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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 interactions between Dmoesin and the TRP channel. (A) Immunoprecipitation of D. melanogaster head extracts using α-phospho-ERM. Extracts were prepared from fly heads of dark-raised and illuminated WT flies and protein complexes were probed with αDmoesin (left) and with αINAD or αTRP (right) in a separate experiment. (bottom) Western blot analysis of the same head extracts probed with the major rhodopsin, αRh1. To detect TRP and INAD proteins in the immune complex, a threefold larger amount of head extracts were used (n = 5). (B) The experiments in A were repeated exactly, except that αTRP was used for the immunoprecipitation from head extracts of WT and trpP343 mutant, and protein complexes were probed with α-phospho-ERM. (bottom) Western blot of the same head extracts probed with αTRP. The two right lanes are Western blots from WT and trpP343 head extract probed with α-phospho-ERM (n = 4).
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fig6: Light- and phosphorylation-dependent interactions between Dmoesin and the TRP channel. (A) Immunoprecipitation of D. melanogaster head extracts using α-phospho-ERM. Extracts were prepared from fly heads of dark-raised and illuminated WT flies and protein complexes were probed with αDmoesin (left) and with αINAD or αTRP (right) in a separate experiment. (bottom) Western blot analysis of the same head extracts probed with the major rhodopsin, αRh1. To detect TRP and INAD proteins in the immune complex, a threefold larger amount of head extracts were used (n = 5). (B) The experiments in A were repeated exactly, except that αTRP was used for the immunoprecipitation from head extracts of WT and trpP343 mutant, and protein complexes were probed with α-phospho-ERM. (bottom) Western blot of the same head extracts probed with αTRP. The two right lanes are Western blots from WT and trpP343 head extract probed with α-phospho-ERM (n = 4).

Mentions: To address the influence of Dmoesin phosphorylation, extracts were immunoprecipitated with an antibody directed against an evolutionarily conserved COOH-terminal peptide of ERM (Polesello et al., 2002) that specifically recognizes the phosphorylated T559 of Dmoesin (designated hereafter as α-phospho-ERM). Protein complexes precipitated with α-phospho-ERM were then analyzed on Western blots probed with αDmoesin. Although Dmoesin was readily detected in extracts of dark-raised flies, Dmoesin staining was strongly reduced in extracts from illuminated WT flies treated under identical conditions (Fig. 6 A, left). In addition, the TRP channel and the INAD scaffold protein that binds to TRP were also detected only in head extracts of dark-raised flies immunoprecipitated with α-phospho-ERM (Fig. 6 A, right). These results are consistent with our previous findings, which demonstrated that Dmoesin only interacts with TRP in dark-raised flies. To support this notion, extracts were immunoprecipitated with αTRP and analyzed on Western blots probed with α-phospho-ERM (Fig. 6 B). Although α-phospho-ERM staining was detected in the protein complexes of dark-raised head extracts precipitated with αTRP, α-phospho-ERM staining was strongly reduced in the protein complexes of illuminated flies (Fig. 6 B, left). In control experiments, no α-phospho-ERM staining was detected in head extracts of dark-raised trpP343 mutant or in NIS-precipitated WT membranes (Fig. 6 B, middle and right).


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 interactions between Dmoesin and the TRP channel. (A) Immunoprecipitation of D. melanogaster head extracts using α-phospho-ERM. Extracts were prepared from fly heads of dark-raised and illuminated WT flies and protein complexes were probed with αDmoesin (left) and with αINAD or αTRP (right) in a separate experiment. (bottom) Western blot analysis of the same head extracts probed with the major rhodopsin, αRh1. To detect TRP and INAD proteins in the immune complex, a threefold larger amount of head extracts were used (n = 5). (B) The experiments in A were repeated exactly, except that αTRP was used for the immunoprecipitation from head extracts of WT and trpP343 mutant, and protein complexes were probed with α-phospho-ERM. (bottom) Western blot of the same head extracts probed with αTRP. The two right lanes are Western blots from WT and trpP343 head extract probed with α-phospho-ERM (n = 4).
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Related In: Results  -  Collection

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fig6: Light- and phosphorylation-dependent interactions between Dmoesin and the TRP channel. (A) Immunoprecipitation of D. melanogaster head extracts using α-phospho-ERM. Extracts were prepared from fly heads of dark-raised and illuminated WT flies and protein complexes were probed with αDmoesin (left) and with αINAD or αTRP (right) in a separate experiment. (bottom) Western blot analysis of the same head extracts probed with the major rhodopsin, αRh1. To detect TRP and INAD proteins in the immune complex, a threefold larger amount of head extracts were used (n = 5). (B) The experiments in A were repeated exactly, except that αTRP was used for the immunoprecipitation from head extracts of WT and trpP343 mutant, and protein complexes were probed with α-phospho-ERM. (bottom) Western blot of the same head extracts probed with αTRP. The two right lanes are Western blots from WT and trpP343 head extract probed with α-phospho-ERM (n = 4).
Mentions: To address the influence of Dmoesin phosphorylation, extracts were immunoprecipitated with an antibody directed against an evolutionarily conserved COOH-terminal peptide of ERM (Polesello et al., 2002) that specifically recognizes the phosphorylated T559 of Dmoesin (designated hereafter as α-phospho-ERM). Protein complexes precipitated with α-phospho-ERM were then analyzed on Western blots probed with αDmoesin. Although Dmoesin was readily detected in extracts of dark-raised flies, Dmoesin staining was strongly reduced in extracts from illuminated WT flies treated under identical conditions (Fig. 6 A, left). In addition, the TRP channel and the INAD scaffold protein that binds to TRP were also detected only in head extracts of dark-raised flies immunoprecipitated with α-phospho-ERM (Fig. 6 A, right). These results are consistent with our previous findings, which demonstrated that Dmoesin only interacts with TRP in dark-raised flies. To support this notion, extracts were immunoprecipitated with αTRP and analyzed on Western blots probed with α-phospho-ERM (Fig. 6 B). Although α-phospho-ERM staining was detected in the protein complexes of dark-raised head extracts precipitated with αTRP, α-phospho-ERM staining was strongly reduced in the protein complexes of illuminated flies (Fig. 6 B, left). In control experiments, no α-phospho-ERM staining was detected in head extracts of dark-raised trpP343 mutant or in NIS-precipitated WT membranes (Fig. 6 B, middle and right).

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