Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Créton, Robbert; Steele, Marjorie E; Jaffe, Lionel F.

doi:10.1016/s0143-4160(97)90071-3

Cited by 19 publications

(19 citation statements)

References 20 publications

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“…Using the same assumptions for embryo volume and water content indicated earlier (Leung et al, 1998) this indicates a final concentration of functional f-aequorin of ~254 nM and f-aequorin-EGFP of ~13 nM at 4 hpf. Our estimate for in vitro f-aequorin reconstitution is thus similar to the 110 nM suggested previously, to be required to image resting levels of Ca 2+ in zebrafish embryos (Créton et al, 1997). However, the luminescent-based value for reconstituted f-aequorin is ~5-fold higher than that estimated for apoaequorin protein expression derived from the Western blot protein quantification.…”

Section: In Vivo Reconstitution Of Aequorin In Aeq-mrna Injected Intasupporting

confidence: 77%

“…This is approximately 70-fold more than the final concentration calculated at the 4 hpf expression and reconstitution peak (i.e., ~254 nM). Créton et al, however, have reported that the estimated in vivo half-life of haequorin (a semi-synthetic aequorin similar to f-aequorin; Shimomura et al, 1989) is only around 3 hours (Créton et al, 1997). In addition, it is clear from Fig.…”

Section: Fig 5 (Right Column)mentioning

confidence: 95%

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Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

Cheung¹,

Webb²,

Meng³

et al. 2006

Int. J. Dev. Biol.

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When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca 2+ signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFPmRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca 2+ . Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca 2+ signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca 2+ signaling during the 24-48 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca 2+ imaging window by an additional 24 hours.

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Section: In Vivo Reconstitution Of Aequorin In Aeq-mrna Injected Intasupporting

confidence: 77%

Section: Fig 5 (Right Column)mentioning

confidence: 95%

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

Cheung¹,

Webb²,

Meng³

et al. 2006

Int. J. Dev. Biol.

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“…The most important of these is aequorin (for a review see Creton and Rizzuto, 1999;Gilland et al, 1999;Kendall and Badminton, 1998;Miller et al, 1994;) a 21 kD protein that can be expressed in cells and may be targeted to specific intracellular organelles (see Badminton et al, 1998), or may be microinjected for studying calcium signaling in early stage embryos (Creton et al, 1997. A drawback of using aequorin in comparison to synthetic calcium indicators is that aequorin is of a one-use nature, and after calcium binding and light emission it is not available for further calcium indication.…”

Section: Direct Measurement Of Intracellular Calciummentioning

confidence: 99%

Study of calcium signaling in non-excitable cells

Brink

Bloemers

Blink

et al. 1999

Microsc. Res. Tech.

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“…The resolution of aequorin imaging (Speksnijder et al, 1990;Swann and Whitaker, 1986) is limited by the small numbers of photons emitted by aequorin, particularly in small cells (Blinks, 1990;Creton et al, 1997). Photons from the micromolar concentrations of fura2 used intracellularly are orders of magnitude more abundant, offering much improved spatial and temporal resolution (Corps et al, 1989;Wier et al, 1988;Williams et al, 1985).…”

Section: Calcium Looks Like a Universal Signalling Response To Hormonmentioning

confidence: 99%

Ways of looking at calcium

Whitaker

1999

Microsc. Res. Tech.

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Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Cited by 19 publications

References 20 publications

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

Study of calcium signaling in non-excitable cells

Ways of looking at calcium

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