Ca2+ Indicators Based on Computationally Redesigned Calmodulin-Peptide Pairs

Palmer, Amy E.; Giacomello, Marta; Kortemme, Tanja; Hires, Samuel Andrew; Lev‐Ram, Varda; Baker, David; Tsien, Roger Y.

doi:10.1016/j.chembiol.2006.03.007

Cited by 457 publications

(483 citation statements)

References 39 publications

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“…D1cpv is a genetically encoded, low affinity, Ca 2+ indicator in which GFP variants, here referred as CFP and YFP, are linked by a modified calmodulin and a calmodulin-binding domain, M13 (18), as in the original cameleon probe (19). Fluorescence resonance energy transfer (FRET) between CFP and YFP is maximal at high [Ca 2+ ] and minimal at low [Ca 2+ ].…”

Section: Resultsmentioning

confidence: 99%

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Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Lissandron

Podini

Pizzo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

116

131

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Taking advantage of a fluorescent Ca 2+ indicator selectively targeted to the trans -Golgi lumen, we here demonstrate that its Ca 2+ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: ( i ) Ca 2+ uptake depends exclusively on the activity of the secretory pathway Ca 2+ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA) is excluded; ( ii ) IP 3 generated by receptor stimulation causes Ca 2+ uptake rather than release; ( iii ) Ca 2+ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans -Golgi Ca 2+ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.

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Section: Resultsmentioning

confidence: 99%

“…Fluorescence resonance energy transfer (FRET) between CFP and YFP is maximal at high [Ca 2+ ] and minimal at low [Ca 2+ ]. Accordingly, the efficiency of FRET, and thus [Ca 2+ ], can be conveniently measured on the basis of the ratio (R) between the intensities of YFP/CFP fluorescence emissions upon selective excitation of CFP (18).…”

Section: Resultsmentioning

confidence: 99%

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Lissandron

Podini

Pizzo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

116

131

View full text Add to dashboard Cite

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“…Endogenous CaM is expected to pose the greatest ''threat'' in this regard because its intracellular concentration can sometimes be Ͼ1 M (31-33). To address this problem, our laboratory has adopted a mutational approach similar to one used recently to modify fluorescent CaM-based calcium sensors (25,34). Second, although the sensors will be targeted to cell cytoplasms, it is possible that some may be trapped in endosomes or other compartments where they cannot function properly.…”

Section: Mri Applications Of Spio-based Calcium Sensorsmentioning

confidence: 99%

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

Atanasijević¹,

Shusteff

Fam

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

227

169

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We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calciumsensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 M Ca 2؉ , suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 M in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens. magnetic resonance ͉ T2 relaxation ͉ signal transduction ͉ molecular imaging ͉ neuroimaging C alcium ions (Ca 2ϩ ) have been a favorite target in molecular imaging studies because of the important role of calcium as a second messenger in cellular signaling pathways. Fluorescent calcium sensors are used widely in optical imaging, both at the cellular level and at the cell population level. Calcium-sensitive dyes have recently been used in conjunction with laser scanning microscopy to follow neural network activity in small, threedimensional brain areas (1) and to characterize patterns of interaction among cells in developing vertebrate embryos (2). Because of the scattering properties of dense tissue, highresolution optical approaches like these are usually limited to superficial regions of specimens and to restricted fields of view (3). To probe calcium dynamics more globally in living systems, a different imaging modality must be used.Magnetic resonance imaging (MRI) is an increasingly accessible technique for imaging opaque subjects at fairly high spatial resolution, and MRI studies of calcium dynamics could, in principle, complement optical approaches by offering both greatly expanded coverage and depth penetration in vivo (4). Calcium isotopes are unsuitable for direct imaging by magnetic resonance, so attempts to sensitize MRI to calcium have focused around the use of molecular imaging agents. Fluorinated derivatives of the bivalent cation chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) have permitted calcium measurements in vivo by 19 F MRI but only at Ϸ10 Ϫ5 the sensitivity of standard MRI methods (5, 6). Two potentially more powerful proton T1 relaxation-promoting contrast agents were subsequently introduced. The paramagnetic ion manganese (Mn 2ϩ ) mimics calcium by entering cells through calcium channels. Because it accumulates much faster than it is removed, Mn 2ϩ produces an ''integral'' of calcium signaling history that can be d...

