Abstract
Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.
Highlights
All five variants could be expressed, and photoconversion was detectable after a 15-s treatment with 100 μM histamine and 4 s of 405 nm photoconversion light (Figure 3)
CaMPARI2-F391W and CaMPARI2, on the other hand, showed high photoconversion rates with 65% and 55%, respectively, without reaching saturation. In nonelicited cells, these variants displayed 13% and 2% photoconversion rates, respectively (PreParabola condition, Figure 4), indicating an already high sensitivity to basal calcium. These results are consistent with published basal cytosolic Ca2+ concentration in mammalian chondrocytes between 100 and 200 nM [67]
Similar results were visible for the CaMPARI2 construct, albeit the base conversion rate was much lower, corresponding to the results described in Figure 4
Summary
Signaling components integrate chemical and physical stimuli to drive developmental and creativecommons.org/licenses/by/ 4.0/). Among these processes are Ca2+ -dependent neurotransmitter release, muscle contraction, or the biological and biochemical processes downstream of membrane receptors, as in the IP3 pathways, which play a role in regulating cell division [1,2,3]. Ca2+ signals vary in their amplitude, temporal and spatial properties, and site of Ca2+. Ca2+ -transporters such as Ca2+ -ATPases ensure a stable cytosolic Ca2+ concentration by pumping Ca2+ ions into the extracellular space or intracellular stores, mainly the endoplasmic reticulum (ER), from where they can be released into the cytosol upon signal activation [3]
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