Fig 1.
The expression pattern of Dync1li1 in the cochlea of wild-type mice.
(A, B) Dync1li1 mRNA (A) and protein (B) expression in P3 mouse cochlea by RT-PCR and Western blotting, respectively. Brain tissue and the HEI-OC1 cell line were used as the positive controls. (C) Whole mount immunofluorescent staining of Dync1li1 in P3 mouse cochlea. The large square image is a single XY slice, the vertical red line shows the position of the orthogonal slice, which is shown on the right side of each panel, and the blue line on the orthogonal line shows the level of the XY slice on the left. (D) Frozen section immunofluorescent staining of Dync1li1 in P3 mouse cochlea. The white boxes and the dotted lines show enlarged images. (E) Whole mount immunofluorescent staining of Dync1li1 in P30 adult mouse cochlea. For all experiments, scale bars are shown in the figure. (F) A diagram of the IHC and OHC in the cochlea.
Fig 2.
Dync1li1 KO results in HC loss and hearing loss in adult mice.
(A) Western blotting of Dync1li1 in P30 mouse cochlea. Gapdh was used as the internal reference. (B) OHC loss (indicated by asterisks) is seen in the apical (APEX), middle (MID), and basal (BASE) turns of P21, P30, and P60 Dync1li1 KO and wild-type (WT) mice cochlea. Myo7a (red) was used as the HC marker. (C, D) Quantification of the OHCs (C) and IHCs (D) in the apical, middle, and basal turns of P21, P30, and P60 Dync1li1 KO and WT mice cochlea. (E, F) Hair bundles were observed by immunofluorescent staining of phalloidin (E) and scanning electron microscopy (F). The enlarged images in the white box in (E) is shown in the lower left corner. (G) The ABR hearing test of Dync1li1 KO mice and control mice at P21, P30, and P60. For all experiments, scale bars and N number are shown in the figure. *p < 0.05, **p < 0.01, n.s. not significant.
Fig 3.
Apoptosis analysis by TUNEL assay in the cochlea of Dync1li1 KO mice at P21 and P30.
(A) TUNEL assay of P21 and P30 Dync1li1 KO mice and WT control mice. Myo7a and DAPI were used as HC and nuclear markers, respectively. Apoptotic cells are indicated by green arrows. (B) Quantification of the mRNA expression of apoptosis related genes in the cochlea of P30 Dync1li1 KO mice and the control mice by qPCR. Red boxes indicate the genes with significant expression differences. For all experiments, scale bars and N number are shown in the figure. **p < 0.01.
Fig 4.
Dync1li1 deficiency affect the integrity of Dynein complex and Golgi apparatus.
(A) Quantification of the mRNA expression of important subunits in dynein complex (Dync1h1, Dync1i1, and Dynll1) in P60 Dync1li1 KO and the control mice by qPCR. N = 3. N refers to 3 independent qPCR experiments were performed. (B, C) Western blotting (B) and quantification of the western blotting (C) of the Dynein subunit in the cochlea of P60 Dync1li1 KO mice. Gapdh was used as the internal reference. N is indicated in the figure. (D, E) TEM of OHCs in P60 Dync1li1 KO and control mice. The Golgi apparatus is indicated by red arrows in (D) and red lines in (E). (F) Quantification of the number of lamellae per Golgi. For all experiments, scale bars are shown on the figure and N is indicated in the figure. *p < 0.05, ***p < 0.001.
Fig 5.
Dync1li1 deficiency induced the accumulation of autophagosomes vacuoles in HCs.
(A) Immunofluorescence of LC3 (green puncta, indicated by white arrows) in HCs of P60 Dync1li1−/−LC3-GFP mice and control mice. Myo7a was used as HC marker. The enlarged images are shown in the lower left corner. (B) Quantification of the number of LC3+ OHCs. N = 3. (C) Western blotting of the LC3 (LC3-I, 16 kDa; LC3-II, 14 kDa.), Rab7, and Sqstm1/p62 in the cochlea. Gapdh was used as the internal reference. (D) Schematic of the role of dynein in mediating autophagosome–lysosome fusion. Rab links autophagosomes to dynein to mediate microtubule-dependent minus-end-directed transportation towards the lysosome. For all experiments, scale bars are shown in the figure. *p < 0.05, **p < 0.01.
Fig 6.
Accumulation of autophagosomes in Dync1li1 KD HEI-OC1 cells.
(A, B) OC1 Cells were transfected with shDync1li1-GFP shRNA, and qPCR analysis (A) and Western blotting (B) were used to test the knockdown efficiency of Dync1li1. shNC-GFP was used as control shRNA. The cells were harvest after 36h transfection. (C, D) shNC-GFP and shDync1li1-GFP were cotransfected with LC3-RFP plasmids into OC1 cells for 24 h and 36 h, respectively. Red dots in (C) indicate the LC3 puncta (autophagic vacuoles), which were quantified in (D). (E) Schematic of the working principle of the RFP-GFP-LC3 plasmid. In a neutral environment (autophagosome), LC3 is expressed with both GFP and RFP fluorescencent proteins, and thus the autophagosome dots (RFP+GFP+) are yellow. In an acidic environment (autolysosome), GFP fluorescence is quenched, and thus the autolysosome dots (RFP+) are red. (F, G) siNC and siDync1li1 siRNA were cotransfected with RFP-GFP-LC3 plasmid for 36 h. Yellow arrow heads and red arrows in (F) indicate the autophagosomes (yellow dots) and the autolysosomes (red dots), respectively. The enlarged images are shown in the upper left in (F). The number of autophagosomes and autolysosomes in the Dync1li1 KD cells and the control cells were quantified in (G). (H, I) siNC and siDync1li1 siRNA were transfected for 24 h and 36 h, respectively. Western blotting (H) and quantification of Dynein subunit proteins after siRNA transfection. Quantification of protein expression levels at 36 h after siRNA transfection (I). (J) Western blotting of LC3, Sqstm1/p62 and Rab7 at 36 h after siRNA transfection. For all experiments, scale bars are shown in the figure. *p < 0.05, **p < 0.01, n.s. not significant.
Fig 7.
Working model of dynein-dependent autolysosome clearance in cochlear HCs.
(A) Under normal conditions, phagophores (derived from the ER, Golgi, mitochondria, etc.) expand and form late autophagosomes, which bind to the dynein complex through Rab7. Late autophagosomes, with both LC3 and Rab7 expressed on their membranes, are transported by dynein along microtubules to lysosomes to form autolysosomes and to be digested. This autophagic flux maintains homeostasis in HCs. (B) When Dync1li1 is knocked out in cochlear HCs, the fusion of late autophagosomes with lysosomes is impaired by the unstable dynein complex. Therefore, large numbers of late autophagosomes are accumulated in the HCs, which disrupts normal autophagic flux and leads to HC apoptosis.