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Junctional cleft [Ca²?]i measurements using novel cleft-targeted Ca²? sensors.

ABSTRACT: RATIONALE:Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. OBJECTIVE:To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. METHODS AND RESULTS:We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. CONCLUSIONS:We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.

SUBMITTER: Despa S

PROVIDER: S-EPMC4132890 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.

Despa Sanda S Shui Bo B Bossuyt Julie J Lang Di D Kotlikoff Michael I MI Bers Donald M DM

Circulation research 20140528 3

<h4>Rationale</h4>Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed.<h4>Objective</h4>To constr ...[more]

PMID: 24871564

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Project description:Flagella and cilia are cellular appendages that inherit essential functions of microbial life including sensing and navigating the environment. In order to propel a swimming microorganism they displace the surrounding fluid by means of periodic motions, while precisely timed modulations of their beating patterns enable the cell to steer towards or away from specific locations. Characterizing the dynamic forces, however, is challenging and typically relies on indirect experimental approaches. Here, we present direct in vivo measurements of the dynamic forces of motile Chlamydomonas reinhardtii cells in controlled environments. The experiments are based on partially aspirating a living microorganism at the tip of a micropipette force sensor and optically recording the micropipette's position fluctuations with high temporal and sub-pixel spatial resolution. Spectral signal analysis allows for isolating the cell-generated dynamic forces caused by the periodic motion of the flagella from background noise. We provide an analytic, elasto-hydrodynamic model for the micropipette force sensor and describe how to obtain the micropipette's full frequency response function from a dynamic force calibration. Using this approach, we measure the amplitude of the oscillatory forces during the swimming activity of individual Chlamydomonas reinhardtii cells of 26 ± 5 pN, resulting from the coordinated flagellar beating with a frequency of 49 ± 5 Hz. This dynamic micropipette force sensor technique generalizes the applicability of micropipettes as force sensors from static to dynamic force measurements, yielding a force sensitivity in the piconewton range. In addition to measurements in bulk liquid environment, we study the dynamic forces of the biflagellated microswimmer in the vicinity of a solid/liquid interface. As we gradually decrease the distance of the swimming microbe to the interface, we measure a significantly enhanced force transduction at distances larger than the maximum extent of the beating flagella, highlighting the importance of hydrodynamic interactions for scenarios in which flagellated microorganisms encounter surfaces.

Dataset Information

Junctional cleft [Ca²?]i measurements using novel cleft-targeted Ca²? sensors.

Publications

Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.

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