XB-IMG-178623
Xenbase Image ID: 178623
![]() |
Figure 3. The Absence of GAS2L2 Causes a Defective Ciliary Beat
(A) Nasal epithelial cells from individual PCD-1367 (family UNC-362, 2-VI) and a control were dissociated into single cells. The HNE cells were conditionally reprogramed and expanded by co-culture with irradiated 3T3J2 fibroblasts for one to two passages in the presence of Y-27632, a selective inhibitor of Rho-associated protein kinase (ROCK). After expansion, we cultured the HNE cells at the ALI to conduct multiple assays.
(B and C) The absence of GAS2L2 in PCD-1367 was confirmed by immunoblot and immunofluorescence.
(D) The CBF in GAS2L2-deficient cells was hyperkinetic in comparison to controls (n = 6; data are represented as the mean + SD; Students t test, p = 0.0002).
(E) High-resolution videos of ciliated cells were observed and showed a normal waveform pattern of ciliary beat in control and GAS2L2-deficient cells. The images represent a time point of the cilia seen in profile at end recovery (left panels) and end effective positions (right panels). For each position, a panel showing the manual tracing of the cilia (highlighted in red) is shown. (n = 4; the scale bar represents 4 m).
(F) Direction of ciliary beat. Four to five cilia were tracked manually in three individual cells. The black dot represents the position at the end recovery phase. The direction of the ciliary beat was plotted on an X-Y graph (the scale bar represents 2 m).
(G) Representative electron micrograph showing the analysis of alignment of the basal body and basal foot in control cells (n = 14) and GAS2L2-deficient cells (n = 22). The arrows in each micrograph are pointing in the direction of the basal foot tip (the scale bar represents 2 m).
(H) The length of the vector (0 < R < 1) for each cell is represented in the graph. The average length of the vector was significantly shorter in GAS2L2-deficient nasal ciliated cells than in control cells (data are expressed as the mean + SD; Students t test, p = 0.0004). Image published in: Bustamante-Marin XM et al. (2019) Copyright © 2019. The Author(s) Larger Image Printer Friendly View |