Figure 2.
Exogenous EGFP-TPRN results in F-actin abnormalities in WT IHCs and in COS-7 cells. (A and B) Similar to endogenous TPRN, transfected FL mouse EGFP-TPRN (green) is localized to stereocilia bases of P2–P4 hair cells (n = 2 cells, upward pointing arrows). (C and G–J) However, excessive expression of EGFP-TPRN transfected into hair cells leads to mislocalization of EGFP-TPRN at stereocilia tips (n = 9 cells) causing abnormal actin remodeling, over-elongation (n = 12 cells), thickening (n = 10 cells), and degeneration of stereocilia (n = 14 cells) (arrowheads in B, C, and G–J). (D–F) Thin abnormally long filamentous structures extend from tips of P4 OHC stereocilia (B, down-pointing arrow), and abnormally long, curvy filaments emanate from the tips of stereocilia of some transfected P3 vestibular hair cells (n = 5 cells) (D–F). (E) Confocal channel showing EGFP-TPRN alone. (F) Phalloidin-stained F-actin. The curled filaments contained EGFP-TPRN stained by phalloidin, indicating abnormal actin remodeling at stereocilia tips. (G–J) Mislocalization of EGFP-TPRN to stereocilia tips in P3 IHC and OHCs (n = 8 cells) (I and J, arrowheads), hair bundle degeneration (n = 14 cells) (G–J), and abnormal actin bundling in hair cell cytoplasm (n = 6 cells) (H, arrows), as well as dotted pattern of EGFP-TPRN in nuclei of hair cells (n = 7 cells) (G, I, and J, arrows) when FL TPRN is overexpressed. (K) Similar patterns of EGFP-TPRN expression showing abnormal actin bundles in the cytoplasm (arrowhead) and dotted pattern in nuclei of transfected COS-7 cells (arrows). Note, the dots of TPRN in the nucleus are smaller when TPRN expression is low and become prominent with higher expression levels of EGFP-TPRN. (L) Three COS-7 cells show different stages of EGFP-TPRN expression (1, 2 and 3): initially dots of TPRN are observed in nuclei (arrow 1). TPRN dots then become short filaments (arrow 2), which coalesce into a long and thick whorl-like bundles (arrow 3), as also shown in Video 1. (M) Overexpressed TPRN260–749 (green) localizes to nuclei (blue DAPI staining, arrows) of COS-7 cells (magenta, phalloidin). Right panel shows TPRN260–749 (green) channel only. (N) FL TPRN (green) co-localizes with F-actin in the cytoplasm of COS-7 cells when NLS of Tprn-GFP cDNA is mutated by replacing Lys(K) at positions 621, 622, and 624 to Ala(A), so the protein sequence was changed from “GSSRKKMKISF” to “GSSRAAMAISF”. (O) In P3 IHCs, EGFP-TPRN1–260 localizes along the stereocilia length and over-elongates stereocilia (n = 3 cells). (P) No nuclear staining is observed (arrow) since TPRN1–260 lacks NLS motif. (Q and R) Overexpression of a truncated EGFP-TPRN1–260 in non-sensory cells of P3 OC explant causes elongation and thickening of apical microvilli (Q), and in the internal sulcus cell area induces additional bundles of microvilli (n = 3 cells) reminiscent of stereocilia bundles of nascent vestibular hair cell (R), which appear limp and lacking the staircase architecture of WT stereocilia bundles. (S and T) Overexpression of a C-terminal fragment EGFP-TPRN730–749 does not alter WT stereocilia bundle morphology and was uniformly distributed along the entire length of P4 stereocilia, the kinocilium (arrow), and cell body (n = 5 cells) of vestibular hair cells (S) and P3 auditory hair cells (T) similar to expression of control EGFP alone in hair cells. In all panels phalloidin (magenta) was used to visualize F-actin. Scale bars are 5 μm.

Exogenous EGFP-TPRN results in F-actin abnormalities in WT IHCs and in COS-7 cells. (A and B) Similar to endogenous TPRN, transfected FL mouse EGFP-TPRN (green) is localized to stereocilia bases of P2–P4 hair cells (n = 2 cells, upward pointing arrows). (C and G–J) However, excessive expression of EGFP-TPRN transfected into hair cells leads to mislocalization of EGFP-TPRN at stereocilia tips (n = 9 cells) causing abnormal actin remodeling, over-elongation (n = 12 cells), thickening (n = 10 cells), and degeneration of stereocilia (n = 14 cells) (arrowheads in B, C, and G–J). (D–F) Thin abnormally long filamentous structures extend from tips of P4 OHC stereocilia (B, down-pointing arrow), and abnormally long, curvy filaments emanate from the tips of stereocilia of some transfected P3 vestibular hair cells (n = 5 cells) (D–F). (E) Confocal channel showing EGFP-TPRN alone. (F) Phalloidin-stained F-actin. The curled filaments contained EGFP-TPRN stained by phalloidin, indicating abnormal actin remodeling at stereocilia tips. (G–J) Mislocalization of EGFP-TPRN to stereocilia tips in P3 IHC and OHCs (n = 8 cells) (I and J, arrowheads), hair bundle degeneration (n = 14 cells) (G–J), and abnormal actin bundling in hair cell cytoplasm (n = 6 cells) (H, arrows), as well as dotted pattern of EGFP-TPRN in nuclei of hair cells (n = 7 cells) (G, I, and J, arrows) when FL TPRN is overexpressed. (K) Similar patterns of EGFP-TPRN expression showing abnormal actin bundles in the cytoplasm (arrowhead) and dotted pattern in nuclei of transfected COS-7 cells (arrows). Note, the dots of TPRN in the nucleus are smaller when TPRN expression is low and become prominent with higher expression levels of EGFP-TPRN. (L) Three COS-7 cells show different stages of EGFP-TPRN expression (1, 2 and 3): initially dots of TPRN are observed in nuclei (arrow 1). TPRN dots then become short filaments (arrow 2), which coalesce into a long and thick whorl-like bundles (arrow 3), as also shown in Video 1. (M) Overexpressed TPRN260–749 (green) localizes to nuclei (blue DAPI staining, arrows) of COS-7 cells (magenta, phalloidin). Right panel shows TPRN260–749 (green) channel only. (N) FL TPRN (green) co-localizes with F-actin in the cytoplasm of COS-7 cells when NLS of Tprn-GFP cDNA is mutated by replacing Lys(K) at positions 621, 622, and 624 to Ala(A), so the protein sequence was changed from “GSSRKKMKISF” to “GSSRAAMAISF”. (O) In P3 IHCs, EGFP-TPRN1–260 localizes along the stereocilia length and over-elongates stereocilia (n = 3 cells). (P) No nuclear staining is observed (arrow) since TPRN1–260 lacks NLS motif. (Q and R) Overexpression of a truncated EGFP-TPRN1–260 in non-sensory cells of P3 OC explant causes elongation and thickening of apical microvilli (Q), and in the internal sulcus cell area induces additional bundles of microvilli (n = 3 cells) reminiscent of stereocilia bundles of nascent vestibular hair cell (R), which appear limp and lacking the staircase architecture of WT stereocilia bundles. (S and T) Overexpression of a C-terminal fragment EGFP-TPRN730–749 does not alter WT stereocilia bundle morphology and was uniformly distributed along the entire length of P4 stereocilia, the kinocilium (arrow), and cell body (n = 5 cells) of vestibular hair cells (S) and P3 auditory hair cells (T) similar to expression of control EGFP alone in hair cells. In all panels phalloidin (magenta) was used to visualize F-actin. Scale bars are 5 μm.

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