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. 2013 Feb 18;200(4):505–522. doi: 10.1083/jcb.201206013

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© 2013 Kuhns et al.

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Figure 6. — Ultrastructural analysis of MARK4 depletion phenotypes. (A) A schematic model depicting the different stages of ciliogenesis, according to Sorokin (1962, 1968). The mother centriole is characterized by the presence of distal (DA) and subdistal appendages (SDA; 0). The initial step of ciliogenesis is the docking of ciliary vesicles (CV) to the mother centriole (1). The CVs increase in size by fusion with nearby vesicles (V; 2) and become invaginated by the ciliary bud (CB), an accumulation of electron-dense material at the distal end of the mother centriole (3). MT doublets start to elongate from the basal body giving rise to the axonemal shaft (AS) and ciliary pocket (CP; 4). The elongated ciliary vesicle fuses with the plasma membrane (PM), and the cilium emerges in the extracellular environment (5). (B and C) Electron micrographs showing serial sections of unstarved RPE1 cells. Cells were seeded 24 h before fixation and cultivated in medium with 10% serum. (D and E) Electron micrographs showing serial sections of RPE1 cells treated with control siRNA. Cells were starved for 24 h to enrich early stages of ciliogenesis. (F–H) Electron micrographs showing serial sections of RPE1 cells treated with control (F) or MARK4 siRNA (G and H). Cells were serum starved for 48 h before fixation for TEM analysis. (I) After control or ODF2 depletion, RPE1 cells were serum starved for 48 h and analyzed by immunofluorescence. Percentages of ciliated cells were determined using polyglutamylated tubulin. Data are means ± SD of three independent experiments. (J) Immunostaining with anti-ODF2 revealed residual low levels of ODF2 at the centrosome in cells treated with ODF2 siRNA. Box and whisker plots show the relative ODF2 intensity at the centrosome after control and ODF2 depletion. One representative experiment out of three is shown. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. A.U., arbitrary unit; polyglu. tub., polyglutamylated tubulin. (K and L) Electron micrographs showing serial sections of RPE1 cells treated with ODF2 siRNA. Cells were serum starved for 48 h before fixation for TEM analysis. (M) Quantification of ultrastructural phenotypes in control, MARK4-, and ODF2-depleted RPE1 cells after the indicated time of serum starvation. For unstarved control cells (0 h), only cells in G1 with unduplicated centrioles were considered. The asterisk indicates that the ODF2-depleted RPE1 cells showed an additional defect in the subdistal appendage formation; subdistal appendages were either completely absent (77%) or grossly impaired (23%).