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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+ response at egg activation detected by transgenic indicator.(A) Time series of a mature oocyte expressing UAS-myrGCaMP5 following the addition of activation buffer, (t = 0′) shows an expected baseline of fluorescence (note: dorsal appendages to the left show typical autofluorescence). After the addition of activation buffer, the mature oocyte swells and a wave of increases Ca2+concentration initiates from the posterior pole (t = 1′06″, arrowhead). The posterior wave propagates across the mature oocyte (t = 3′50″, arrowhead). Following a brief period when the whole cell has an increase in Ca2+ (t = 8′20″), a slower recovery commences (t = 16′05″) and leaves the cell a similar Ca2+ concentration as prior to addition of activation buffer (t = 19′25″) and no oscillations are detected. (B) Time series of a mature oocyte not expressing UAS-myrGCamp-5 (without the tub-GAL4VP16 driver) or (C) wild-type following the addition of activation buffer. Corresponding bright-field images show the mature oocytes swelling and fluorescent images show no change in fluorescence, as expected (n = 7). (D) Mature oocytes expressing tub-GAL4VP16 and UAS-myrGCamp-5 following the addition of Schneider's Insect media does not show the mature oocyte swelling and an increase in Ca2+ is not detected (n = 7). Scale bars A–D = 100 µm. Max projection A = 41 µm, B = 40.3 µm, C = 43.7 µm, D = 33.2 µm.
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f02: A rapidly propagating Ca2+ response at egg activation detected by transgenic indicator.(A) Time series of a mature oocyte expressing UAS-myrGCaMP5 following the addition of activation buffer, (t = 0′) shows an expected baseline of fluorescence (note: dorsal appendages to the left show typical autofluorescence). After the addition of activation buffer, the mature oocyte swells and a wave of increases Ca2+concentration initiates from the posterior pole (t = 1′06″, arrowhead). The posterior wave propagates across the mature oocyte (t = 3′50″, arrowhead). Following a brief period when the whole cell has an increase in Ca2+ (t = 8′20″), a slower recovery commences (t = 16′05″) and leaves the cell a similar Ca2+ concentration as prior to addition of activation buffer (t = 19′25″) and no oscillations are detected. (B) Time series of a mature oocyte not expressing UAS-myrGCamp-5 (without the tub-GAL4VP16 driver) or (C) wild-type following the addition of activation buffer. Corresponding bright-field images show the mature oocytes swelling and fluorescent images show no change in fluorescence, as expected (n = 7). (D) Mature oocytes expressing tub-GAL4VP16 and UAS-myrGCamp-5 following the addition of Schneider's Insect media does not show the mature oocyte swelling and an increase in Ca2+ is not detected (n = 7). Scale bars A–D = 100 µm. Max projection A = 41 µm, B = 40.3 µm, C = 43.7 µm, D = 33.2 µm.

Mentions: While injected dyes allowed us to initially observe and make preliminary characterisation of the calcium response we found it difficult to thoroughly analyse speed and magnitude. To overcome this and in order to further characterise the Ca2+ transient during activation by standard hypotonic activation buffer, we used the genetically encoded Ca2+ indicator Ca2+-sensitive GFP (GCaMP) (Nakai et al., 2001) under the control of a germ line specific GAL4 driver. We found that using the myristoylated variant of the indicator (Melom and Littleton, 2013), targeting it to the inner leaflet of the plasma membrane, resulted in a dramatic increase in the signal-to-noise ratio between the GFP and auto-fluorescent yolk granules in the cytoplasm of the egg. This approach showed a similar transient Ca2+ increase to that detected with injected dyes (Fig. 1B, Fig. 2A; supplementary material Movies 1–3). Using UAS-myrGCaMP5, we could clearly see that the Ca2+ transient propagated as a wave (Fig. 2A, arrowheads) and subsequently returned to basal levels (Fig 2A). Initiation of the wave following the addition of activation buffer was observed on average at 1′15″ ± 17.5″ (n = 26, SEM) and the completion of the wave resulting in the whole oocyte showed an increase in intracellular calcium after 3′ 35″ ± 41.1″ (n = 16, SEM). Recovery begins 4′06″ ± 50.6″ (n = 10, SEM) later and took 5′33″ ± 50″ (n = 10, SEM) to complete. The majority of waves initiated from the posterior pole (69%) versus the anterior (18%) or lateral cortex (13%) (n = 29). Even with prolonged observations, we did not detect any further transients or oscillating waves after ex-vivo activation (Fig. 2A, t = 70′) or in the early embryo (0–2 hours) (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+ response at egg activation detected by transgenic indicator.