Margiotta et al. · Frontiers in Physiology · 2020

Cryptochromes Mediate Intrinsic Photomechanical Transduction in Avian Iris and Somatic Striated Muscle

Margiotta Joseph F., Howard Marthe J.

The study

What was asked, and what was found

Margiotta and Howard, Frontiers in Physiology, 2020 asked which cell in the bird eye senses light when an isolated iris constricts, and which photopigment does the sensing. The iris of many non mammalian species contracts within seconds of illumination without any input from the retina or the brain, and in chicken this response had been attributed to the cryptochromes rather than to melanopsin. The authors traced the response to the striated muscle fibre itself and then tested the attribution directly.

Six AUMsilence sdASOs were made, three against chicken CRY1 and three against CRY2, each 21 nucleotides long, and added straight to the culture medium of chicken pectoral myotubes for 24 to 48 hours with no transfection reagent. Four had no effect on the transcript. The other two, named CRY1 ASO 1.3 and CRY2 ASO 2.3, lowered CRY1 by 87 ± 6% and CRY2 by 91 ± 5% against untreated controls. Neither touched the related CRY4 transcript, which matters because CRY4 also binds flavin adenine dinucleotide and could have explained the result. AUM BioTech is named as the supplier.

With both transcripts down, the calcium response to 435 nm blue light was gone. Peak change in Rhod-2 fluorescence fell from 135 ± 33% to −1 ± 27% in pectoral myotubes and from 57 ± 21% to −47 ± 19% in iris myotubes. Two controls make the case: a scramble oligonucleotide of the same length and chemistry left the light response untouched at 128 ± 6% against 129 ± 24% in untreated cells, and the response to potassium depolarisation was unchanged after knockdown, so only the light sensing step was removed. The same result in pectoral muscle as in iris led the authors to conclude that light driven contraction is a general feature of developing striated muscle.

Key findings

Two of six AUMsilence sdASOs lowered chicken CRY1 and CRY2 transcripts by 87% and 91% in pectoral myotube cultures, with no effect on the related CRY4 transcript.(Discussion)
Knocking down both cryptochrome transcripts abolished the calcium response of iris and pectoral myotubes to 435 nm blue light.(Results, Cryptochromes 1 and 2 Mediate Myotube PMT, Figures 5D to 5F)
A scramble control oligonucleotide of matched length and chemistry left the light response intact, so the loss was sequence specific.(Figure 5 caption, panel G left)
The cells still responded normally to potassium depolarisation after knockdown, so the machinery downstream of the light sensor was untouched.(Results, Cryptochromes 1 and 2 Mediate Myotube PMT)
Treated myotubes looked no different from untreated ones, so the oligonucleotides did not visibly harm the differentiated muscle fibres.(Results, Cryptochromes 1 and 2 Mediate Myotube PMT)
Because the same result held in pectoral muscle as in iris, the authors conclude that light driven contraction is a general property of developing striated muscle rather than something peculiar to the eye.(Abstract)

Product: AUMsilence sdASO
Targets: CRY1 · chicken · mRNACRY2 · chicken · mRNACRY4 · chicken · mRNA, tested for off target knockdown and not silenced
Models: chicken pectoral muscle myotube cultures from embryonic day 11 to 12 embryos · primary cellschicken iris sphincter muscle myotube cultures from embryonic day 13 to 14 embryos · primary cellswhole chicken iris preparations, used for the pharmacology rather than for oligonucleotide treatment · ex vivo tissue
Delivery: added straight to the culture medium at 2 to 5 μM and incubated for 24 to 48 hours, then washed off before imaging. The two controls in Figure 5G ran 25 hours. No transfection reagent is described.
Readouts: semi-quantitative RT-PCR, Rhod-2 calcium imaging, myotube contraction measurement, iris sphincter open area measurement, immunolabelling and confocal microscopy
Measured: knockdown of CRY1 transcript by CRY1 ASO 1.3: 87 ± 6 % (pectoral myotube cultures, versus untreated controls, N = 4 experiments)knockdown of CRY2 transcript by CRY2 ASO 2.3: 91 ± 5 % (pectoral myotube cultures, versus untreated controls, N = 3 experiments)candidate oligonucleotides that lowered the transcript: 2 of 6 oligonucleotides (three against CRY1 and three against CRY2, screened at 5 μM)peak light response of pectoral myotubes, change in Rhod-2 fluorescence relative to background: 135 ± 33 in controls versus −1 ± 27 after treatment (both oligonucleotides together, dual excitation 435 and 535 nm light, n = 11 control and n = 13 treated)peak light response of iris myotubes, change in Rhod-2 fluorescence relative to background: 57 ± 21 in controls versus −47 ± 19 after treatment (both oligonucleotides together, n = 6 control and n = 5 treated)peak light response after the scramble control oligonucleotide: 128 ± 6 with scramble versus 129 ± 24 untreated (pectoral myotubes, scramble at 5 μM for 25 h, n = 6 and n = 12)calcium response to potassium depolarisation after knockdown, a specificity control: 464 ± 151 treated versus 522 ± 192 untreated (pectoral myotubes, focal application of 100 mM KCl, n = 5 and n = 11)knockdown of the two transcripts by the two chosen oligonucleotides: 87 ± 6% for CRY1 and 91 ± 5% for CRY2 (CRY1 ASO 1.3 and CRY2 ASO 2.3, against untreated controls, N = 4 and N = 3 experiments respectively)
Figures: Figure 5 Cryptochrome knockdown measured by RT-PCR, the loss of the blue light calcium response in pectoral and iris myotubes, and the scramble and depolarisation controls.
Citation: Margiotta et al. Cryptochromes Mediate Intrinsic Photomechanical Transduction in Avian Iris and Somatic Striated Muscle. Frontiers in Physiology 2020;11:128. doi:10.3389/fphys.2020.00128

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