Fig 1: sDAP depletion eliminates the anchoring of a subset of microtubules around the mother centriole.(a) Representative immunofluorescence images of a-tubulins in WT and CEP128–/– RPE-1 cells. Centrin was the centriole marker whereas SCLT1 was the mother-centriole-specific marker. (Inset) Magnified images showing the organization of a-tubulin fibers around the centriole. (b) Analysis of mean a-tubulin intensity around mother and daughter centrioles in WT and CEP128–/– cells. NS, not significant. (c) (Left) dSTORM images revealing fewer a-tubulin fibers arranged around the CEP128–/– centriole (white dotted line) as compared to the WT centriole. (Right) Representative dSTORM images of a-tubulin fibers in WT and CEP128–/–centrioles. SCLT1 is co-stained as a marker for the axial centriole view (green dashed line). (d) Statistical analysis counting the number of a-tubulin fibers per radian (rad) around the WT and CEP128–/–centrioles (n = 5 centrioles each), *p<0.05. Figure 5—figure supplement 1 details the analysis approach. (e) Representative lateral dSTORM images revealing the organization of centrosomal a-tubulin fibers around the WT and CEP128–/–centrioles in the longitudinal direction. (f) dSTORM images in panel (e) aligned and combined using SCLT1 as a position reference. (g) Statistical analysis counting the number of a-tubulin fibers at various longitudinal positions around the WT and CEP128–/–centrioles (WT, n = 78 data points; CEP128–/–, n = 57 data points, p<0.01). Figure 5—figure supplement 2 shows the analysis strategy. Bars: (a, c) (WF images) = 2 µm; (c, e, f) = 200 nm.
Fig 2: Super-resolved localizations of subdistal appendage (sDAP) proteins exhibit distinct radial distributions.(a) Axial-view direct stochastic optical reconstruction microscopy (dSTORM) images of CEP170 under serum-supplied (FBS+) and serum-starved (FBS–) conditions. (b) Image analysis revealing that, under the proliferating condition (FBS+), CEP170 exhibits a relatively random radial distribution compared to that under the resting (FBS–) G0 phase. Each color represents a data value for different centrioles. (c) Statistical analysis of the radial distribution of CEP170 under FBS+ and FBS– conditions. n = 10 centrioles for both conditions, p<0.05. (d) Axial-view dSTORM images of CEP128 under serum-supplied (FBS+) and serum-starved (FBS–) conditions. (e) Image analysis revealing that CEP128 rings are less organized under the proliferating condition (FBS+) than under the resting phase (FBS–). (f) Statistical analysis showing the completeness of the ring-shaped occupancy of CEP128 under FBS+ and FBS– conditions. **p<0.01. (g) Representative dSTORM super-resolution images of axial-view centrioles showing the radial distribution of the distal appendage (DAP) protein SCLT1, various sDAP proteins, and the centriole proximal-end (Prox. end) protein CNAP1, which were not resolvable under wide-field (WF) imaging. ‘Overhang structures’ (arrowheads) were sometimes observed in the ninein and CEP170 rings. (h) (Left) Mean diameter analysis revealing size differences among the proteins in panel (g). Supplementary file 1 lists the dimensional details. The diameters of ODF2 and CNAP1 were similar to that of the centriole wall measured from the electron microscopy (EM) images (dotted line). (Right) A schematic figure summarizing previous studies on the hierarchical assembly of sDAPs. (i) Serial transmission EM (TEM) sections of an RPE-1 mother centriole. The centriole, reconstituted by TEM analysis with serial sectioning, reveals an approximately nine-fold distribution of sDAP in RPE-1 cells. Asterisks and the arrowhead indicate sDAPs and DAP, respectively. Bars: panels (a, b, d, e, g) = 200 nm; panel (i) = 100 nm.
