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The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

Cerny AC, Altendorfer A, Schopf K, Baltner K, Maag N, Sehn E, Wolfrum U, Huber A - PLoS Genet. (2015)

Bottom Line: TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid.In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant.In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosensorics, Institute of Physiology, University of Hohenheim, Stuttgart, Germany.

ABSTRACT
Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

No MeSH data available.


Related in: MedlinePlus

Photoreceptors of the ttd14P75L mutant undergo light-enhanced, but Rh1-independent degeneration.(A) Transmission electron microscopy of tangential sections through eyes of wild type flies and homozygous mutant eye clones of ttd14P75L mutant flies. Flies were assayed after eclosion (1 d), kept in the dark for 7 days followed by 16 hours orange light (7 d dark 16 h light), kept in the dark for 21 d, or subjected to a 12 hours light / 12 hours dark cycle for 21 d (21 d dark light). Flies were either raised on normal food (upper panels) or on vitamin A-deprived diet (low vitA; lower panels). 1–7 denotes rhabdomeres of photoreceptor cells R1 to R7. N, nucleus. Scale bar: 2.5 μm. Wild type flies display a normal morphology of the rhabdomeres at all conditions analyzed, except that vitamin A-deprived flies have smaller rhabdomeres due to the reduced amount of rhodopsin. Degeneration of inner and outer photoreceptor cells is obvious in ttd14P75L mutants exposed to a light/dark cycle for 21 days. Interestingly, R7 cells show signs of cell death while R1-6 cells are not affected (arrowhead) in ttd14P75L mutants kept in the dark for 21 days. (B) Time course of photoreceptor degeneration in the ttd14P75L mutant raised on normal food or on vitamin A-deprived diet (low vitA). Fluorescent water immersion images of wild type flies and ttd14P75L mutant eye clones, which express TRP-eGFP as a fluorescent marker for rhabdomeres in photoreceptor cells R1-6, were scored for the presence of rhabdomeres (see Material and Methods). 100% represents fully intact rhabdomeres. 3 ommatidia from five flies each were analyzed. Error bars denote SEM. Flies were kept in a 12 hours light / 12 hours dark cycle for the indicated number of days. Examples of original images used for this analysis are shown in S3 Fig.
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pgen.1005578.g007: Photoreceptors of the ttd14P75L mutant undergo light-enhanced, but Rh1-independent degeneration.(A) Transmission electron microscopy of tangential sections through eyes of wild type flies and homozygous mutant eye clones of ttd14P75L mutant flies. Flies were assayed after eclosion (1 d), kept in the dark for 7 days followed by 16 hours orange light (7 d dark 16 h light), kept in the dark for 21 d, or subjected to a 12 hours light / 12 hours dark cycle for 21 d (21 d dark light). Flies were either raised on normal food (upper panels) or on vitamin A-deprived diet (low vitA; lower panels). 1–7 denotes rhabdomeres of photoreceptor cells R1 to R7. N, nucleus. Scale bar: 2.5 μm. Wild type flies display a normal morphology of the rhabdomeres at all conditions analyzed, except that vitamin A-deprived flies have smaller rhabdomeres due to the reduced amount of rhodopsin. Degeneration of inner and outer photoreceptor cells is obvious in ttd14P75L mutants exposed to a light/dark cycle for 21 days. Interestingly, R7 cells show signs of cell death while R1-6 cells are not affected (arrowhead) in ttd14P75L mutants kept in the dark for 21 days. (B) Time course of photoreceptor degeneration in the ttd14P75L mutant raised on normal food or on vitamin A-deprived diet (low vitA). Fluorescent water immersion images of wild type flies and ttd14P75L mutant eye clones, which express TRP-eGFP as a fluorescent marker for rhabdomeres in photoreceptor cells R1-6, were scored for the presence of rhabdomeres (see Material and Methods). 100% represents fully intact rhabdomeres. 3 ommatidia from five flies each were analyzed. Error bars denote SEM. Flies were kept in a 12 hours light / 12 hours dark cycle for the indicated number of days. Examples of original images used for this analysis are shown in S3 Fig.

