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Staurosporine-induced collapse of cochlear hair bundles.

Goodyear RJ, Ratnayaka HS, Warchol ME, Richardson GP - J. Comp. Neurol. (2014)

Bottom Line: Staurosporine exposure results in the fusion of the hair bundle's stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle's rootlets from the actin cores of the stereocilia.It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine.Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Sussex Neuroscience and School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK.

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Activated caspase-3 labeling and TUNEL in staurosporine-treated cultures. Phalloidin (red) and anti-activated caspase-3 (green) staining following treatment with control medium (A) or medium containing 10 nM staurosporine (C) or 0.5 mM neomycin (E) for 18 hours. Images A–C are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. Caspase-3 positive hair cells (arrowheads in E) are only observed in the neomycin condition. Corresponding DAPI-stained nuclei are shown in (B,D,F). Although hair-cell nuclei of staurosporine-treated cultures have a normal appearance, those exposed to neomycin are often shrunken and condensed (arrows in F). Phalloidin staining (red) and TUNEL (green) following treatment with control medium (G,H) or medium containing 10 nM staurosporine (I,J) or 0.5 mM neomycin (K,L) for 48 hours. Images are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. TUNEL-positive hair-cell nuclei are abundant following neomycin but not staurosporine. A magenta-green version of this figure is available as Supporting Figure 1. Scale bar = 20 μm.
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fig07: Activated caspase-3 labeling and TUNEL in staurosporine-treated cultures. Phalloidin (red) and anti-activated caspase-3 (green) staining following treatment with control medium (A) or medium containing 10 nM staurosporine (C) or 0.5 mM neomycin (E) for 18 hours. Images A–C are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. Caspase-3 positive hair cells (arrowheads in E) are only observed in the neomycin condition. Corresponding DAPI-stained nuclei are shown in (B,D,F). Although hair-cell nuclei of staurosporine-treated cultures have a normal appearance, those exposed to neomycin are often shrunken and condensed (arrows in F). Phalloidin staining (red) and TUNEL (green) following treatment with control medium (G,H) or medium containing 10 nM staurosporine (I,J) or 0.5 mM neomycin (K,L) for 48 hours. Images are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. TUNEL-positive hair-cell nuclei are abundant following neomycin but not staurosporine. A magenta-green version of this figure is available as Supporting Figure 1. Scale bar = 20 μm.

Mentions: Cochlear cultures were treated for 48 hours with 10 nM staurosporine and then fixed and sectioned in order to see if the drug caused the loss of hair cells. Inner and outer hair cells and their hair bundles were readily visible in Toluidine-blue stained 1-μm thick sections from the apical and basal coils of control cultures (Fig. 6A,B). Although hair bundles were not seen in the staurosporine-exposed cultures, hair-cell bodies remained within the epithelium and had nuclei of normal appearance (Fig. 6C,D) in both the apical and the basal coils. One of the most rapid known effects of ototoxins like the aminoglycoside antibiotics is to cause the externalization of phosphatidylserine at the hair cell’s apical pole (Goodyear et al., 2008). Cultures were therefore treated with 10 nM staurosporine for 8 hours at 37°C and then exposed to Alexa-Fluor 488-conjugated annexin V to determine if phosphatidylserine was present on the external surface of the hair cells. Control cultures did not label with annexin V (not shown), nor did cultures that were exposed to 10 nM staurosporine (Fig. 6E,F). By contrast, exposure to 2 mM neomycin, a known ototoxin that selectively kills hair cell in cochlear cultures, elicited PS externalization in both the apical and basal coils of staurosporine-treated cultures within 10 minutes (Fig. 6I,J). To confirm hair cells were not undergoing apoptosis in response to staurosporine treatment, cultures were treated with 10 nM staurosporine or 0.5 mM neomycin for 18 or 48 hours, fixed, and either double-labeled with the nuclear dye DAPI and antibodies to activated caspase-3, or subjected to TUNEL (Fig. 7). In the cultures treated with neomycin, a proportion of the hair cells were stained by anti-activated caspase-3 (Fig. 7E) and the hair-cell nuclei were of a condensed morphology (Fig. 7F) and positive for TUNEL (Fig. 7K,L). In the staurosporine-treated cultures, activated caspase-3 staining was not observed in hair cells (Fig. 7C), and the hair-cell nuclei were of normal morphology (i.e., not fragmented or condensed) (Fig. 7D) and were not TUNEL-positive (Fig. 7I,J).


Staurosporine-induced collapse of cochlear hair bundles.

