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A single and rapid calcium wave at egg activation in Drosophila.

York-Andersen AH, Parton RM, Bi CJ, Bromley CL, Davis I, Weil TT - Biol Open (2015)

Bottom Line: Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation.We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave.We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

No MeSH data available.


Related in: MedlinePlus

A rapidly propagating Ca2+ wave at egg activation.(A–C) Mature oocytes microinjected with Calcium Green-1 and Texas Red Dextrans and left for dyes to diffuse for 45 minutes. (A) Addition of activation buffer results in a rapid increase in intracellular Ca2+ within the first 90 seconds followed by a slower recovery. Graph displays a single representative egg chamber imaged in a single cortical plane in order to calculate relative increase in calcium in the entire egg. Plotted values are the average for the entire egg chamber. (B) Time series showing propagation of the Ca2+ transient at high temporal resolution, cross-section images displayed every 10 seconds. (C) The addition of Schneider's Insect medium (t = 0′) shows no propagating Ca2+ response. Scale bars A–C = 100 µm. Time in minutes (′) and seconds (″).
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f01: A rapidly propagating Ca2+ wave at egg activation.(A–C) Mature oocytes microinjected with Calcium Green-1 and Texas Red Dextrans and left for dyes to diffuse for 45 minutes. (A) Addition of activation buffer results in a rapid increase in intracellular Ca2+ within the first 90 seconds followed by a slower recovery. Graph displays a single representative egg chamber imaged in a single cortical plane in order to calculate relative increase in calcium in the entire egg. Plotted values are the average for the entire egg chamber. (B) Time series showing propagation of the Ca2+ transient at high temporal resolution, cross-section images displayed every 10 seconds. (C) The addition of Schneider's Insect medium (t = 0′) shows no propagating Ca2+ response. Scale bars A–C = 100 µm. Time in minutes (′) and seconds (″).

Mentions: Although mutations in the Ca2+ signalling pathway have been shown to disrupt egg activation (Horner et al., 2006), to date, Ca2+ signalling events have not been recorded during Drosophila egg activation. To investigate this we monitored the change in intracellular Ca2+ by ratiometric imaging on ex vivo isolated mature oocytes (stage 14). Prior to addition of activation buffer, both Calcium Green-1 Dextran (10 kDa) and Texas Red Dextran (10 kDa) were micro-injected in equal concentrations and allowed to evenly diffuse through the cytoplasm (45 minutes) to provide a baseline measure of intracellular Ca2+ concentration. Upon activation, we observed a consistent, rapidly propagating increase in intracellular Ca2+, followed by a slower “recovery phase”, decrease (Fig. 1A,B, n = 8). These events were concomitant with the physical “swelling” response normally associated with activation (data not shown). To confirm that the change in Ca2+ was due to activation rather than simply mechanical perturbation, Schneider's Insect medium was applied to the post-injected egg chambers (Fig. 1C, n = 8). In these cases there was no Ca2+ wave or swelling, however, there was a small local Ca2+ elevation associated with the injection site, which did not propagate. We verified that the ratio imaging method was responding to Ca2+ by artificially elevating the Ca2+ concentration locally through microinjection of CaCl2 (data not shown).


A single and rapid calcium wave at egg activation in Drosophila.

York-Andersen AH, Parton RM, Bi CJ, Bromley CL, Davis I, Weil TT - Biol Open (2015)

A rapidly propagating Ca2+ wave at egg activation.(A–C) Mature oocytes microinjected with Calcium Green-1 and Texas Red Dextrans and left for dyes to diffuse for 45 minutes. (A) Addition of activation buffer results in a rapid increase in intracellular Ca2+ within the first 90 seconds followed by a slower recovery. Graph displays a single representative egg chamber imaged in a single cortical plane in order to calculate relative increase in calcium in the entire egg. Plotted values are the average for the entire egg chamber. (B) Time series showing propagation of the Ca2+ transient at high temporal resolution, cross-section images displayed every 10 seconds. (C) The addition of Schneider's Insect medium (t = 0′) shows no propagating Ca2+ response. Scale bars A–C = 100 µm. Time in minutes (′) and seconds (″).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400597&req=5

f01: A rapidly propagating Ca2+ wave at egg activation.(A–C) Mature oocytes microinjected with Calcium Green-1 and Texas Red Dextrans and left for dyes to diffuse for 45 minutes. (A) Addition of activation buffer results in a rapid increase in intracellular Ca2+ within the first 90 seconds followed by a slower recovery. Graph displays a single representative egg chamber imaged in a single cortical plane in order to calculate relative increase in calcium in the entire egg. Plotted values are the average for the entire egg chamber. (B) Time series showing propagation of the Ca2+ transient at high temporal resolution, cross-section images displayed every 10 seconds. (C) The addition of Schneider's Insect medium (t = 0′) shows no propagating Ca2+ response. Scale bars A–C = 100 µm. Time in minutes (′) and seconds (″).
Mentions: Although mutations in the Ca2+ signalling pathway have been shown to disrupt egg activation (Horner et al., 2006), to date, Ca2+ signalling events have not been recorded during Drosophila egg activation. To investigate this we monitored the change in intracellular Ca2+ by ratiometric imaging on ex vivo isolated mature oocytes (stage 14). Prior to addition of activation buffer, both Calcium Green-1 Dextran (10 kDa) and Texas Red Dextran (10 kDa) were micro-injected in equal concentrations and allowed to evenly diffuse through the cytoplasm (45 minutes) to provide a baseline measure of intracellular Ca2+ concentration. Upon activation, we observed a consistent, rapidly propagating increase in intracellular Ca2+, followed by a slower “recovery phase”, decrease (Fig. 1A,B, n = 8). These events were concomitant with the physical “swelling” response normally associated with activation (data not shown). To confirm that the change in Ca2+ was due to activation rather than simply mechanical perturbation, Schneider's Insect medium was applied to the post-injected egg chambers (Fig. 1C, n = 8). In these cases there was no Ca2+ wave or swelling, however, there was a small local Ca2+ elevation associated with the injection site, which did not propagate. We verified that the ratio imaging method was responding to Ca2+ by artificially elevating the Ca2+ concentration locally through microinjection of CaCl2 (data not shown).

Bottom Line: Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation.We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave.We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

No MeSH data available.


Related in: MedlinePlus