Consistent with this report, we observed that DZIP1 localized to the basal body in all cilia examined (S4A and S4B Fig)

Consistent with this report, we observed that DZIP1 localized to the basal body in all cilia examined (S4A and S4B Fig). in hearing (left panel) and touch sensitivity (right panel). Expression of CEP290 rescued these defects. For the hearing assay, 5 larvae as a group, and at least 5 groups of flies were tested. For the touch sensitivity assay, = 50. (F) Testes of WT flies and mutants. The mitochondrial protein DJ was used to label sperm cysts. Compared to those in WT flies and mutants, sperm cysts in cep290mutants were severely defective in elongation. The arrow indicates that this cysts failed to elongate. Bar, 200 m. (G) Male fertility assay in WT flies and mutants. We first rescued the severely uncoordinated phenotype of mutants by expressing CEP290 driven by males were completely infertile, while more than 65% of males were fertile. = 50. (H) EM images of testis cross sections from WT flies and mutants. There were 64 spermatids in each cyst in WT flies, whereas axonemes were almost completely lost in mutants. The arrows show defective axonemes. (I) EM images of longitudinal sections of auditory cilia in WT flies and mutants. Bars, 200 nm. Numerical data for panels D, E, and G can be found in the file S1 Data. CDS, coding sequence; CEP290, centrosomal protein 290; DBB, distal basal body; DJ, Donjuan; EM, electron microscope; PBB, proximal basal body; DZIP1 is usually orthologous to both DZIP1 and DZIP1L. (C) Schematic representation of the protein structure of DZIP1 and human DZIP1 and DZIP1L. All share highly conserved domain Mouse monoclonal to CD106(FITC) name that includes Dzip_like-N and ZnF_C2H2. (D) Table showing the sequence similarity and identity between DZIP1 and human DZIP1 and DZIP1L.(TIF) pbio.3001034.s002.tif (26M) GUID:?CAA8F01E-6EC9-4AE2-84E6-AE97822F2796 S3 Fig: CEP290CDZIP1 interaction analysis. (A) DZIP1 did not interact with CEP290-N ( aa 1C887) in the GST pull-down assay. (B) The GST pull-down assay was used to p-Hydroxymandelic acid narrow down the conversation region between CEP290 and DZIP1. DZIP1-N (aa 1C293) interacts with both CEP290 N (aa 401C650) and CEP290 N (aa 651C887), and DZIP1-C (aa 294C737) interacts with CEP290 N (aa 651C887). Uncropped immunoblots can be found in S2 Raw Image. CEP290, centrosomal protein 290; DZIP1, DAZ interacting zinc finger protein 1; GST, glutathione S-transferase.(TIF) pbio.3001034.s003.tif (31M) GUID:?F8E741F8-087D-4BAA-AE0F-00F4AEABC94B S4 Fig: The subcellular localization of DZIP1 in mutants. (A) Generation of the deletion mutant. Schematic of the gene. The gRNA target sites are represented by scissors. has a deletion of cDNA from nt 1,069 to 1 1,529, resulting in a frameshift and a premature stop codon and leading to the loss of the C-terminus. (B) Genotyping of the mutant. The sizes of the PCR products are as follows: 1,070 bp in flies and 609 bp in flies. (C) Anti-DZIP1 staining confirmed that DZIP1, at least the C-terminus of DZIP1, was completely lost in mutants. Bars, 1 m. (D) mutant flies showed defects in hearing and touch sensitivity, but expression of DZIP1-GFP driven by its own promoter rescued these defects. The retraction index is usually shown as the median and interquartile range. Numerical data can be found in the file S1 Data. The bars and error bars represent the p-Hydroxymandelic acid means and SDs, respectively. = 50. (E) was used to mark auditory cilia. Cilia completely disappeared in mutants. (F) EM images of cross sections of cilia showing that no axonemes existed in mutants. The black arrows indicate ciliary axonemes in WT flies. Bars, 200 nm. DZIP1, DAZ interacting zinc finger protein 1; EM, electron microscope; GFP, green fluorescent protein; gRNA, guide RNA.(TIF) pbio.3001034.s005.tif (31M) GUID:?904DDFCB-25A2-4F1B-9089-EAB28989EEF8 S6 Fig: Subcellular localization of Rab8 in sensory cilia and phenotype of mutants. (A) Rab8-GFP and Rab8Q67L-GFP colocalized with DZIP1 at the ciliary base in auditory cilia and olfactory cilia. Notably, Rab8Q67L-GFP was frequently observed in cilia. Bar, 1 m. (B) 3D-SIM images of the colocalization of Rab8Q67L-GFP and DZIP1 at the base of auditory cilia. Rab8 Q67L-GFP surrounded DZIP1 at TZ and expanded into cilia. Bar, 1 m. (C) DZIP1 was critical for Rab8-GFP localization at the ciliary base of olfactory cilia. Live imaging p-Hydroxymandelic acid showed that Rab8-GFP exhibited a clear 2-dot pattern at the ciliary base in WT flies, but the signal was dispersed in mutants. Interestingly, these dispersed signals completely disappeared in our immunofluorescence assay, most likely due to fixation. Bars, 5 m. (D) DZIP1 was required.