Figure 1.
Molecular characterizations and subcellular localizations of mammalian Kif6 and Kif9. (A) Schematics of full-length murine Kif6 and Kif9. The motor and coiled-coil domains were identified using the Conserved Domain Search Service of NCBI and SMART (Simple Modular Architecture Research Tool) (http://smart.embl.de). (B) Conservation of Kif6 and Kif9 across species with motile cilia and/or flagella in the evolution. The phylogenetic tree and taxonomic groups were based on literature (Cetkovic et al., 2018; Mukherjee and Brocchieri, 2013). (C) mRNA expression profiles of Kif6 and Kif9 during multiciliation. The gene expression profiles were obtained from our previous cDNA microarray analysis of mTECs cultured at the air–liquid interface (ALI) for the indicated days (Xu et al., 2015). Expression patterns of genes crucial for cilia formation (Ift88 and Ift122) and BB amplification (Cep152) are listed for comparison (Klena and Pigino, 2022; Zhao et al., 2013). (D) Expression patterns of Kif6 and Kif9 during multiciliation of mTECs. Multicilia formation is indicated by increased levels of Ift81, Bbs3, and acetylated tubulin (Ac-tub) (Klena and Pigino, 2022). Gapdh served as loading control. (E and F) Expression patterns of Kif6 and Kif9 in mouse tissues (E) and cultured cells (F). Tissues from a 2-month-old male mouse were dissected and lysed. mEPCs were harvested on day 8 post serum starvation. RPE1, NIH3T3, and IMCD3 cells were serum-starved for 48 h to induce primary (immotile) cilia formation. Gapdh or β-actin served as loading control. (G) Experimental scheme for H, J, and K. Precursors of mEPCs were isolated from P0 mouse brain and serum-starved on day 0 to induce differentiation into multiciliated cells. To express GFP-tagged proteins, the cells were infected with adenovirus on day 1. The cells were fixed on day 8 for immunofluorescence (IF) staining and imaged by confocal microscopy (H and J) or 3D-SIM (K). (H) Localizations of endogenous Kif6 and Kif9 in motile cilia. Ac-tub labeled ciliary axonemes. Arrowheads indicate typical cilia. (I) Kif6 and Kif9 were not detected in primary cilia. IMCD3 cells were serum-starved to induce primary ciliogenesis and fixed at day 2 after serum starvation for IF, followed by confocal microscopy. The nucleus was visualized by DAPI, a DNA-specific dye. Cilia pointed by arrowheads were zoomed in 2 × to show details. (J) Localizations of exogenous Kif6-GFP and Kif9-GFP in multicilia. (K) Typical 3D-SIM images of endogenous Kif9 and Kif6. Ac-tub and Hydin served as axoneme and CP markers, respectively. Cilia pointed by arrowheads were magnified by 200% to show details. Corresponding line scans at the indicated regions of the magnified insets showed colocalizations of Kif9 with Hydin and of Kif6 with Ac-tub. Illustrations are provided to aid comprehension. Source data are available for this figure: SourceData F1.

Molecular characterizations and subcellular localizations of mammalian Kif6 and Kif9. (A) Schematics of full-length murine Kif6 and Kif9. The motor and coiled-coil domains were identified using the Conserved Domain Search Service of NCBI and SMART (Simple Modular Architecture Research Tool) (http://smart.embl.de). (B) Conservation of Kif6 and Kif9 across species with motile cilia and/or flagella in the evolution. The phylogenetic tree and taxonomic groups were based on literature (Cetkovic et al., 2018; Mukherjee and Brocchieri, 2013). (C) mRNA expression profiles of Kif6 and Kif9 during multiciliation. The gene expression profiles were obtained from our previous cDNA microarray analysis of mTECs cultured at the air–liquid interface (ALI) for the indicated days (Xu et al., 2015). Expression patterns of genes crucial for cilia formation (Ift88 and Ift122) and BB amplification (Cep152) are listed for comparison (Klena and Pigino, 2022; Zhao et al., 2013). (D) Expression patterns of Kif6 and Kif9 during multiciliation of mTECs. Multicilia formation is indicated by increased levels of Ift81, Bbs3, and acetylated tubulin (Ac-tub) (Klena and Pigino, 2022). Gapdh served as loading control. (E and F) Expression patterns of Kif6 and Kif9 in mouse tissues (E) and cultured cells (F). Tissues from a 2-month-old male mouse were dissected and lysed. mEPCs were harvested on day 8 post serum starvation. RPE1, NIH3T3, and IMCD3 cells were serum-starved for 48 h to induce primary (immotile) cilia formation. Gapdh or β-actin served as loading control. (G) Experimental scheme for H, J, and K. Precursors of mEPCs were isolated from P0 mouse brain and serum-starved on day 0 to induce differentiation into multiciliated cells. To express GFP-tagged proteins, the cells were infected with adenovirus on day 1. The cells were fixed on day 8 for immunofluorescence (IF) staining and imaged by confocal microscopy (H and J) or 3D-SIM (K). (H) Localizations of endogenous Kif6 and Kif9 in motile cilia. Ac-tub labeled ciliary axonemes. Arrowheads indicate typical cilia. (I) Kif6 and Kif9 were not detected in primary cilia. IMCD3 cells were serum-starved to induce primary ciliogenesis and fixed at day 2 after serum starvation for IF, followed by confocal microscopy. The nucleus was visualized by DAPI, a DNA-specific dye. Cilia pointed by arrowheads were zoomed in 2 × to show details. (J) Localizations of exogenous Kif6-GFP and Kif9-GFP in multicilia. (K) Typical 3D-SIM images of endogenous Kif9 and Kif6. Ac-tub and Hydin served as axoneme and CP markers, respectively. Cilia pointed by arrowheads were magnified by 200% to show details. Corresponding line scans at the indicated regions of the magnified insets showed colocalizations of Kif9 with Hydin and of Kif6 with Ac-tub. Illustrations are provided to aid comprehension. Source data are available for this figure: SourceData F1.

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