Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25 ms, reached 50% F-max with similar to 1200 effectively absorbed photons and saturated (Delta F/F-0 similar to 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (Delta F/F-0 similar to 4), long latency (similar to 200ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP(3) receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to < 10 nM (Delta F/F-0 similar to 0.1), and unlikely to play any role in phototransduction. (c) 2017 The Authors. Published by Elsevier Ltd.

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Asteriti S;
2017

Abstract

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25 ms, reached 50% F-max with similar to 1200 effectively absorbed photons and saturated (Delta F/F-0 similar to 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (Delta F/F-0 similar to 4), long latency (similar to 200ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP(3) receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to < 10 nM (Delta F/F-0 similar to 0.1), and unlikely to play any role in phototransduction. (c) 2017 The Authors. Published by Elsevier Ltd.
Calcium imaging
Genetically encoded calcium indicators
InsP(3)
Na/Ca exchanger
Phospholipase C
Phototransduction
TRP channels
Animals
Drosophila Proteins
Drosophila melanogaster
Microscopy, Fluorescence
Photoreceptor Cells, Invertebrate
Type C Phospholipases
Calcium Signaling
File in questo prodotto:
File Dimensione Formato  
1-s2.0-S0143416016302287-main.pdf

accesso aperto

Descrizione: Articolo principale
Tipologia: Versione dell'editore
Licenza: Creative commons
Dimensione 2.75 MB
Formato Adobe PDF
2.75 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/1049775
Citazioni
  • ???jsp.display-item.citation.pmc??? 7
  • Scopus 10
  • ???jsp.display-item.citation.isi??? 11
social impact