectively. Statistical analysis performed using the binned signal intensity from the designated “inner” (yellow), “edge” (orange) and “outer” (red) regions of sphere for each timepoint (see Figure S5A for additional details); *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 by unpaired t-test.(C) Representative images of low-percentage NT or TTC5 KD Halo-NLS-expressing neurospheres overlaid with track trajectories color-coded by track mean speed (also see Movie S3). Scale bar: 500 µm. Tracks were analyzed for motility parameters shown in (D).(D) Cumulative distribution functions (CDF) for i) track mean speed, ii) track displacement and iii) confinement ratio (a metric for efficiency of motion as the ratio of displacement over total distance traveled) with dotted lines representing profiles for individual neurospheres and solid lines their average; n = 4 NT, 4 TTC5 KD neurospheres comprising 1653 and 1397 tracked nuclei respectively; median and 95% confidence interval indicated on graph and calculated based on corresponding scatterplots in Figure S5B; ****, p < 0.0001 by Kolmogorov-Smirnov test.

Fig 4: Autoregulation-defective TTC5 mutant R147A phenocopies TTC5 KD morphology and motility defects.(A) Structure of the TTC5 pocket (PDB 6T598), highlighting R147 (cyan) whose mutation to alanine abolishes binding of the MREI tubulin motif 8.(B) RT-qPCR for TUBB and TUBA1A mRNA expression in WT (grey) and R147A KI clone # 1 (cyan) upon colchicine treatment (Methods) in D14 i3Neurons (fold change relative to the untreated control following normalization to HPRT).(C) Representative images of mScarlet and EB1-EGFP-expressing WT and R147A KI clone #1 and 2 neurospheres at D8 (maximum-intensity projection). Scale bar: 1 mm.(D) Axonal outgrowth for the WT clone and two R147A KI clones at D4, D6 and D8; n = 16 spheres for WT and 8 spheres each for R147A KI clone #1 (circle) and #2 (triangle). Error bars, mean +/− SD; ****, p < 0.0001 by Welch’s t-test.(E-F) Average EB1-EGFP comet density (E) and comet velocity (F) per growth cone for WT and R147A KI clone #1 (data for additional clones shown in Figure S8A). Dots represent average values from individual growth cones from four independent experiments; n = 87 and 76 growth cones for WT and R147A KI, respectively; error bars and symbol indicate the paired means of each experiment +/− SEM; *, p < 0.05, **, p < 0.01 by paired t-test.(G) Arborization metrics averaged per neuron as in Figures 2A, 2B. Dots represent individual neurons from D12 WT (gray, n = 93), and R147A KI (cyan, n = 188 pooled between R147 KI clone #1 and clone #2) i3Neurons from two independent experiments (circle/triangle symbol); error bars indicate mean +/− SD; *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 by Welch’s t-test.(H) CDFs for i) track mean speeds, ii) track displacement and iii) confinement ratio of tracked nuclei in glial migration assay for Halo-NLS WT clone (n = 3 neurospheres, 1403 nuclei) and R147A KI clone #1 (n = 2 neurospheres, 1820 nuclei) and R147A KI clone #2 (n = 2 neurospheres, 1264 nuclei) (also see Movie S4); dotted lines represent individual neurospheres, solid lines, combined average; median and 95% confidence interval indicated on graph and calculated based on corresponding scatterplots in Figure S8B with statistical significance calculated via Kruskal-Wallis test; **** p < 0.0001.(I) Representative images of Halo-NLS- expressing WT control and R147A KI neurospheres at D9 and D12 showing nuclear distribution over time (maximum-intensity projection). Scale bar: 500 µm.(J) Representative images of TTC5 KD neurospheres at D8 expressing WT TTC5-mNG (purple) or R147A TTC5-mNG (cyan) (maximum-intensity projection). Scale bar: 1 mm.(K) Axonal outgrowth for neurospheres in (J) with comparative inclusion of TTC5 KD neurospheres expressing mNG alone (teal); n = 7 spheres/condition. Error bars, mean +/−SD; *, p < 0.05 by one-way ANOVA with Tukey’s correction for multiple comparisons.(L) CDF for mean speeds of tracked nuclei in glial migration assay for Halo-NLS-expressing TTC5 KD neurons upon reintroduction of either WT TTC5-mNG (purple, n = 4 neurospheres, 3013 nuclei) or R147A TTC5-mNG (cyan, n = 4 neurospheres, 3258 nuclei); dotted lines represent individual neurospheres, solid lines, combined average; median and 95% confidence interval indicated on graph and calculated based on corresponding scatterplots in Figure S8C with statistical significance calculated via Kruskal-Wallis test; **** p < 0.0001.

Fig 5: Loss of tubulin autoregulation mediator SCAPER recapitulates TTC5-dependent neuronal phenotypes.(A) Quantification of arborization metrics for NT (gray) and SCAPER KD (magenta) neurons (n = 102, 70 neurons for NT and SCAPER KD, respectively) compiled across two independent experiments (circle/triangle symbol).(B) Representative images of mScarlet-expressing NT and SCAPER KD neurospheres at D8 (maximum-intensity projected). Scale bar: 1 mm.(C) NT (gray) and SCAPER KD (magenta) neurospheres were assayed longitudinally at D4, D6 and D8 post-differentiation for axonal outgrowth (n = 8 spheres NT and 8 spheres SCAPER KD). Error bars indicate the mean +/− SD; ** p<0.01, *** p<0.001 by Welch’s t-test.(D) CDF for i) mean speeds, ii) track displacement and iii) confinement ratio of tracked nuclei in glial migration assay for Halo-NLS NT (n = 4 neurospheres, 2249 nuclei) and SCAPER KD (n = 4 neurospheres, 4277 nuclei); dotted line represents individual neurospheres, solid lines, combined average; ****, p < 0.0001 by Kruskal-Wallis test based on corresponding scatterplots in Figure S9B.