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Calaxin calcium-binding activity is dispensable for KV ciliary movement. (A) Classification of KV cilia. KV cilia of WT embryos, calaxin−/− embryos, and calaxin−/− embryos injected with calaxin WT (WT mRNA) or E130A mRNA (E130A mRNA) were recorded by a bright-field microscope (AF6000B; Leica) equipped with a high-speed camera (HAS-L1; DITECT) at 1,000 fps at the 8–10 somite stage. KV ciliary motility was classified into normal conically rotating movement and abnormal movement, including irregular movement and quiescence. Both WT and E130A mRNA restored the irregular movement of calaxin−/− KV cilia. All data were collected from n = 30 cilia from 6 embryos (WT), 30 cilia from 5 embryos (calaxin−/−), 17 cilia from 3 embryos (WT mRNA), and 33 cilia from 4 embryos (E130A mRNA). (B) Kymographs of KV cilia. Normal regularly rotating cilia from WT, calaxin−/−, and calaxin−/− injected with WT or E130A mRNA embryos and an irregularly moving cilium from calaxin−/− embryo are shown. (C) Beat frequencies of KV cilia. Normal conically rotating cilia shown in (A) were subjected to the analysis. calaxin−/− cilia showed a slower beating frequency. Both calaxin WT and E130A mRNA injected into calaxin−/− embryo restored the motility to the same extent. All data are shown with a mean (bar graphs) + SE (error bars). Scale bar, 100 ms. n = 29 cilia from 6 embryos (WT), seven cilia from 3 embryos (calaxin−/−), 16 cilia from 3 embryos (calaxin−/− + WT mRNA), and 30 cilia from 4 embryos (calaxin−/− + E130A mRNA). (D) Laterality of the calaxin−/− and Tg E130A embryos. calaxin−/− and Tg E130A adults were crossed to obtain embryos. The direction of the heart looping was observed at the 30–33 hpf stage to determine the laterality, followed by genotyping. calaxin−/− embryos exhibited laterality randomization as observed before, whereas Tg E130A showed normal laterality. n = 12 embryos for each genotype.
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