Goodyear RJ, Ratnayaka HS, Warchol ME, Richardson GP - J. Comp. Neurol. (2014)

Activated caspase-3 labeling and TUNEL in staurosporine-treated cultures. Phalloidin (red) and anti-activated caspase-3 (green) staining following treatment with control medium (A) or medium containing 10 nM staurosporine (C) or 0.5 mM neomycin (E) for 18 hours. Images A–C are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. Caspase-3 positive hair cells (arrowheads in E) are only observed in the neomycin condition. Corresponding DAPI-stained nuclei are shown in (B,D,F). Although hair-cell nuclei of staurosporine-treated cultures have a normal appearance, those exposed to neomycin are often shrunken and condensed (arrows in F). Phalloidin staining (red) and TUNEL (green) following treatment with control medium (G,H) or medium containing 10 nM staurosporine (I,J) or 0.5 mM neomycin (K,L) for 48 hours. Images are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. TUNEL-positive hair-cell nuclei are abundant following neomycin but not staurosporine. A magenta-green version of this figure is available as Supporting Figure 1. Scale bar = 20 μm.
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fig07: Activated caspase-3 labeling and TUNEL in staurosporine-treated cultures. Phalloidin (red) and anti-activated caspase-3 (green) staining following treatment with control medium (A) or medium containing 10 nM staurosporine (C) or 0.5 mM neomycin (E) for 18 hours. Images A–C are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. Caspase-3 positive hair cells (arrowheads in E) are only observed in the neomycin condition. Corresponding DAPI-stained nuclei are shown in (B,D,F). Although hair-cell nuclei of staurosporine-treated cultures have a normal appearance, those exposed to neomycin are often shrunken and condensed (arrows in F). Phalloidin staining (red) and TUNEL (green) following treatment with control medium (G,H) or medium containing 10 nM staurosporine (I,J) or 0.5 mM neomycin (K,L) for 48 hours. Images are confocal Z-compressions encompassing a depth of 15 μm below the apical surface and are taken from the mid-apical region. TUNEL-positive hair-cell nuclei are abundant following neomycin but not staurosporine. A magenta-green version of this figure is available as Supporting Figure 1. Scale bar = 20 μm.
Mentions: Cochlear cultures were treated for 48 hours with 10 nM staurosporine and then fixed and sectioned in order to see if the drug caused the loss of hair cells. Inner and outer hair cells and their hair bundles were readily visible in Toluidine-blue stained 1-μm thick sections from the apical and basal coils of control cultures (Fig. 6A,B). Although hair bundles were not seen in the staurosporine-exposed cultures, hair-cell bodies remained within the epithelium and had nuclei of normal appearance (Fig. 6C,D) in both the apical and the basal coils. One of the most rapid known effects of ototoxins like the aminoglycoside antibiotics is to cause the externalization of phosphatidylserine at the hair cell’s apical pole (Goodyear et al., 2008). Cultures were therefore treated with 10 nM staurosporine for 8 hours at 37°C and then exposed to Alexa-Fluor 488-conjugated annexin V to determine if phosphatidylserine was present on the external surface of the hair cells. Control cultures did not label with annexin V (not shown), nor did cultures that were exposed to 10 nM staurosporine (Fig. 6E,F). By contrast, exposure to 2 mM neomycin, a known ototoxin that selectively kills hair cell in cochlear cultures, elicited PS externalization in both the apical and basal coils of staurosporine-treated cultures within 10 minutes (Fig. 6I,J). To confirm hair cells were not undergoing apoptosis in response to staurosporine treatment, cultures were treated with 10 nM staurosporine or 0.5 mM neomycin for 18 or 48 hours, fixed, and either double-labeled with the nuclear dye DAPI and antibodies to activated caspase-3, or subjected to TUNEL (Fig. 7). In the cultures treated with neomycin, a proportion of the hair cells were stained by anti-activated caspase-3 (Fig. 7E) and the hair-cell nuclei were of a condensed morphology (Fig. 7F) and positive for TUNEL (Fig. 7K,L). In the staurosporine-treated cultures, activated caspase-3 staining was not observed in hair cells (Fig. 7C), and the hair-cell nuclei were of normal morphology (i.e., not fragmented or condensed) (Fig. 7D) and were not TUNEL-positive (Fig. 7I,J).

Bottom Line: Staurosporine exposure results in the fusion of the hair bundle's stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle's rootlets from the actin cores of the stereocilia.It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine.Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Sussex Neuroscience and School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK.

Show MeSH
Related in: MedlinePlus