Leggere JC , Hibbard JVK , Papoulas O , Pearson CG , Marcotte EM , Wallingford JB .

F31 DE029114 NIDCR NIH HHS, R01 HD085901 NICHD NIH HHS , R35 GM122480 NIGMS NIH HHS , R35 GM140813 NIGMS NIH HHS

	FIGURE 1:. Ift122 Tetrahymena DIFFRAC detects specific and predicted changes in protein abundance and organization. (A) Experimental and computational pipeline overview. Samples of whole-cell wild-type or Ift122-null strains were lysed under nondenaturing conditions. Size-exclusion HPLC and subsequent mass spectrometry were used to determine the identity and abundance of proteins in fractions with high protein content. Differential abundance between the wild-type and null samples was calculated for every protein observed, for every fraction in which it was observed in either sample. P-values and FDRs were calculated for the largest (positive or negative) z-score for each protein, and 617 orthogroups which rose above the FDR cutoff of 0.1% were further analyzed. (B–E) Elution profiles show nonciliary and select ciliary protein complexes are intact in the Ift122 mutant, but IFT-A abundance and assembly are drastically perturbed. Profiles display elution behavior of select orthogroup in wild-type (black) and Ift122-null (blue) samples. Each row plots abundance of an individual orthogroup across all fractions analyzed. Members of known protein complexes are grouped together. (B) The abundance of each observed IFT-A subunit is drastically decreased. Additionally, the later elution of IFT144 and IFT121 in the mutant suggests the remaining subunits are present in a smaller, lower molecular weight complex in the Ift122∆ sample. (C and D) Abundance and assembly the IFT-B and BBSome complexes are unaltered in the Ift122 mutant. For each complex, subunits coelute and do not have significantly altered abundance or elution patterns in the Ift122∆ sample. (E) The negative control CCT complex has consistent protein composition and abundance in the absence of Ift122. Individual complex members behave similarly in the two samples (compare black and blue line for CCT8) and subunits behave similarly to each other (compare black line of CCT8 to black line of CCT2, or blue line of CCT8 to blue line of CCT2).
	FIGURE 2:. Differential abundance analysis identifies 617 orthogroups as significantly changed, including known IFT-A cargos. (A) Differential abundance of orthogroups in wild-type versus Ift122∆ proteomes. Plot of the fold change in abundance versus p-value for the maximum per fraction z-score calculated for every orthogroups observed in the experiment. Orthogroups that surpass the FDR = 0.001 (0.1%) cutoff are plotted as blue dots, while those that do not are plotted in gray. IFT-A orthogroups are labeled in dark blue, IFT-B orthogroups are labeled in gray, and known ciliary proteins are labeled in orange. Orthogroups corresponding to proteins further studied in this study are labeled in magenta. One of the highest significance orthogroups, ENOG502RZNF, does not have any predicted vertebrate homologues, but the Tetrahymena paralogues in this orthogroup are poorly characterized and are primarily annotated as putative ATPases. (B) Elution profiles of established IFT-A cargoes The known IFT-A cargo PKHD1 has reduced abundance in the Ift122∆ sample. In contrast, the orthogroups containing Cnga2 and OGT show increased abundance in the null strain. Additionally, Cnga2 elutes at higher molecular weight peak compared with the control sample, suggesting potential aggregation or accumulation of a larger protein complex. (C) Known and predicted ciliary proteins are enriched among DIFFRAC hits [OR] Coverage of known and predicted ciliary proteins among DIFFRAC hits Proportion of observed orthogroups that contain one or more vertebrate homologues identified by CiliaCarta as known or probable ciliary proteins. Categories are: “Gold Standard” proteins, for which significant experimental evidence of ciliary localization or function exists; “Gene Ontology” proteins which have previously been annotated with cilia-related GO-term; and proteins were “Predicted” to be ciliary using CiliaCarta’s Bayesian model.
	FIGURE 3:. Ift122 knockdown in Xenopus recapitulates evolutionarily conserved IFT-A loss of function phenotypes, which are rescued by Ift122 overexpression. (A) RT-PCR validation of Ift122 morpholino mediated knockdown. Dose curve of morpholino targeting the first splice junction of Ift122 pre-mRNA. Embryos were injected at the four-cell stage and mRNA was extracted at stage 25. Subsequent RT-PCR amplification of the MO target region shows a dose-dependent decrease in Ift122 transcript levels. Because the 6 ng dose resulted in a robust reduction of transcripts but not a decrease in embryo viability (which was observed for the 12 ng dose – data NS), 6 ng injections were used for all following experiments. (B−D) Increase in ciliary IFT-B localization and tip accumulation upon Ift122 knockdown in epidermal MCC cilia. (B) Representative images of Ift46 ciliary localization in control, Ift122 knockdown, and rescue embryos. Note loss of cilia numbers and accumulation of Ift46 in morphants of rescue of both phenotypes. All fusion constructs