Real Time Imaging Of Intracellular Store Ca²⁺ Release

Calcium ions are ubiquitous second messengers in cells and are involved in the secretion of neurotransmitters in neurons.Cytoplasmic Ca²⁺transients in neurons are mediated by extracellular Ca²⁺influx via calcium channels on the cell membrane,or Ca²⁺-mediated release from intracellular Ca²⁺stores such as ER,mitochondria,endolysosomes,and secretory vesicles.Despite the current advances in Ca2+imaging,the"fast""calcium signal and slow""off""feature requirements make it challenging to directly observe Ca²⁺release from individual calcium stores.In this study,the intracellular Ca²⁺imaging method was innovated to obtain super-fast and high-resolution local intracellular calcium signals,providing a fast and efficient new research method for intracellular calcium signal research.?1?We used the LAMP-EGFP as lysosome indicator,EGTA as intracellular calcium ion chelating agent,Rhod2 for calcium indicator,and the Confocal line-scan mode,realized the calcium release real-time imaging mediated lysosome of TRPA1.The calcium signal is not dependent on the extracellular calcium ions,but the GPN can completely block the TRPA1 was mediated partial calcium signals,shows that this method can be real-time detection of intracellular calcium lysosome release release;?2?In the hippocampus neurons,we used Mitotracker green as mitochondria indicator,in a similar pattern of Confocal line scan,we real-time monitoring to the mitochondrial calcium release,FCCP mediated the calcium release can occur in the neuron cell body area,but also in neuronal axons and dendrites and synapses,empty mitochondrial calcium library can block FCCP mediated partial calcium signals;?3?We using EGTA as intracellular calcium ion chelating agent,Rhod2 for calcium indicator,using Confocal scanning mode,we also observed that high extracellular calcium ions caused by potassium stimulate internal flow signals,TRPA1 mediated extracellular calcium partial calcium signals caused by internal flow,etc.,removal of extracellular calcium ions can completely block the partial calcium signals,shows that our research method is not only suitable for intracellular stores calcium release,also can be applied to calcium ion channels located in the cell membrane.In this study,we skillfully adopted the Confocal line scan mode to achieve the direct visualization of local calcium signals with millisecond accuracy of different types and single organelles at the level of living cell organelles.It is reasonable to believe that this method can be extended to almost all intracellular calcium stores.At the same time,different calcium secretion patterns were found in different individual mitochondria.During the scanning process,suggested that the same organelle calcium pool also has different regulation modes and response pathways,which need to be further studied.In addition,the interference effect of fluorescence energy transfer?FRET?effect in high time-space resolution real-time quantitative imaging was found,laying a foundation for further optimization and reform of imaging equipment and other related fields.This method can be used to screen out unknown calcium pool or calcium flow area in cells.In a word,this method provides a more convenient and rapid technical means for calcium imaging,and has a great development and application prospect,which is worthy of further promotion.