Figure 5.
SRPS Majewski Type Fibroblasts Have a Severely Reduced Number and Structurally Abnormal Cilia
The cells were grown on coverslips or on cell-culture dishes with gas-permeable base to confluence and then serum starved for 5 days, fixed, and treated for immunofluorescence analyses or TEM.
(A, E) Immunofluorescence for acetylated α-tubulin (red), pericentrin (green), and DAPI (blue) shows normal presentation of cilia in control fibroblast cells (A), but missing or severely shortened cilia with normal appearance of the basal body and increased cytoplasmatic acetylated α-tubulin in P1 fibroblast cells (E).
(J) Only 27% of P1 cells have verifiable cilia, in contrast to 92% of control cells (t test, p value 4 × 10−10).
(B, F) Immunofluorescence for α-tubulin (red), NEK1 (green), and DAPI (blue) suggests the presence of the 63 kDa isoform NEK1 seen by immunoblotting in the PCM in control and P1 cells (white bar represents 5μm). The latter indicates presence of a physiological NEK1 isoform not affected by the nonsense mutation. Such an isoform lacking the N-terminal region would not be able to maintain cilial formation because of lack of the kinase domain.
(K) Cilia length was measured at 6.2 ± 1.2 μm on average (median 6.2 μm, 25th centile 5.3 μm, 75th centile 6.8 μm) in control cells but only 1.7 ± 0.7 μm (median 1.6 μm, 25th centile 1.2 μm, 75th centile 2.2 μm) in P1 fibroblast cells (t test, p value 1.7 × 10−69).
(C, D, G) Electron micrographs illustrating representative images from normal control fibroblast cells (C, D) and P1 fibroblast cells (F). (C) shows a primary cilia in full length, whereas in (D) the TEM image illustrates a primary cilia at the beginning of ciliogenesis (stage 2).41 In contrast, in P1 fibroblast cells (G), we found only centrioles at stage 1 of ciliogenesis with an abnormal microtubule growth (arrow) but no enveloping ciliary pocket around.
(H) Imaris three-dimensional construction of normal cilia reveals the location of NEK1 at the basal body and within the cilium, suggesting participation in the intraflagellular transport.
(I) In contrast, P1 cell cilia are shortened, with a broad base and a thin tip.