Mentions: In order to assess if the decline of the ERG amplitude was associated with morphological alterations in the rhabdomeres, we assessed rhabdomeral structure in wild type and ttd14P75L mutant eye clones for up to 21 days in a 12 hours light / 12 hours dark cycle by transmission electron microscopy and by monitoring TRP-eGFP fluorescence (Fig 7). While no obvious changes could be detected in wild type eyes, electron microscopy revealed severe degeneration of photoreceptor cells in ttd14P75L mutant flies kept in a 12 hours light / 12 hours dark cycle for 21 days. Most rhabdomeres of photoreceptor cells R1-6 and also the rhabdomere of R7 were absent in these flies. Degeneration was much less pronounced when the ttd14P75L mutant was kept in the dark for 21 days. In these flies the R7 cell was affected frequently while almost all rhabdomeres of R1-6 cells remained intact. These findings show that degeneration of photoreceptor cells in the ttd14P75L mutant is enhanced by light and affects inner and outer photoreceptor cells. Of note, no signs of degeneration of inner or outer photoreceptor cells were detected in ttd14P75L mutant flies kept in the dark for 7 days followed by 16 h orange light, a condition in which TRPL failed to recycle back to the rhabdomere. This finding suggests that the TRPL trafficking defect in the ttd14P75L mutant is not a result of photoreceptor cell degeneration. As observed before Vitamin A deprivation resulted in diminished rhabdomere size [23]. However, degeneration was also observed in vitamin A-deprived ttd14P75L mutant flies showing that a reduction of the rhodopsin content cannot rescue the degeneration phenotype. Using water immersion microscopy with ttd14P75L mutant eye clones that express TRP-eGFP as a rhabdomeral marker we analyzed the time course for the loss of rhabdomeres both under regular and vitamin A-deprived conditions (Fig 7B and S4 Fig). In flies kept in a light-dark cycle first signs of degeneration were detected after seven days while almost all rhabdomeres were lost after 21 days (Fig 7B). Vitamin A-deprivation had a small but not statistically significant effect on the degeneration time course and slightly slowed down the speed of degeneration in the ttd14P75L mutant. Taken together, loss of functional TTD14 in the ttd14P75L mutant resulted in late onset light-dependent, but Rh1-independent retinal degeneration.


The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

Cerny AC, Altendorfer A, Schopf K, Baltner K, Maag N, Sehn E, Wolfrum U, Huber A - PLoS Genet. (2015)

Photoreceptors of the ttd14P75L mutant undergo light-enhanced, but Rh1-independent degeneration.(A) Transmission electron microscopy of tangential sections through eyes of wild type flies and homozygous mutant eye clones of ttd14P75L mutant flies. Flies were assayed after eclosion (1 d), kept in the dark for 7 days followed by 16 hours orange light (7 d dark 16 h light), kept in the dark for 21 d, or subjected to a 12 hours light / 12 hours dark cycle for 21 d (21 d dark light). Flies were either raised on normal food (upper panels) or on vitamin A-deprived diet (low vitA; lower panels). 1–7 denotes rhabdomeres of photoreceptor cells R1 to R7. N, nucleus. Scale bar: 2.5 μm. Wild type flies display a normal morphology of the rhabdomeres at all conditions analyzed, except that vitamin A-deprived flies have smaller rhabdomeres due to the reduced amount of rhodopsin. Degeneration of inner and outer photoreceptor cells is obvious in ttd14P75L mutants exposed to a light/dark cycle for 21 days. Interestingly, R7 cells show signs of cell death while R1-6 cells are not affected (arrowhead) in ttd14P75L mutants kept in the dark for 21 days. (B) Time course of photoreceptor degeneration in the ttd14P75L mutant raised on normal food or on vitamin A-deprived diet (low vitA). Fluorescent water immersion images of wild type flies and ttd14P75L mutant eye clones, which express TRP-eGFP as a fluorescent marker for rhabdomeres in photoreceptor cells R1-6, were scored for the presence of rhabdomeres (see Material and Methods). 100% represents fully intact rhabdomeres. 3 ommatidia from five flies each were analyzed. Error bars denote SEM. Flies were kept in a 12 hours light / 12 hours dark cycle for the indicated number of days. Examples of original images used for this analysis are shown in S3 Fig.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4624897&req=5

