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Far-red fluorescent genetically encoded calcium ion indicators
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Far-red fluorescent genetically encoded calcium ion indicators
Rochelin Dalangin1, Mikhail Drobizhev2, Rosana S. Molina2, Abhi Aggarwal3, Ronak Patel3,
Ahmed S. Abdelfattah3, Yufeng Zhao1, Jiahui Wu1, Kaspar Podgorski3, Eric R. Schreiter3,
Thomas E. Hughes2, Robert E. Campbell1,4*, Yi Shen1*
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
2Department of Cell Biology & Neuroscience, Montana State University, Bozeman, Montana,
USA
3Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
4Department of Chemistry, The University of Tokyo, Tokyo, Japan
*Correspondence should be addressed to Y.S. (yi.shen@ualberta.ca) or R.E.C.
(robert.e.campbell@ualberta.ca)
1 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Rochelin Dalangin1, Mikhail Drobizhev2, Rosana S. Molina2, Abhi Aggarwal3, Ronak Patel3,
Ahmed S. Abdelfattah3, Yufeng Zhao1, Jiahui Wu1, Kaspar Podgorski3, Eric R. Schreiter3,
Thomas E. Hughes2, Robert E. Campbell1,4*, Yi Shen1*
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
2Department of Cell Biology & Neuroscience, Montana State University, Bozeman, Montana,
USA
3Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
4Department of Chemistry, The University of Tokyo, Tokyo, Japan
*Correspondence should be addressed to Y.S. (yi.shen@ualberta.ca) or R.E.C.
(robert.e.campbell@ualberta.ca)
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The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Abstract
Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for
functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we
report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and
FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized
these far-red GECIs as purified proteins and assessed their performance when expressed in
cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644
nm, respectively, display large responses to Ca2+ (ΔF/F0 = 6 for FR-GECO1a, 18 for
FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs
also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for
Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.
2 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for
functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we
report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and
FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized
these far-red GECIs as purified proteins and assessed their performance when expressed in
cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644
nm, respectively, display large responses to Ca2+ (ΔF/F0 = 6 for FR-GECO1a, 18 for
FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs
also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for
Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.
2 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Introduction
A property of most mammalian tissues is that they are most transparent to wavelengths of
light between ~600 nm and ~1300 nm (the range often referred to as the “optical window”)1,2.
This wavelength range falls between the absorbance profile of hemoglobin, which
predominates at wavelengths below 600 nm, and the absorbance profile of water, which
predominates at wavelengths greater than 1300 nm. Due to the greater tissue transparency in
this wavelength range, fluorescent probes that absorb and emit efficiently within the optical
window are highly desirable for in vivo imaging. In addition, fluorescent probes with longer
excitation wavelengths are associated with lower phototoxicity and autofluorescence, reduced
crosstalk with green fluorescent indicators, and better spectral compatibility with blue or cyan
light-activated optogenetics tools.
Realization of the advantages of fluorophores that excite and emit within the optical
window has been a driving force for molecular tool engineers to shift the excitation and
emission wavelengths of genetically encodable fluorophores, such as standard red
fluorescent proteins (RFP) with excitation maxima (λex) at 550 to 580 nm and emission (λem) at
580 to 620 nm, into the far-red region of the spectrum. This longstanding effort has yielded a
plethora of far-red FPs (Figure 1A) with λex > 580 nm and λem > 620 nm (Refs. 3–12). Efforts
to engineer biliverdin (BV)-binding FPs, that fluoresce at even further red-shifted wavelengths,
have resulted in near infrared (NIR) FPs with λex > 640 nm and λem > 670 nm (Refs. 13–15).
The key difference between these two classes of genetically encodable fluorophores is that
the red and far-red FPs autocatalytically form their own chromophore and are homologous
with GFP, but genetically encoded NIR FPs use the biliverdin cofactor as chromophore.
Genetically encodable fluorophores can be engineered into genetically encoded
indicators. One of the most important examples is the genetically encoded Ca2+ indicator
(GECI) which can be used for detection and imaging of cell signalling and neuronal activities.
The latest generation of red GECIs for neuronal activity detection include jRCaMP1a/b,
jRGECO1a, K-GECO1, and XCaMP-R, all with single-photon excitation and emission peaks
outside of the optical window16–18. Among currently available RFP-based GECIs, the most
red-shifted variant that uses a fluorescent protein with an autocatalytic chromophore is
CAR-GECO1 (λex ~ 560 nm, λem ~ 609 nm)19. Near-infrared GECI indicator NIR-GECO1 (λex ~
678 nm, λem ~ 704 nm) based on the BV-binding FP mIFP has been recently reported.
3 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
A property of most mammalian tissues is that they are most transparent to wavelengths of
light between ~600 nm and ~1300 nm (the range often referred to as the “optical window”)1,2.
