Expanded dynamic range of fluorescent indicators�for Ca⁺² by circularly permuted yellow�fluorescent proteins

Ketaki Nitin Mhatre MSc.

AIM

• Optimization of Ca+2 indicator, Yellow Chameleon (YC) (CFP+ CaM+ a glycylglycine linker+ the CaM-binding peptide of myosin light-chain kinase (M13)+ YFP)

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• Use of Flourescence Resonance Energy Transfer (FRET) technology.

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• Several variants of YC: different composition of Ca sensing domain, resistant to acidification, quick maturation and enhanced brightness.

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• Venus YFP variant shortcomings: poor dynamic range, low signal-to-noise ratios.�
• Generation of circularly permuted YFPs (cpYFPs)�
• Study of the properties of circularly permuted YC variants

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(Miyawaki A. et.al., 1997)

Steps involved in the experiment

• Gene Construction.�
• Protein Expression, in Vitro Spectroscopy, Ca⁺², and pH Titrations.�
• Cell Culture and Transfection.�
• Production of Transgenic Mice.�
• Slice Preparation.

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• Imaging.

Results

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(A) The three-dimensional structure of GFP with the positions of the original (Met-1) and new N termini are indicated.

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(B) Domain structures of YC3.12, YC3.20, YC3.30,YC3.60, YC3.70, and YC3.90. XCaM, Xenopus CaM; E104Q, mutation of the conserved bidentate glutamate (E104) at position 12 of the third Ca⁺² binding loop to glutamine.

(C) Emission spectra of YC variants (excitation at 435 nm) at zero and saturated Ca⁺².

Schematic structures and spectral properties of YC3.12 and the new YC variants:

Anisotropy study and Titrations:

Table:1 Ca⁺² responses of the conventional and new YC variants

Table:2 Affinities for Ca⁺² of YC3.60 and its derivatives

A: Fluorescence anisotropy of YC variants at zero and saturated Ca⁺²

B: Ca⁺² titration curves of YC2.60 (triangles), YC3.60 (circles), and YC4.60 (squares) at pH 7.4.

C: pH titration curves of YC3.60 at zero and saturated Ca⁺².

Comparative study : measurements of Ca⁺² dynamics in HeLa cells expressing YC3.60 and YC3.12.

(A)Typical Ca⁺² transients reported by YC3.60 in HeLa cells induced with 30 μM ATP. Inset : The same graph with the ordinate expanded. (Lower) Changes in fluorescence intensities of CFP and cp173Venus.

�(B)Typical Ca⁺² transients reported by YC3.12 in HeLa cells induced with 30 μM ATP

Inset : The same graph with the ordinate expanded. (Lower) Changes in fluorescence intensities of CFP and Venus.

(A) A series of confocal pseudocolored ratio images showing propagation of [Ca⁺²]c.

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(B) A real-color image of the HeLa cells. (Scale bar 10 m.)

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(C) Time courses of changes in [Ca⁺²]c in the six ROIs indicated in B.

Confocal Ca⁺² imaging in cytosol using YC 3.6.

Plasma membrane targeted expression: Confocal Ca⁺² imaging beneath the plasma membrane by using YC3.60pm.�

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D) A real-color image of a HeLa cell expressing YC3.60pm.

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(E) The histamine-induced change in [Ca⁺²]pm in the peripheral region indicated by a circle in D.

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(F) A series of confocal pseudocolored ratio images showing changes in [Ca⁺²] in filopodial structures.

Expression in Transgenic mice: Fast Ca2 imaging of a hippocampal brain slice from a YC3.60 pm producing transgenic mouse.

(A) A low-magnification image of brains of a WT mouse and a transgenic line. (480nm)�

(B) A high magnification fluorescence image in the CA1 region.�

(C) A bright-field image of hippocampal slice.�

(D) A series of pseudocolored images showing a sensitised emission by YFP at 100 Hz.

(E) The field potential (f.p.) change induced by tetanus.

(F) The time course of [Ca⁺²]pm observed in area CA1 of the transgenic line.�

(G) The time course of [Ca⁺²]pm observed in area DG of the transgenic line. �

(H) The time course of [Ca⁺²]pm observed in area CA1 of a WT mouse.

Discussion

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• Fluorescence efficiency of cpVenus variants: F46L mutation and position of new N and C termini.�
• YC3.6: Larger dynamic Range, enhanced signal –to-noise ratio, targeted expression.

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• Transgenic Expression: reduced dynamic range due to YC3.6 expressed in all the cells and presence of CaM and CaM binding protein.�
• Further studies: Single Molecule detection and spectroscopy.

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• Not the end: Optimisation required for different application.

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References

• Tsien RY. , Bacskai BJ , Adams SR. FRET for studying intracellular signalling. Trends Cell Biol. 1993 Jul;3(7):242-5.

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• Topell SS, Hennecke JJ, Glockshuber R Circularly permuted variants of the green fluorescent protein. FEBS Lett. 1999 Aug 27;457(2):283-9.

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• Miyawaki A , Llopis J , Heim R , McCaffery JM. , Adams JA. , Ikura M. , Tsien RY . Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997 Aug 28;388(6645):882-7.

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• Tsien R . The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44.

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• Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki . A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol. 2002 Jan;20(1):87-90.

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• Miyawaki A, Griesbeck O, Heim R., Tsien RY. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2135-40.

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• Nagai T, Sawano A, Park ES, Miyawaki A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A.2001 Mar 13;98(6):3197-202.

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• Baird GS., Zacharias DA., Tsien RY. Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci U S A.1999 Sep 28;96(20):11241-6.

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• Montero M., Lobatón CD., Gutierrez-Fernández S., Moreno A., Alvarez J. Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks. J Biol Chem. 2003 Dec 12;278(50):49972-9.

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Thank You

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