Fig 3: The majority of ?-tubulins around the mother centriole are not associated with microtubule anchoring at sDAPs in the G0 phase.(a) (Left) Representative dSTORM images revealing the longitudinal positions of ?-tubulins with respect to CEP170. (Right) Scatter plot comparing the longitudinal positions of ?-tubulin with that of CEP170 (n > 7 centrioles); the longitudinal position of SCLT1 is set as zero. (b) (Left) Representative dSTORM images revealing the radial distribution of ?-tubulins. SCLT1 is the marker for an axial centriole view. (Right) Mean diameter analysis revealing that the radial distribution of ?-tubulins is similar to that of SCLT1. The dotted line indicates the diameter of the centriole wall measured in EM images. (c) (Top) Representative lateral two-color dSTORM images revealing the organization of centrosomal ?-tubulin in the WT and CEP128–/– centrioles. (Bottom) Magnified images of the inset in the top row. (d) Statistical analysis measuring ?-tubulin intensity around sDAPs (insets in panel [c]) in the WT and CEP128–/– centrioles (both WT and CEP128–/–, n = 5 centrioles), **, p<0.01. (e) Two-color dSTORM images in panel (c) aligned and combined according to the longitudinal position of ?-tubulin relative to SCLT1 (n = 5 centrioles). (f) Statistical analysis of ?-tubulin intensity in the insets in panel (e) revealing that ?-tubulins are distributed towards the centriole distal end upon CEP128 depletion. (g) A model speculating on the role of sDAPs in microtubule anchoring. The loss of CEP128 relaxes the distribution of ?-tubulins toward the centriole distal end, whereas microtubules fail attach to the centriole at sDAPs upon CEP128 depletion. Bars = 200 nm.
Fig 4: DAP and sDAP integrity regulates ODF2, CEP89, and ninein localization differentially.(a) Immunoblotting confirming knockout of CEP83 or CEP128 in RPE-1 cells. WT, wild-type RPE-1 cells; CEP128–/–, CEP128 knockout RPE-1 cells. (b, c) Representative two-color dSTORM images of (b) the N-terminus of ODF2 and (c) the C-terminus of ODF2 with SCLT1 in WT cells and CEP128–/– cells. (d) Two-color dSTORM images in panels (b) and (c) aligned and combined according to their longitudinal positions relative to SCLT1 (n = 5 centrioles per group). (e) (Left) Representative dSTORM images of ODF2 in WT and CEP83 knockout RPE-1 cells (CEP83–/–). (Right) Intensity profile of WT and CEP83–/– cells (WT, n = 7 centrioles; CEP83–/–, n = 6 centrioles) showing that ODF2 becomes a single-layer structure when CEP83 is depleted. (f) Representative two-color dSTORM images of CEP89 and FBF1 in WT and CEP128–/–cells. (g) (Left) Two-color dSTORM images in panel (e) aligned and combined according to their longitudinal positions relative to FBF1 (WT, n = 5 centrioles; CEP128–/–, n = 6 centrioles). (Right) Intensity profile of the images in the left panel showing that the lower layer of CEP89 is absent in the CEP128–/– cells. (h) Representative two-color dSTORM images of ninein and CP110 in WT, CEP83–/–, and CEP83–/– cells stably expressing wild-type CEP83 protein (CEP83–/–Rescue). (i) (Left, top) Two-color dSTORM images in panel (g) aligned and combined according to their longitudinal positions relative to CP110 (WT, n = 5 centrioles; CEP83–/–, n = 6 centrioles; CEP83–/–Rescue, n = 5 centrioles). (Left, bottom) Magnified images of the insets in the top row. (Right) Histograms for the images on the left revealing that ninein is distributed towards the centriole distal end in CEP83–/– cells as compared to that in WT and CEP83–/–Rescue cells. (j) TEM of the mother centriole of WT and CEP83–/–RPE-1 cells. sDAPs in the WT and CEP83–/–cells are marked by blue and red arrowheads, respectively. (k) Cartoon model illustrating the changes in sDAP protein localization upon CEP128 depletion and the variations of sDAP structure upon CEP83 depletion. Bars = 200 nm.
Fig 5: Analysis of efficacy in mono- and bi-allelic gene knockouts with a variety of deletion lengths using the CRISPR-del pipeline. (A) Schematic representation of the CEP128 gene and the longest transcript variant annotated in genome databases. The target positions of sgRNAs and the expected lengths of large deletions are shown. Black and red arrows indicate locations of primers to detect WT and the deleted regions, respectively. (B) Summary for the efficiency of mono- and bi-allelic deletions within CEP128 gene. (C) A graph showing a relationship between the length and the frequency of chromosomal deletion from C. (D) Genomic PCR for detection of WT and the 440-kb deleted alleles of CEP128 gene using the indicated primers. (E) Sequencing result of the CEP128 deleted alleles in the 440-kb deleted clone #1. (F) Western blotting to analyze the protein expression of CEP128 in the lysate of WT cells and the 440-kb deleted clones. HSP90 was used as loading control. Images shown in D,F are representative of three experimental repeats. The experiment in E was performed once as it shows the result of DNA sequencing, and the possibility that it might change with the number of trials was not considered.
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