pgen.1005578.g007: Photoreceptors of the ttd14P75L mutant undergo light-enhanced, but Rh1-independent degeneration.(A) Transmission electron microscopy of tangential sections through eyes of wild type flies and homozygous mutant eye clones of ttd14P75L mutant flies. Flies were assayed after eclosion (1 d), kept in the dark for 7 days followed by 16 hours orange light (7 d dark 16 h light), kept in the dark for 21 d, or subjected to a 12 hours light / 12 hours dark cycle for 21 d (21 d dark light). Flies were either raised on normal food (upper panels) or on vitamin A-deprived diet (low vitA; lower panels). 1–7 denotes rhabdomeres of photoreceptor cells R1 to R7. N, nucleus. Scale bar: 2.5 μm. Wild type flies display a normal morphology of the rhabdomeres at all conditions analyzed, except that vitamin A-deprived flies have smaller rhabdomeres due to the reduced amount of rhodopsin. Degeneration of inner and outer photoreceptor cells is obvious in ttd14P75L mutants exposed to a light/dark cycle for 21 days. Interestingly, R7 cells show signs of cell death while R1-6 cells are not affected (arrowhead) in ttd14P75L mutants kept in the dark for 21 days. (B) Time course of photoreceptor degeneration in the ttd14P75L mutant raised on normal food or on vitamin A-deprived diet (low vitA). Fluorescent water immersion images of wild type flies and ttd14P75L mutant eye clones, which express TRP-eGFP as a fluorescent marker for rhabdomeres in photoreceptor cells R1-6, were scored for the presence of rhabdomeres (see Material and Methods). 100% represents fully intact rhabdomeres. 3 ommatidia from five flies each were analyzed. Error bars denote SEM. Flies were kept in a 12 hours light / 12 hours dark cycle for the indicated number of days. Examples of original images used for this analysis are shown in S3 Fig.
Mentions: In order to assess if the decline of the ERG amplitude was associated with morphological alterations in the rhabdomeres, we assessed rhabdomeral structure in wild type and ttd14P75L mutant eye clones for up to 21 days in a 12 hours light / 12 hours dark cycle by transmission electron microscopy and by monitoring TRP-eGFP fluorescence (Fig 7). While no obvious changes could be detected in wild type eyes, electron microscopy revealed severe degeneration of photoreceptor cells in ttd14P75L mutant flies kept in a 12 hours light / 12 hours dark cycle for 21 days. Most rhabdomeres of photoreceptor cells R1-6 and also the rhabdomere of R7 were absent in these flies. Degeneration was much less pronounced when the ttd14P75L mutant was kept in the dark for 21 days. In these flies the R7 cell was affected frequently while almost all rhabdomeres of R1-6 cells remained intact. These findings show that degeneration of photoreceptor cells in the ttd14P75L mutant is enhanced by light and affects inner and outer photoreceptor cells. Of note, no signs of degeneration of inner or outer photoreceptor cells were detected in ttd14P75L mutant flies kept in the dark for 7 days followed by 16 h orange light, a condition in which TRPL failed to recycle back to the rhabdomere. This finding suggests that the TRPL trafficking defect in the ttd14P75L mutant is not a result of photoreceptor cell degeneration. As observed before Vitamin A deprivation resulted in diminished rhabdomere size [23]. However, degeneration was also observed in vitamin A-deprived ttd14P75L mutant flies showing that a reduction of the rhodopsin content cannot rescue the degeneration phenotype. Using water immersion microscopy with ttd14P75L mutant eye clones that express TRP-eGFP as a rhabdomeral marker we analyzed the time course for the loss of rhabdomeres both under regular and vitamin A-deprived conditions (Fig 7B and S4 Fig). In flies kept in a light-dark cycle first signs of degeneration were detected after seven days while almost all rhabdomeres were lost after 21 days (Fig 7B). Vitamin A-deprivation had a small but not statistically significant effect on the degeneration time course and slightly slowed down the speed of degeneration in the ttd14P75L mutant. Taken together, loss of functional TTD14 in the ttd14P75L mutant resulted in late onset light-dependent, but Rh1-independent retinal degeneration.

Bottom Line: TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid.In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant.In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosensorics, Institute of Physiology, University of Hohenheim, Stuttgart, Germany.

ABSTRACT
Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

No MeSH data available.


Related in: MedlinePlus