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Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors.

Chorna-Ornan I, Tzarfaty V, Ankri-Eliahoo G, Joel-Almagor T, Meyer NE, Huber A, Payre F, Minke B - J. Cell Biol. (2005)

Bottom Line: Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin.The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration.Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

ABSTRACT
Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

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Light- and phosphorylation-dependent movement of Dmoesin from the rhabdomere to the cell body. (A–F) Intracellular distribution of Dmoesin-GFP protein fusions, as observed in confocal micrograph cross sections of living D. melanogaster retinae of the transgenic lines: UAS Dmoesin-WT-GFP (A and B), UAS Dmoesin-T559A -GFP (C and D), and UAS Dmoesin-T559D-GFP (E and F). Green indicates GFP fluorescence. The strong autofluorescence of the rhabdomeres (red) allows localizing Dmoesin distribution with respect to photoreceptor compartments. Dark-raised flies were kept in obscurity (A, C, and E) or submitted to blue light illumination (B, D, and F). In flies expressing Dmoesin-WT-GFP (A and B), the fluorescent protein moves from the rhabdomere and cortical actin regions to the cell body of photoreceptors in response to light. Almost all Dmoesin-T559A-GFP (C and D) proteins accumulate outside of the rhabdomeres, independently of the illumination regime, and a significant fraction of Dmoesin-T559D-GFP (E and F) was observed in the rhabdomeres and cortical actin regions regardless of illumination regime. (G) The histogram plots the ratio of the number of green (GFP) to red (autofluorescence) pixels in the rhabdomere and cortical actin regions, as defined by the area that displays autofluorescence. P < 0.01; n = 20 for each fly strain. The error bars are SEM.
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fig7: Light- and phosphorylation-dependent movement of Dmoesin from the rhabdomere to the cell body. (A–F) Intracellular distribution of Dmoesin-GFP protein fusions, as observed in confocal micrograph cross sections of living D. melanogaster retinae of the transgenic lines: UAS Dmoesin-WT-GFP (A and B), UAS Dmoesin-T559A -GFP (C and D), and UAS Dmoesin-T559D-GFP (E and F). Green indicates GFP fluorescence. The strong autofluorescence of the rhabdomeres (red) allows localizing Dmoesin distribution with respect to photoreceptor compartments. Dark-raised flies were kept in obscurity (A, C, and E) or submitted to blue light illumination (B, D, and F). In flies expressing Dmoesin-WT-GFP (A and B), the fluorescent protein moves from the rhabdomere and cortical actin regions to the cell body of photoreceptors in response to light. Almost all Dmoesin-T559A-GFP (C and D) proteins accumulate outside of the rhabdomeres, independently of the illumination regime, and a significant fraction of Dmoesin-T559D-GFP (E and F) was observed in the rhabdomeres and cortical actin regions regardless of illumination regime. (G) The histogram plots the ratio of the number of green (GFP) to red (autofluorescence) pixels in the rhabdomere and cortical actin regions, as defined by the area that displays autofluorescence. P < 0.01; n = 20 for each fly strain. The error bars are SEM.

Mentions: To directly visualize intracellular movements of Dmoesin in photoreceptors upon illumination, we made use of transgenic lines that allow the expression of functional Dmoesin fused to GFP (Polesello et al., 2002). Dmoesin-GFP fusions were expressed under the control of the Rh1-Gal4 driver, which is specific to mature peripheral R1–6 photoreceptors. Upon application of long wavelength excitation light that elicits a strong autofluorescence of the rhabdomeres (without excitation of the GFP), locations and dimensions of the rhabdomeres in each ommatidium are readily observed in the living retina. The typical structure of the ommatidium is visible as seven red circles, representing the R1–6 peripheral rhabdomeres and the smaller R7 rhabdomere, at the center. Live retinae were dissected under dim red light, and the subcellular localization of Dmoesin-GFP was examined with confocal microscopy. Fig. 7 A shows a representative image of ommatidia from dark-raised flies expressing Dmoesin-WT-GFP, which localizes to the rhabdomeres and cortical actin of R1–6 photoreceptors. Because of variability in the expression levels of Dmoesin-GFP in the various ommatidia, some photoreceptor cells did not express Dmoesin (Fig. 7 A). In the photoreceptor cells that did express Dmoesin-GFP, the intense fluorescence of the GFP masked the weaker red autofluorescence and the merged images appeared green. When the level of Dmoesin-GFP in the rhabdomeres was reduced (Fig. 7 F) a yellow color appeared in the merged images. The lack of green fluorescence from the central R7 rhabdomere (which does not express Dmoesin-GFP) provides an internal negative control. After illumination a marked redistribution of Dmoesin-GFP is observed, with the green fluorescence moving from the rhabdomeres to the cell body region (Fig. 7 B). Although there are some differences in the detailed localization and movement of the native Dmoesin (Fig. 2) and Dmoesin-WT-GFP, the results of Fig. 7 confirm our interpretation and clearly indicate that illumination induces a redistribution of the Dmoesin protein from the rhabdomere to the cytoplasm of the cell body.


Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors.

