Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano

Abstract

We report a new set of ultra-sensitive Ca 2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca 2+ -sensing domain of a genetically encoded Ca 2+ indicator, YC2.60 or YC3.60. Their high Ca 2+ affinities (K d = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca 2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multi-cellular networks.3To decipher the principles of information processing in multi-cellular networks, such as a brain or developing embryo, it is essential to record cellular activity with fine We therefore generated YCs with a longer linker, containing 3 to 8 amino acids (designated 3 to 8) (Fig. 1a). The Ca 2+ affinity gradually increased as the linker was elongated and we named the resulting series of sensors, high-affinity yellow cameleons or "YC-Nano". For the 3 (Gly-Gly-Ser) linker the K d was 50 nM (YC-Nano50) and for the 4 (Gly-Gly-Gly-Ser) linker the K d was 30 nM (YC-Nano30). YCs with a 5 to 8 5 linker also had a higher affinity than YC2.60. The lowest K d (= 15 nM) was achieved with a 5 linker (Gly-Gly-Gly-Gly-Ser) (YC-Nano15); this indicator had the highest affinity of any GECI reported so far 11 (Fig. 1b, Supplementary Table 1 and Supplementary Fig. 1). Linker elongation was also effective for YC3.60, yielding YC-Nano140 (4; Gly-Gly-Gly-Ser, K d = 140 nM) and YC-Nano65 (5;Gly-Gly-Gly-Gly-Ser, K d = 65 nM) (Fig. 1b, Supplementary Fig. 1 and Supplementary Table 1).Kinetic measurement by stopped-flow fluorometry of YC-Nano140 and YC3.60 revealed that only the rate constant for the on reaction was increased, while that of the off reaction remained unchanged (Supplementary Fig. 2 Fig. 3). Supplementary Fig. 4). To verify the advantages of YC-Stimulation with 10 M cAMP yielded large FRET signal changes, assessed by ratiometric wide-field imaging of aggregation-competent cells (Fig. 1c). The YFP/CFP for the YC-Nano15-and YC2.60-expressing cells changed from 5.0 to 9.5 (R = 4.5) and 2.0 to 6.2 (R = 4.2), respectively. We next determined the amplitude of the Ca The increased signal strength achieved by optimizing the K d also allowed us to perform Ca 2+ imaging on a large spatial scale. The field of view for imaging could be expanded to a millimeter-sized network that included 100,000-Dictyostelium cells, in which the aggregation wave was clearly visible as a rotating spiral (Supplementary Fig. 5, Supplementary Video 2, R Nano15 = 0.6), indicating that YC-Nano15, unlike YC2.60, was useful for detecting multi-cellular network activity in self-organized signaling dynamics (R YC2.60 = 0.15, Supplementary Fig. 6, Supplementary Video 3).We next tested the performance of YC-Nano in a neuronal system (Supplementary Note 3 and Supplementary Figure 7). For this, we examined YC-Nano's sensitivity for the subtle Ca 2+ transient triggered by a single action potential (AP). YC-Nano15 and 7 YC3...

Behaviour-dependent recruitment of long-range projection neurons in somatosensory cortex

“…For the construction of the pAAV-EF1a-YCNano140 viral construct, a YC-Nano140 insert with 59 BamHI and 39 EcoRI restriction sites was generated by polymerase chain reaction (PCR) amplification from a pcDNA3-YC-Nano140 vector 8 and subcloned into an pAAV-EF1a-dioeYFP plasmid 31 . The pAAV-pgk-Cre construct has been described previously 3 .…”

“…2b-f). For calcium imaging, we injected into S1 an AAV6 expressing yellow-cameleon Nano140 (YC-Nano140) 8 , a sensitive genetically encoded calcium indicator that reliably reports actionpotential firing ( Supplementary Fig. 3).…”

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

“…FRET sensors include yellow cameleon 3.60 (refs. 16,17), D3cpv 18 , yellow cameleon Nano 19 and TN-XXL 20 .…”

Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane

“…This biosensor, Cameleon-Nano, achieved a higher Ca 2+ affinity, which resulted in approximately a 50-fold reduction in its Kd value (to 15 nM) compared with the classical Cameleon protein. 11 This biosensor can detect fluctuations in the basal calcium concentration in D. discoideum that have never been detected by previous GECIs. Alongside the evolution of Cameleon, an alternative FRET-based calcium sensor has also been developed whose calcium-sensing domain is troponin C, called TN-XXL.…”