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“…Moreover, it displays a low-Ca 2+ affinity with a K d value of 60 µM and has proven to be an ideal probe for measuring the Ca 2+ concentration in the endoplasmic reticulum. The binding interface of CaM and M13 was further reengineered by computationally designing complementary bumps and holes (Palmer et al 2006). Another attempt to eliminate the sensitivity of genetically encoded Ca 2+ indicators to endogenous CaM and CaM-binding proteins involved the use of troponin C proteins from skeletal and cardiac muscles (Heim and Griesbeck 2004).…”

Section: Discussionmentioning

confidence: 99%

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Miyawaki¹,

Nagai²,

Mizuno³

2013

Cold Spring Harb Protoc

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Green fluorescent protein (GFP)-based fluorescent indicators for Ca 2+ offer significant promise for monitoring Ca 2+ in previously unexplored organisms, tissues, and submicroscopic environments because they are genetically encoded, function without cofactors, can be targeted to any intracellular location, and are bright enough for single-cell imaging. These probes use simple GFP variants, circularly permuted GFP (cpGFP), in which the amino and carboxyl portions have been interchanged and reconnected by short spacers between the original termini, or pairs of GFP variants that permit fluorescence resonance energy transfer (FRET). Yellow cameleons (YCs) use FRET between cyanand yellow-emitting variants of Aequorea GFP (cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP], respectively). YCs are composed of a linear combination of CFP, calmodulin (CaM), a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and YFP. Binding of Ca 2+ to the CaM moiety of the YC initiates an intramolecular interaction between the CaM and the M13 domains, causing the chimeric protein to shift from an extended conformation to a more compact one, thereby increasing the efficiency of FRET from CFP to YFP. This technique is amenable to emission ratioing, which is more quantitative than single-wavelength monitoring. YC3.60 was engineered to enhance its performance as a fluorescent Ca 2+ indicator and here we describe the use of this cameleon to image rapid changes in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) within HeLa cells. FRET imaging is performed using a laser-scanning confocal microscope. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. ReagentsCa 2+ -free medium For fast and simultaneous acquisition of cpVenus173 and CFP images from HeLa cells expressing YC3.60, this camera, which comprises three charge-coupled device (CCD) chips (RGB: red, green, and blue) and a prism, can be used. The cpVenus173 and CFP images are captured by the G and B chips, respectively. In addition, to improve spatial resolution along the z-axis, a spinning-disk unit (e.g., CSU21 from Yokogawa Electric Corporation) can be placed in front of the camera.Diode-pumped solid-state laser (430 nm) (Melles Griot) Glass-bottomed dishes (35 mm) (Matsunami Glass Ind., Ltd., D111300) Image acquisition software to control the Ashura 3CCD camera (e.g., . These include intensity of the laser, scanning speed, binning, and sensitivity of the camera.In our experience, the intensity of the laser should be attenuated greatly using neutral-density filters for Ca 2+ imaging on a timescale of subseconds. Photobleaching does not happen with a laser that is sufficiently attenuated.6. Observe fluorescence signals from CFP and cp173Venus by the blue and green channels of the 3CCD camera, respectively. 7. Set 4 × 4 binning for high-sensitive fluorescence ...

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Ca2+ Indicators Based on Computationally Redesigned Calmodulin-Peptide Pairs

Cited by 457 publications

References 39 publications

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

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Ca2+ Indicators Based on Computationally Redesigned Calmodulin-Peptide Pairs

Cited by 457 publications

References 39 publications

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

Imaging Intracellular Free Ca2+ Concentration Using Yellow Cameleons

Contact Info

Product

Resources

About

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons