Optimizing precision nanoparticle delivery for magneto-mechanically-based calcium modulation
Optimizing precision nanoparticle delivery for magneto-mechanically-based calcium modulation
复制标题
优化基于磁机械的钙调制的精密纳米颗粒输送
DOI：
10.1016/j.bpj.2022.11.1375
复制
发表时间：
2023
影响因子：
3.4
通讯作者：
Kunze, Anja
中科院分区：
文献类型：
--
作者：
Beck, Connor L.;Kunze, Anja
Monday, February 20, 2023 233a functionalized MNPs (incubated for 4 h and 24 h) over 12 min, where a magnetic field actuated 40-80 pN forces on the MNPs from 4 min to 8 min. We decomposed the mean somatic calcium signals into a resting concentration (lower envelope) and calcium events (magnitude and frequency) and correlated them with the Sørenson-Dice. All data was compared with the Mann-Whitney U test (N= 4 cultures, single neuron cells: control= 2262, 4 h= 2424, 24= 1062). Our results showed that both incubation times provided increases in resting concentration and calcium event frequency and decreased event magnitude and synchronous correlation in comparison to the control. Although similar, we observed that the 4 h incubation time increased the calcium event response with higher frequency and decreased event magnitudes and synchronous correlation when compared to the 24 h incubation. Altogether, our results show that shorter MNPs incubation times can make calcium influx more precise and potentially overcome adverse effects stemming from protein turnover.1140-Pos Photo-inactivation disrupts rapid fluorescence readout of jGCaMP8 calcium indicators tethered to Orai1 channels Joseph L. Dynes, Andriy V. Yeromin, Michael D. Cahalan. Physiology and Biophysics, University of California Irvine, Irvine, CA, USA. We sought to optimize indicator response time in channel-tethered genetically encoded Ca2þ indicators (GECIs) using the jGCaMP8 series indicators released by the HHMI Genie Project. These indicators possess< 10 ms rise times, substantially narrowing the temporal resolution gap between electrical and optical recording of Ca2þ currents. DNA encoding jGCaMP8f was inserted downstream of the Orai1 coding sequence to monitor Orai1 Ca2þ channel activity using TIRF microscopy combined with whole-cell recording. The use of non-inactivating channel currents provided a steady and electrically controlled source of Ca2þ to compare current and fluorescence readouts of Orai1 channel activity in transfected HEK 293A cells. Non-inactivating test pulses to À40 mV produced fluorescence traces that unexpectedly declined by $25% over 100 ms before reaching a stable plateau. Moreover, using an Orai1 Y80E mutant that lacks fast Ca2þ-dependent inactivation, fluorescence traces declined by $50% at À100 mV. Testing of Orai1-jGCaMP8f using unroofed cells demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photo-inactivation spontaneously recovered over 5 minutes, and recovery was accelerated in the absence of Ca2þ. Mutational analysis of residues near the tri-peptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased ($90%) photo-inactivation reminiscent of fluorescent protein fluorophore cis-trans photo-switching. Indeed, 405 nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photorecovery, and simultaneous illumination with 405 and 488 nm light blocked photo-inactivation. Subsequent mutagenesis produced a variant that lacks photo-inactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2þ signals using jGCaMP8 indicators, suggest strategies to avoid photoswitching, and serve as a starting point to produce more photo-stable, and thus more accurate, GECI derivatives.
