Complexity and Versatility of Calcium Signaling and Dynamics

The calcium ion is a universal second messenger mediating a wide range of dynamic cellular functions, such as exocytosis, contraction, transcription, and proliferation. A hallmark of calcium signaling is the transient pulsing dynamics of cytosolic calcium concentration, which is versatile tool regulating cellular processes. The aim of this thesis is to study the calcium signals and dynamics primarily related to mechanotransduction within rod photoreceptors, primary hippocampal neurons and glioblastoma (GBM) cells. To study calcium dynamics in rods of Xenopus laevis, which are composed of an inner segment, IS and an outer segment, OS (IS+OS), we used the fluorescent calcium dye CaSiR-1 AM working in the near-infrared (NIR) (excitation at 650 and emission at 664 nm), which allowed us investigate calcium dynamics with an unprecedented accuracy and most importantly in semi dark-adapted conditions. The experiments showed: there are spontaneous calcium flares in functional OSs, and these flares are highly localized and are more pronounced at the OS tip; a bright flash of light at 488 nm induces a drop in intracellular calcium concentration at the OS base but often a flare at the OS tip. We also have investigated mechanosensitivity in the rods by combining electrophysiology, optical tweezers (OTs),and biochemistry, and we show that mechanical stimulation of the order of 10 pN applied briefly to either the OS or IS evokes calcium transients. The pN force evoked calcium transients can be inhibited by application of GsMTx-4 whereas spontaneous calcium flares can be seen at tip of OS. In primary hippocampal neurons, we employed an Oscillatory Optical Tweezers (OOT) to exert a local indentation with pN forces. We found that single local indentation evokes a transient intracellular calcium change, whereas repeated mechanical stimulations induce a more sustained and variable calcium response, which can be inhibited by the GsMTx-4 treatment. Moreover, we observed a mechanically evoked activation of the CaMKII and small G protein RhoA. Lastly, with glioblastoma cells, we investigated the calcium flares and its coupling with chloride dynamics in both flat and round GBM cells undergoing the mitosis primarily by using multi-channels imaging of calcium and chloride, ratiometric calcium imaging. The preliminary data showed that in normal flat cells, global calcium flares can last stably for at least one hour without significantly changing the morphology of the cell, and more importantly are able to activate chloride signals which can be unexpectedly localized. And within round cells undergoing mitosis, instead of finding calcium flares at the initial phase before the cell divides into two, calcium flares were frequently seen at the end of mitosis, which can be further increased by Piezo 1 agonist Yoda 1 to activate favorable chloride influx signals.