Fig 2. Ciliary pattern of paramecia depleted for TZ proteins.

(A) Ciliary pattern of paramecia treated with control RNAi or TZ^RNAi (CEP290, RPGRIP1L, and NPHP4). Cells were immunostained by the monoclonal anti-mono-glycylated tubulin TAP952 (magenta, cilia tip labelling) and the polyclonal anti-poly-glutamylated tubulin (polyE) antibodies (green, decorating BB and cilia). These TZ-protein–depleted paramecia display a ciliary pattern similar to that of control paramecia. (B) Control, TMEM107-depleted, and TMEM216-depleted paramecia stained for cilia using TAP952 (magenta) and the poly-E tubulin (green). Control paramecia show the usual ciliary pattern with long cilia (10 μm) and a few short growing cilia indicated by asterisks. A large increase in short (about 4 μm) or tiny cilia is observed in TMEM107- and TMEM216-depleted cells. Bar = 10 μm. (C) Bar plot showing the mean percentage of short cilia in Control (n = 60 cells, 3 independent replicates), TMEM107 (n = 50 cells, 3 independent replicates), and TMEM216-depleted (n = 42 cells, 3 independent replicates) cells. Error bars show the SEM. Statistical significance was assessed by an unpaired t test, two-sided p < 0.0001****. Source data can be found in S2 Data. (D–G) Ciliary shedding in TMEM107- or TMEM216-depleted cells. (D) Quantification of free cilia in culture medium: dot plot showing the number of free cilia found in the culture medium (about 10 microscope fields were analyzed per experiment; see Materials and Methods). Two independent replicates. Cilia were labelled using ID5 and poly-E antibodies. Error bars represent the standard deviation. Statistical significance was assessed by an unpaired t test. ***p = 0.0004, ****p < 0.0001. Source data can be found in S2 Data. (E) Effect of the increase of ciliary beating forces on ciliary shedding quantification of the mean number of bald cells (<25% cilia per cell) after 1 h in 10% PEG for control (n = 410 cells, 8 independent replicates), TMEM107^RNAi (n = 316, 5 independent replicates), TMEM216^RNAi (n = 303 cells, 5 independent replicates), CEP290^RNAi (n = 291, 4 independent replicates), RPGRIP1L^RNAi (n = 204, 3 independent replicates), and NPHP4^RNAi (n = 196, 3 independent replicates). Errors bars represent the standard deviation. Statistical significance was assessed by unpaired two-sided χ² test, ****p < 0.0001. Confidence interval 95% source data can be found in S2 Data. (F) Effect of inhibition of ciliary beating on ciliary shedding; quantification of the mean percentage of short cilia in Control (n = 60 cells, 2 independent replicates), TMEM216^RNAi (n = 42 cells, 2 independent replicates), DNAH2^RNAi (n = 15 cells, 2 independent replicates), and TMEM216-DNAH2^RNAi (n = 16 cells, 2 independent replicates) paramecia. DNAH2^RNAi cells present a percentage of short cilia slightly higher than the controls. Impairing ciliary beating of TMEM216^RNAi cells by DNAH2 decreases this percentage. Statistical significance was assessed by unpaired two-sided t test. p-Values: *p = 0.0306, ****p < 0.0001. Source data can be found in S2 Data. (G) EM images of ciliary defects induced by TMEM107^RNAi and TMEM216^RNAi. Control^RNAi basal bodies showing a two-BB unit, with one unciliated and one ciliated BB. The length of the TZ is indicated by a red arrow. Cilia are either severed at the level of the axosomal plate as shown in TMEM107^RNAi or have been severed and are in a regrowth process as in TMEM216^RNAi. Note that the length of the TZ corresponds to the length of TZ of ciliated BB. BB, basal body; CEP290, centrosomal protein of 290 kDa; DNAH2, dynein axonemal heavy chain 2; NPHP4, Nephronophtysis 4; ns, nonsignificant; PEG, Polyethylene glycol; polyE, anti-poly-glutamylated tubulin; RNAi, RNA interference; RPGRIP1L, Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein; TMEM216, Transmembrane protein 216; TZ, transition zone.