Figure 3.
Kif6-GFP puncta move with or without accompanying Ift27-2Halo puncta along DMTs. (A) Typical 3D-SIM images of Kif6-GFP and IFT proteins (Ift56 and Ift88). Cilia cropped from Fig. S2 B were magnified by 400% to show details. Ac-tub marked ciliary axoneme. Line scans at the indicated regions (I–IV) help to identify Kif6-GFP puncta with (arrowheads) or without (arrows) an obviously colocalized IFT particle. (B–F) Ciliary trafficking of fluorescent particles. mEPCs were infected with adenovirus to express Kif6-GFP and Ift27-2Halo as illustrated in Fig. 2 A, followed by dual-color live imaging at 14 fps using GI-SIM. GFP- or Halo-positive puncta in the first 200 frames of Video 4 were projected to show trafficking trajectories (projected images). Typical trajectories (I–II) were magnified by 300% so that the cilia could be roughly outlined (dashed green lines). Corresponding representative image sequences are presented, in which typical, clearly traceable puncta are denoted to show anterograde (arrowheads) or retrograde (arrows) movements of Ift27+ Kif6+ duo-particles (B), Kif6+ particles (C), or Ift27+ particles (C). For each cilium, the corresponding kymograph is also presented. The ratios (D) and velocities (E) of these three types of trafficking particles were quantified from 45 cilia in four mEPCs. The velocities for each particle type with the indicated numbers (E) are presented as mean ± SD. These data were statistically analyzed using an unpaired two-tailed Student’s t test. Refer to Fig. S2 D for additional examples of bidirectional movements of Kif6+ or Ift27+ puncta. The illustration (F) is provided to aid comprehension. (G) Prediction of T104 and F105 as critical residues for nucleotide-binding in the motor domain of Kif6. Superposition of Kif6 (AlphaFold prediction, blue, highlighting the side chains of residues T104 and F105) and the structure of AMPPNP-bound Kif4 (PDB 3ZFD, gray) (Cao et al., 2017) indicate that both motor domains share the same overall conformation. AMPPNP is an analogue of ATP. (H) The AlphaFold predictions of 3D structures of Kif6 and Kif6-mut. pTM: the predicted template modeling (pTM). A pTM score above 0.5 indicates that the overall predicted fold of the protein is likely similar to the actual structure. (I) Kif6 bearing T104A/F105A mutations did not localize to ependymal cilia. mEPCs were infected with adenovirus on day −1 to express GFP-tagged Kif6 and Kif6-mut, a T104A/F105A mutant, and fixed on day 8 for fluorescent microscopy. Ac-tub served as a ciliary marker. Nuclei were stained with DAPI. Cilia-containing and cell body-containing zones of the same microscopic field are presented separately to show fluorescent signals in the two regions. Arrows point to GFP-positive cells.

Kif6-GFP puncta move with or without accompanying Ift27-2Halo puncta along DMTs. (A) Typical 3D-SIM images of Kif6-GFP and IFT proteins (Ift56 and Ift88). Cilia cropped from Fig. S2 B were magnified by 400% to show details. Ac-tub marked ciliary axoneme. Line scans at the indicated regions (I–IV) help to identify Kif6-GFP puncta with (arrowheads) or without (arrows) an obviously colocalized IFT particle. (B–F) Ciliary trafficking of fluorescent particles. mEPCs were infected with adenovirus to express Kif6-GFP and Ift27-2Halo as illustrated in Fig. 2 A, followed by dual-color live imaging at 14 fps using GI-SIM. GFP- or Halo-positive puncta in the first 200 frames of Video 4 were projected to show trafficking trajectories (projected images). Typical trajectories (I–II) were magnified by 300% so that the cilia could be roughly outlined (dashed green lines). Corresponding representative image sequences are presented, in which typical, clearly traceable puncta are denoted to show anterograde (arrowheads) or retrograde (arrows) movements of Ift27+ Kif6+ duo-particles (B), Kif6+ particles (C), or Ift27+ particles (C). For each cilium, the corresponding kymograph is also presented. The ratios (D) and velocities (E) of these three types of trafficking particles were quantified from 45 cilia in four mEPCs. The velocities for each particle type with the indicated numbers (E) are presented as mean ± SD. These data were statistically analyzed using an unpaired two-tailed Student’s t test. Refer to Fig. S2 D for additional examples of bidirectional movements of Kif6+ or Ift27+ puncta. The illustration (F) is provided to aid comprehension. (G) Prediction of T104 and F105 as critical residues for nucleotide-binding in the motor domain of Kif6. Superposition of Kif6 (AlphaFold prediction, blue, highlighting the side chains of residues T104 and F105) and the structure of AMPPNP-bound Kif4 (PDB 3ZFD, gray) (Cao et al., 2017) indicate that both motor domains share the same overall conformation. AMPPNP is an analogue of ATP. (H) The AlphaFold predictions of 3D structures of Kif6 and Kif6-mut. pTM: the predicted template modeling (pTM). A pTM score above 0.5 indicates that the overall predicted fold of the protein is likely similar to the actual structure. (I) Kif6 bearing T104A/F105A mutations did not localize to ependymal cilia. mEPCs were infected with adenovirus on day −1 to express GFP-tagged Kif6 and Kif6-mut, a T104A/F105A mutant, and fixed on day 8 for fluorescent microscopy. Ac-tub served as a ciliary marker. Nuclei were stained with DAPI. Cilia-containing and cell body-containing zones of the same microscopic field are presented separately to show fluorescent signals in the two regions. Arrows point to GFP-positive cells.

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