(A) Time series of a mature oocyte expressing UAS-myrGCaMP5 following the addition of activation buffer, (t = 0′) shows an expected baseline of fluorescence (note: dorsal appendages to the left show typical autofluorescence). After the addition of activation buffer, the mature oocyte swells and a wave of increases Ca2+concentration initiates from the posterior pole (t = 1′06″, arrowhead). The posterior wave propagates across the mature oocyte (t = 3′50″, arrowhead). Following a brief period when the whole cell has an increase in Ca2+ (t = 8′20″), a slower recovery commences (t = 16′05″) and leaves the cell a similar Ca2+ concentration as prior to addition of activation buffer (t = 19′25″) and no oscillations are detected. (B) Time series of a mature oocyte not expressing UAS-myrGCamp-5 (without the tub-GAL4VP16 driver) or (C) wild-type following the addition of activation buffer. Corresponding bright-field images show the mature oocytes swelling and fluorescent images show no change in fluorescence, as expected (n = 7). (D) Mature oocytes expressing tub-GAL4VP16 and UAS-myrGCamp-5 following the addition of Schneider's Insect media does not show the mature oocyte swelling and an increase in Ca2+ is not detected (n = 7). Scale bars A–D = 100 µm. Max projection A = 41 µm, B = 40.3 µm, C = 43.7 µm, D = 33.2 µm.
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Related In: Results  -  Collection

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f02: A rapidly propagating Ca2+ response at egg activation detected by transgenic indicator.(A) Time series of a mature oocyte expressing UAS-myrGCaMP5 following the addition of activation buffer, (t = 0′) shows an expected baseline of fluorescence (note: dorsal appendages to the left show typical autofluorescence). After the addition of activation buffer, the mature oocyte swells and a wave of increases Ca2+concentration initiates from the posterior pole (t = 1′06″, arrowhead). The posterior wave propagates across the mature oocyte (t = 3′50″, arrowhead). Following a brief period when the whole cell has an increase in Ca2+ (t = 8′20″), a slower recovery commences (t = 16′05″) and leaves the cell a similar Ca2+ concentration as prior to addition of activation buffer (t = 19′25″) and no oscillations are detected. (B) Time series of a mature oocyte not expressing UAS-myrGCamp-5 (without the tub-GAL4VP16 driver) or (C) wild-type following the addition of activation buffer. Corresponding bright-field images show the mature oocytes swelling and fluorescent images show no change in fluorescence, as expected (n = 7). (D) Mature oocytes expressing tub-GAL4VP16 and UAS-myrGCamp-5 following the addition of Schneider's Insect media does not show the mature oocyte swelling and an increase in Ca2+ is not detected (n = 7). Scale bars A–D = 100 µm. Max projection A = 41 µm, B = 40.3 µm, C = 43.7 µm, D = 33.2 µm.
Mentions: While injected dyes allowed us to initially observe and make preliminary characterisation of the calcium response we found it difficult to thoroughly analyse speed and magnitude. To overcome this and in order to further characterise the Ca2+ transient during activation by standard hypotonic activation buffer, we used the genetically encoded Ca2+ indicator Ca2+-sensitive GFP (GCaMP) (Nakai et al., 2001) under the control of a germ line specific GAL4 driver. We found that using the myristoylated variant of the indicator (Melom and Littleton, 2013), targeting it to the inner leaflet of the plasma membrane, resulted in a dramatic increase in the signal-to-noise ratio between the GFP and auto-fluorescent yolk granules in the cytoplasm of the egg. This approach showed a similar transient Ca2+ increase to that detected with injected dyes (Fig. 1B, Fig. 2A; supplementary material Movies 1–3). Using UAS-myrGCaMP5, we could clearly see that the Ca2+ transient propagated as a wave (Fig. 2A, arrowheads) and subsequently returned to basal levels (Fig 2A). Initiation of the wave following the addition of activation buffer was observed on average at 1′15″ ± 17.5″ (n = 26, SEM) and the completion of the wave resulting in the whole oocyte showed an increase in intracellular calcium after 3′ 35″ ± 41.1″ (n = 16, SEM). Recovery begins 4′06″ ± 50.6″ (n = 10, SEM) later and took 5′33″ ± 50″ (n = 10, SEM) to complete. The majority of waves initiated from the posterior pole (69%) versus the anterior (18%) or lateral cortex (13%) (n = 29). Even with prolonged observations, we did not detect any further transients or oscillating waves after ex-vivo activation (Fig. 2A, t = 70′) or in the early embryo (0–2 hours) (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