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. 2018 Apr 17;9:1504. doi: 10.1038/s41467-018-03719-6

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Schematic summary and sensor performance for GCaMP and GCaMP-X. a GCaMP defects and advantages of GCaMP-X. In neurons, GCaMP sensors behave as CaM-like proteins perturbing Ca_V1 gating and distorting Ca²⁺ dynamics (1). The abnormal enhancement of Ca_V1 by cytosolic GCaMP underlies overgrown neurites. Different from endogenous CaM, GCaMP itself could no longer properly translocate into the nucleus in response to membrane excitation, which also dominant-negatively impairs the acute mobility of endogenous CaM. Once present in the nucleus (by accumulation or NLS), nuclear GCaMP perturbs transcription signaling and gene expression potentially through its aberrant CaM motif, impairing general health of neurons including Ca²⁺ signals (reflected as abnormal sensor readouts) and neurite outgrowth (2). The new GCaMP-X sensors are designed with apoCaM protection and explicit localization (e.g., GCaMP-X_C for cytosolic Ca²⁺) to eliminate GCaMP perturbations on Ca_V1 gating (3) and signaling (4). b Sensor performance of GCaMP-X_C validated by single-AP like Ca²⁺ transients in HEK293 cells. Briefly, glass electrodes formed the Giga-Ohm seal with cell membrane (resting); then 3 ms ZAP for break-in generated a rapid Ca²⁺ influx due to transient membrane rupture, immediately followed by a fast decay arising from strong Ca²⁺ chelators of 10 mM BAPTA included in the pipette solution. Key indices of peak ΔF/F₀, rise time t_r, decay time t_d and SNR were compared between GCaMP6m and GCaMP6m-X_C. Epi-fluorescence images were acquired at the sampling rate of 100 Hz or higher. c–e Sensor performance of GCaMP-X_C validated with mechanosensing outer hair cells. Cells were transfected with GCaMP6m or GCaMP6m-X_C at P1 by electroporation, and cultured for 1 day (DIV 1) or 3 days (DIV 3). Fluid-jet pulses of 100, 300 or 500 ms were applied to cells (c). Representative images, normalized fluorescence changes (ΔF/F₀) indicative of Ca²⁺ dynamics and statistical summary of peak ΔF/F₀ were compared between DIV 1 (d) and DIV 3 (e) cells expressing GCaMP6m or GCaM6m-X_C. Standard error of the mean (S.E.M.) and Student’s t-test (two-tailed unpaired with criteria of significance: *p < 0.05; **p < 0.01, and ***p < 0.001) were calculated when applicable, and n.s. denotes “not significant”