Chorna-Ornan I, Tzarfaty V, Ankri-Eliahoo G, Joel-Almagor T, Meyer NE, Huber A, Payre F, Minke B - J. Cell Biol. (2005)

Light- and phosphorylation-dependent movement of Dmoesin from the rhabdomere to the cell body. (A–F) Intracellular distribution of Dmoesin-GFP protein fusions, as observed in confocal micrograph cross sections of living D. melanogaster retinae of the transgenic lines: UAS Dmoesin-WT-GFP (A and B), UAS Dmoesin-T559A -GFP (C and D), and UAS Dmoesin-T559D-GFP (E and F). Green indicates GFP fluorescence. The strong autofluorescence of the rhabdomeres (red) allows localizing Dmoesin distribution with respect to photoreceptor compartments. Dark-raised flies were kept in obscurity (A, C, and E) or submitted to blue light illumination (B, D, and F). In flies expressing Dmoesin-WT-GFP (A and B), the fluorescent protein moves from the rhabdomere and cortical actin regions to the cell body of photoreceptors in response to light. Almost all Dmoesin-T559A-GFP (C and D) proteins accumulate outside of the rhabdomeres, independently of the illumination regime, and a significant fraction of Dmoesin-T559D-GFP (E and F) was observed in the rhabdomeres and cortical actin regions regardless of illumination regime. (G) The histogram plots the ratio of the number of green (GFP) to red (autofluorescence) pixels in the rhabdomere and cortical actin regions, as defined by the area that displays autofluorescence. P < 0.01; n = 20 for each fly strain. The error bars are SEM.
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Related In: Results  -  Collection

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fig7: Light- and phosphorylation-dependent movement of Dmoesin from the rhabdomere to the cell body. (A–F) Intracellular distribution of Dmoesin-GFP protein fusions, as observed in confocal micrograph cross sections of living D. melanogaster retinae of the transgenic lines: UAS Dmoesin-WT-GFP (A and B), UAS Dmoesin-T559A -GFP (C and D), and UAS Dmoesin-T559D-GFP (E and F). Green indicates GFP fluorescence. The strong autofluorescence of the rhabdomeres (red) allows localizing Dmoesin distribution with respect to photoreceptor compartments. Dark-raised flies were kept in obscurity (A, C, and E) or submitted to blue light illumination (B, D, and F). In flies expressing Dmoesin-WT-GFP (A and B), the fluorescent protein moves from the rhabdomere and cortical actin regions to the cell body of photoreceptors in response to light. Almost all Dmoesin-T559A-GFP (C and D) proteins accumulate outside of the rhabdomeres, independently of the illumination regime, and a significant fraction of Dmoesin-T559D-GFP (E and F) was observed in the rhabdomeres and cortical actin regions regardless of illumination regime. (G) The histogram plots the ratio of the number of green (GFP) to red (autofluorescence) pixels in the rhabdomere and cortical actin regions, as defined by the area that displays autofluorescence. P < 0.01; n = 20 for each fly strain. The error bars are SEM.
Mentions: To directly visualize intracellular movements of Dmoesin in photoreceptors upon illumination, we made use of transgenic lines that allow the expression of functional Dmoesin fused to GFP (Polesello et al., 2002). Dmoesin-GFP fusions were expressed under the control of the Rh1-Gal4 driver, which is specific to mature peripheral R1–6 photoreceptors. Upon application of long wavelength excitation light that elicits a strong autofluorescence of the rhabdomeres (without excitation of the GFP), locations and dimensions of the rhabdomeres in each ommatidium are readily observed in the living retina. The typical structure of the ommatidium is visible as seven red circles, representing the R1–6 peripheral rhabdomeres and the smaller R7 rhabdomere, at the center. Live retinae were dissected under dim red light, and the subcellular localization of Dmoesin-GFP was examined with confocal microscopy. Fig. 7 A shows a representative image of ommatidia from dark-raised flies expressing Dmoesin-WT-GFP, which localizes to the rhabdomeres and cortical actin of R1–6 photoreceptors. Because of variability in the expression levels of Dmoesin-GFP in the various ommatidia, some photoreceptor cells did not express Dmoesin (Fig. 7 A). In the photoreceptor cells that did express Dmoesin-GFP, the intense fluorescence of the GFP masked the weaker red autofluorescence and the merged images appeared green. When the level of Dmoesin-GFP in the rhabdomeres was reduced (Fig. 7 F) a yellow color appeared in the merged images. The lack of green fluorescence from the central R7 rhabdomere (which does not express Dmoesin-GFP) provides an internal negative control. After illumination a marked redistribution of Dmoesin-GFP is observed, with the green fluorescence moving from the rhabdomeres to the cell body region (Fig. 7 B). Although there are some differences in the detailed localization and movement of the native Dmoesin (Fig. 2) and Dmoesin-WT-GFP, the results of Fig. 7 confirm our interpretation and clearly indicate that illumination induces a redistribution of the Dmoesin protein from the rhabdomere to the cytoplasm of the cell body.

Bottom Line: Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin.The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration.Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

ABSTRACT
Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

Show MeSH
Related in: MedlinePlus