were injected as mRNAs. CAAX-RFP labels both cell and ciliary membrane, IFT-B is labeled with Ift46-mNG, and the phenotypes were rescued with the addition of Ift1222-FLAG mRNA. (C) The average intensity of Ift46-mNG per cilium is significantly increased upon MO-mediated knockdown of Ift122. This change is rescued by addition of Ift122-FLAG mRNA. (D) The tip accumulation of Ift46 in Ift122 KD cilia is rescued by addition of Ift122-FLAG mRNA. 1 corresponds to the distal-most Ift46-mNG signal (toward the ciliary tip), 0 corresponds to the proximal-most point measured (toward the ciliary base). Points show the intensity of Ift46-mNG at a single, min-max scaled position along a single cilium. Min−max scaling was used for position normalization. Lines are smoothed conditional means calculated using locally estimated scatterplot smoothing (LOESS).
	FIGURE 4:. DIFFRAC identifies IFT-A-mediated ciliary exit and entry cargo. (A−C) Ak8 localization increases along the entire length Ift122 KD cilia, suggesting IFT-A is required for ciliary exit of Ak8. (A) Representative images. Membrane is labeled with CAAX-RFP, and Ak8 is visualized with a GFP-tagged construct. (B) The average GFP-Ak8 intensity/CAAX-RFP intensity per cilium increased from 0.492 ± 0.187 in control to 0.91 ± 0.377 in Ift122 KD (***p < 0.001, Wilcoxon rank sum test with continuity correction; Control, n = 134 cilia from 29 cells, N = 6; Ift122 KD, n = 145 cilia from 41 cells, N = 6). Data represent mean ± S.D. (C) Ak8-GFP intensity versus scaled position along cilium. In contrast to the tip accumulation of Ift46-mNG, the intensity of GFP-Ak8 increases evenly along the length of the cilium in the Ift122 KD. (1) denotes the distal-most Ak8-GFP signal (toward the ciliary tip), (0) denotes the proximal-most point measured (toward the ciliary base). Points show the intensity of GFP-Ak8 at a single, min−max scaled position along a single cilium. Lines are smoothed conditional means calculated using a general additive model (GAM). The smoothed means are calculated from all data points, but points above the 99th percentile of GFP-Ak8 intensity are not shown. (D−F) Novel ciliary protein Pip5kl1 has increased ciliary localization upon Ift122 KD, suggesting IFT-A regulates its entry into the cilium. (D) Representative images. Membrane is labeled with CAAX-RFP, and Pip5kl1 is visualized with an n-terminal GFP-tagged construct. GFP-Pip5kl1 localizes along the entirety of the ciliary membrane. (E) Quantification of average GFP-Pip5kl1 intensity per cilium. GFP-Pip5kl1/CAAX-RFP is reduced from 0.812 ± 0.33 in control to 0.147 ± 0.0864 in Ift122 KD (***p < 0.001, Wilcoxon rank sum test with continuity correction; Control, n = 135 cilia from 33 cells, N = 6; Ift122 KD, n = 135 cilia from 41 cells, N = 7). Data represent mean ± S.D. (F) Pip5kl1 localization along the cilium: GFP-Pip5kl1 intensity versus scaled position along the cilium. From proximal (0) to distal (1) in relation to the cell body. Points show the intensity of GFP-Pip5kl1 at a single, min−max scaled position along a single cilium. Lines are smoothed conditional means calculated using a GAM. The smoothed means are calculated from all data points, but points above the 99th percentile of GFP-Pip5kl1 intensity are not shown.
	FIGURE 5:. Ift122 regulates both the ciliary and subapical localization of novel ciliary protein Ccdc157. (A and B) Ccdc157 displays variable ciliary localization that is significantly increased upon Ift122 knockdown. (A) Representative images showing increased localization of GFP-Ccdc157 in Ift122 KD cilia. (B) Knockdown of Ift122 increases average GFP-Ccdc157/CAAX-RFP per cilium from 0.0957 ± 0.0503 in control to 0.196 ± 0.125 in Ift122 KD (***p < 0.001, Wilcoxon rank sum test with continuity correction; Control, n = 155 cilia from 34 cells, N = 6; Ift122 KD, n = 191 cilia from 41 cells, N = 6). Data represent mean±s.d. Statistics are calculated from all data points, but outliers are not shown. (C and D) Ccdc157 has robust peri-basal body localization in wildtype cells but is lost from this region in Ift122 KD MCCs. (C) Representative images of the polarized, peri-basal body localization of GFP-Ccdc157. Basal bodies are labeled with centrin-RFP. Average intensity of GFP-Ccdc157 per basal body is significantly reduced in Ift122 KD MCCs, as quantified in (D). Means are calculated from all data points, but outliers are not shown. (***P < 0.001, Wilcoxon rank sum test with continuity correction; Control, n = 4768 basal bodies from 30 cells, N = 6; Ift122 KD, n = 3820 basal bodies from 30 cells, N = 6).
	FIGURE 6:. Pccb localization near basal bodies in Ift122-dependent. (A) Pccb-GFP (green) is enriched near basal bodies labeled by Centrin-RFP (magenta). Orthogonal views reveal Pccb is present at and below the position of the basal bodies. (B) Ift122 KD severely deplete GFP-Pccb intensity at basal bodies (marked by centrin-RFP) (***p < 0.001, Wilcoxon rank sum test with continuity correction; Control, n = 3462 basal bodies from 24 cells, N = 4; Ift122 KD, n = 3540 basal bodies from 24 cells, N = 4). Data represent mean ± s.d.

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