FRET‐based genetically encoded sensors allow high‐resolution live cell imaging of Ca<sup>2+</sup> dynamics

Krebs, Melanie; Held, Katrin; Binder, Andreas; Hashimoto, Kenji; Herder, Griet Den; Parniske, Martin; Kudla, Jörg; Schumacher, Karin

doi:10.1111/j.1365-313x.2011.04780.x

Cited by 241 publications

(284 citation statements)

References 72 publications

(83 reference statements)

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“…Also distinct from a typical signal peptide, TTS does not serve as a transmemrbane helix, can work in the middle or in the C-terminus of the protein, and would not be cleaved. Such sequences would be a useful tool to target other proteins to the tonoplast in genetic engineering processes [32]. Future work will be required to fully characterize this elusive pathway for protein trafficking in plant cells.…”

Section: Discussionmentioning

confidence: 99%

“…Both types of YFP-TTS fusions were associated with vacuolar membrane, similar to the N-terminal fusion of CBL2-YFP, suggesting that TTS works regardless of its location in the protein (Supplementary information, Figure S4). Interestingly, we further found that TTS was capable of directing different soluble proteins to the vacuolar membrane [32] (Supplementary information, Figure S5A), but it failed to change the subcellular localization of proteins with multiple transmembrane domains (Supplementary information, Figure S5B). CBL2 and CBL3 proteins both bind Ca 2+ Although deduced amino acid sequence and structural analysis suggest that all CBL proteins including CBL2 and CBL3 contain putative EF-hand motifs that contribute to calcium binding [10,33], experimental evidence for the calcium-binding property of CBL2 and CBL3 is limited.…”

Section: Cbl2 and Cbl3 Exhibit Overlapping But Distinct Expression Pamentioning

confidence: 99%

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Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

et al. 2012

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Plant responses to developmental and environmental cues are often mediated by calcium (Ca 2+ ) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H + -ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.

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Section: Discussionmentioning

confidence: 99%

Section: Cbl2 and Cbl3 Exhibit Overlapping But Distinct Expression Pamentioning

confidence: 99%

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

et al. 2012

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“…To obtain a specific measure of free Ca 2+ concentration in the mitochondrial matrix in planta, we used the genetically encoded sensor Yellow Cameleon 3.6 (YC3.6; Nagai et al, 2004;Palmer and Tsien, 2006;Krebs et al, 2012), which was recently adapted for dynamic measurement of Ca 2+ dynamics in the mitochondrial matrix of Arabidopsis (Loro et al, 2012;Wagner et al, 2015). Reversible Ca 2+ binding by the sensor induces a conformational change that can be detected by a change in the Förster resonance energy transfer (FRET) efficiency between the two fluorophores that are part of the probe (ECFP and cpVenus) such that the FRET ratio (cpVenus:ECFP) increases with increasing free Ca 2+ concentration, thereby providing a quantitative readout.…”

Section: Generation Of Sensor Lines For In Vivo Measurements Of Matrimentioning

confidence: 99%

The EF-Hand Ca²⁺ Binding Protein MICU Choreographs Mitochondrial Ca²⁺ Dynamics in Arabidopsis

et al. 2015

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Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca 2+ signaling may play a central role in this process. Free Ca 2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca 2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca 2+ uniporter machinery in mammals. MICU binds Ca 2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca 2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca 2+ in the matrix. Furthermore, Ca 2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca 2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca 2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca 2+ uptake by moderating influx, thereby shaping Ca 2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca 2+ signaling in plants.

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“…Such studies trace back historically to 3D reconstructions of fixed images at sequential time points, which were used to understand basic subcellular architecture in plants (Donohoe et al, 2013, Staehelin, 1997. Live imaging of FP lines now permits experimental studies of whole organ growth, interactions among neighboring cells, behavior of individual cytoskeletal elements and the ability to trace movement of proteins between cells (Grossmann et al, 2012, Gutierrez et al, 2009, Krebs et al, 2012, Lindeboom et al, 2013, Mathur et al, 2012, Sampathkumar et al, 2011, Teh et al, 2013. With the advent of microfluidic devices, FP markers can be used as biosensors for hormone induction studies during whole root or shoot growth.…”

Section: Live Cell Imaging Using Fluorescent Protein Markersmentioning

confidence: 99%

Fluorescent protein marker lines in maize: generation and applications

Wu¹,

Luo²,

Zadrozny³

et al. 2013

Int. J. Dev. Biol.

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Fluorescent proteins (FP) have significantly impacted the way that we study plants in the past two decades. In the post-genomics era, these FP tools are in higher demand by plant scientists for studying the dynamics of protein localization, function, and interactions, and to translate sequence information to biological knowledge that can benefit humans. Although FP tools have been widely used in the model plant Arabidopsis, few FP resources have been developed for maize, one of the most important food crops worldwide, and an ideal species for genetic and developmental biology research. In an effort to provide the maize and cereals research communities with a comprehensive set of FP resources for different purposes of study, we generated more than 100 stable transformed maize FP marker lines, which mark most compartments in maize cells with different FPs. Additionally, we are generating driver and reporter lines, based on the principle of the pOp-LhG4 transactivation system, allowing specific expression or mis-expression of any gene of interest to precisely study protein functions. These marker lines can be used not only for static protein localization studies, but will be useful for studying protein dynamics and interactions using kinetic microscopy methods, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET). All of the constructs and maize marker lines are publicly available through our website, http://maize. jcvi.org/cellgenomics/index.php

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FRET‐based genetically encoded sensors allow high‐resolution live cell imaging of Ca²⁺ dynamics

Cited by 241 publications

References 72 publications

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

The EF-Hand Ca²⁺ Binding Protein MICU Choreographs Mitochondrial Ca²⁺ Dynamics in Arabidopsis

Fluorescent protein marker lines in maize: generation and applications

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FRET‐based genetically encoded sensors allow high‐resolution live cell imaging of Ca2+ dynamics

Cited by 241 publications

References 72 publications

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Fluorescent protein marker lines in maize: generation and applications

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Product

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FRET‐based genetically encoded sensors allow high‐resolution live cell imaging of Ca²⁺ dynamics

The EF-Hand Ca²⁺ Binding Protein MICU Choreographs Mitochondrial Ca²⁺ Dynamics in Arabidopsis