This wavelength range falls between the absorbance profile of hemoglobin, which
predominates at wavelengths below 600 nm, and the absorbance profile of water, which
predominates at wavelengths greater than 1300 nm. Due to the greater tissue transparency in
this wavelength range, fluorescent probes that absorb and emit efficiently within the optical
window are highly desirable for in vivo imaging. In addition, fluorescent probes with longer
excitation wavelengths are associated with lower phototoxicity and autofluorescence, reduced
crosstalk with green fluorescent indicators, and better spectral compatibility with blue or cyan
light-activated optogenetics tools.
Realization of the advantages of fluorophores that excite and emit within the optical
window has been a driving force for molecular tool engineers to shift the excitation and
emission wavelengths of genetically encodable fluorophores, such as standard red
fluorescent proteins (RFP) with excitation maxima (λex) at 550 to 580 nm and emission (λem) at
580 to 620 nm, into the far-red region of the spectrum. This longstanding effort has yielded a
plethora of far-red FPs (Figure 1A) with λex > 580 nm and λem > 620 nm (Refs. 3–12). Efforts
to engineer biliverdin (BV)-binding FPs, that fluoresce at even further red-shifted wavelengths,
have resulted in near infrared (NIR) FPs with λex > 640 nm and λem > 670 nm (Refs. 13–15).
The key difference between these two classes of genetically encodable fluorophores is that
the red and far-red FPs autocatalytically form their own chromophore and are homologous
with GFP, but genetically encoded NIR FPs use the biliverdin cofactor as chromophore.
Genetically encodable fluorophores can be engineered into genetically encoded
indicators. One of the most important examples is the genetically encoded Ca2+ indicator
(GECI) which can be used for detection and imaging of cell signalling and neuronal activities.
The latest generation of red GECIs for neuronal activity detection include jRCaMP1a/b,
jRGECO1a, K-GECO1, and XCaMP-R, all with single-photon excitation and emission peaks
outside of the optical window16–18. Among currently available RFP-based GECIs, the most
red-shifted variant that uses a fluorescent protein with an autocatalytic chromophore is
CAR-GECO1 (λex ~ 560 nm, λem ~ 609 nm)19. Near-infrared GECI indicator NIR-GECO1 (λex ~
678 nm, λem ~ 704 nm) based on the BV-binding FP mIFP has been recently reported.
3 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Although the spectrum of NIR-GECO1 lies well within the optical window, the molecular
brightness of NIR-GECO1 is relatively dim20.
In an effort to achieve the ideal compromise between red-shifts in the excitation and
emission maxima and brightness, we have undertaken the development of two bright
intensiometric far-red fluorescent GECI variants, FR-GECO1a (λex ~ 596 nm, λem ~ 642 nm)
and FR-GECO1c (λex ~ 596 nm, λem ~ 646 nm), based on the recently engineered far-red FP
mKelly1 (λex ~ 596 nm, λem ~ 656 nm) and mKelly2 (λex ~ 598 nm, λem ~ 649 nm)12. These
genetically encoded far-red Ca2+ indicators will open new avenues for multicolor Ca2+ imaging
in combination with other optogenetic indicators and actuators, as well as functional Ca2+
imaging in deep tissue in vivo.
4 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
brightness of NIR-GECO1 is relatively dim20.
In an effort to achieve the ideal compromise between red-shifts in the excitation and
emission maxima and brightness, we have undertaken the development of two bright
intensiometric far-red fluorescent GECI variants, FR-GECO1a (λex ~ 596 nm, λem ~ 642 nm)
and FR-GECO1c (λex ~ 596 nm, λem ~ 646 nm), based on the recently engineered far-red FP
mKelly1 (λex ~ 596 nm, λem ~ 656 nm) and mKelly2 (λex ~ 598 nm, λem ~ 649 nm)12. These
genetically encoded far-red Ca2+ indicators will open new avenues for multicolor Ca2+ imaging
in combination with other optogenetic indicators and actuators, as well as functional Ca2+
imaging in deep tissue in vivo.
4 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Results
Protein engineering
Initial efforts to engineer a far-red Ca2+ indicator followed two parallel strategies. The first
strategy was to graft key mutations for far-red fluorescence onto existing red GECIs. We used
R-GECO1 (Ref. 21), CH-GECO1 (Ref. 22) and K-GECO1 (Ref. 17) as templates and
introduced key mutations from E2-Crimson (λex ~ 611 nm, λem ~ 646 nm, tetrameric)5,
RDSmCherry (λex ~ 600 nm, λem ~ 630 nm)7, and mNeptune (λex ~ 600 nm, λem ~ 650 nm)4,
respectively (Figure 1A, Supplementary Table 1). Unfortunately, complete loss of
fluorescence or no substantial spectral red-shifts were observed in these designed
prototypes. The second strategy was de novo engineering of a GECI starting from far-red FP
scaffolds. However, our attempts to engineer GECI based on mNeptune4 and mCardinal8 did
not yield fluorescent prototypes. As both mNeptune and mCardinal retained weak
dimerization tendencies4,8,12,23, we suspected that the failure of these prototypes to fluoresce
could be due to the circular permutation site and/or insertion site of calmodulin and its binding
peptide overlapping with the oligomerization interface, possibly disrupting dimerization that
was crucial to the proper folding and/or chromophore maturation of these proteins.
mKelly1 and mKelly2 are far-red FP variants of mCardinal that were engineered to
have increased monomericity by a combination of deletion of the C-terminal “tail”, directed
evolution, and consensus design12 (Figure 1A). We rationalized that the enhanced
monomericity of mKelly1 and mKelly2 might facilitate circular permutation and further
engineering to create GECIs. We thus circularly permuted (cp) mKelly2, the brighter of the
two mKelly variants, at Thr143 (numbering according to the crystal structure for mCardinal8;
PDB ID: 4OQW). This designed cpmKelly2 was then used to replace the cpFusionRed RFP in
K-GECO1 (Ref. 17). K-GECO1 was chosen as the starting scaffold because the use of the
ckkap peptide instead of RS20 as the calmodulin binding peptide yielded a sensor with high
sensitivity, strong affinity, good linearity and fast kinetics. Additionally, it is currently the only
GECI engineered using a FP variant derived from eqFP578, from which mKelly2 was also
derived (Figure 1A). This initial construct resulted in a dimly fluorescent variant we named
FR-GECO0.1 (Figure 1B). Optimization of linker1 (the amino acids linking the ckkap peptide
to cpmKelly2) led to the identification of an improved, but still dim, variant, FR-GECO0.2, with
λex = 586 nm, λem = 632 nm and ~7-fold increase of fluorescence upon Ca2+ addition. We then
5 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
Protein engineering
Initial efforts to engineer a far-red Ca2+ indicator followed two parallel strategies. The first
strategy was to graft key mutations for far-red fluorescence onto existing red GECIs. We used
R-GECO1 (Ref. 21), CH-GECO1 (Ref. 22) and K-GECO1 (Ref. 17) as templates and
introduced key mutations from E2-Crimson (λex ~ 611 nm, λem ~ 646 nm, tetrameric)5,
RDSmCherry (λex ~ 600 nm, λem ~ 630 nm)7, and mNeptune (λex ~ 600 nm, λem ~ 650 nm)4,
respectively (Figure 1A, Supplementary Table 1). Unfortunately, complete loss of
fluorescence or no substantial spectral red-shifts were observed in these designed
prototypes. The second strategy was de novo engineering of a GECI starting from far-red FP
scaffolds. However, our attempts to engineer GECI based on mNeptune4 and mCardinal8 did
not yield fluorescent prototypes. As both mNeptune and mCardinal retained weak
dimerization tendencies4,8,12,23, we suspected that the failure of these prototypes to fluoresce
could be due to the circular permutation site and/or insertion site of calmodulin and its binding
peptide overlapping with the oligomerization interface, possibly disrupting dimerization that
was crucial to the proper folding and/or chromophore maturation of these proteins.
mKelly1 and mKelly2 are far-red FP variants of mCardinal that were engineered to
have increased monomericity by a combination of deletion of the C-terminal “tail”, directed
evolution, and consensus design12 (Figure 1A). We rationalized that the enhanced
monomericity of mKelly1 and mKelly2 might facilitate circular permutation and further
engineering to create GECIs. We thus circularly permuted (cp) mKelly2, the brighter of the
two mKelly variants, at Thr143 (numbering according to the crystal structure for mCardinal8;
PDB ID: 4OQW). This designed cpmKelly2 was then used to replace the cpFusionRed RFP in
K-GECO1 (Ref. 17). K-GECO1 was chosen as the starting scaffold because the use of the
ckkap peptide instead of RS20 as the calmodulin binding peptide yielded a sensor with high
sensitivity, strong affinity, good linearity and fast kinetics. Additionally, it is currently the only
GECI engineered using a FP variant derived from eqFP578, from which mKelly2 was also
derived (Figure 1A). This initial construct resulted in a dimly fluorescent variant we named
FR-GECO0.1 (Figure 1B). Optimization of linker1 (the amino acids linking the ckkap peptide
to cpmKelly2) led to the identification of an improved, but still dim, variant, FR-GECO0.2, with
λex = 586 nm, λem = 632 nm and ~7-fold increase of fluorescence upon Ca2+ addition. We then
5 of 34
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 15, 2020.;https://doi.org/10.1101/2020.11.12.380089doi:bioRxiv preprint
100%
