Spatiotemporal Characteristics of Intercellular Calcium Wave Communication in Micropatterned Assemblies of Single Cells

Abstract

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 μm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity.

Stimulation of Local Cytosolic Calcium Release by Photothermal Heating for Studying Intra‐ and Intercellular Calcium Waves

“…Thus, the most likely reason is intercellular communication by secreted chemical messenger molecules. Indeed, it is known that cells may secrete ATP upon stimulation, [ 31a,35 ] which acts as a paracrine transmitter of intercellular Ca 2+ waves to excite increased [Ca 2+ ] i in other cells, [ 36 ] which has been also demonstrated here in the above described control experiments, cf. Figures S22 and S23, Supporting Information.…”

“…This can be attributed to the diffusion of released free Ca 2+ , or Ca 2+ induced Ca 2+ release [ 30 ] in other part of the cell, or mixed effects. The decay of [Ca 2+ ] i of a stimulated cell to its baseline has been reported to happen within 1 min, [ 31 ] which was much slower in our results. Of note, the photobleaching effect under continuous imaging can be ignored by using the LSM880 set‐up due to its low excitation power (Table S1, Supporting Information).…”

“…For those cells with a similar distance to the excited cell, the response was first detected in cells that were located opposite to the excited region, in agreement with other reports. [ 31a ] We also noticed that the part of responding cells close to the irradiated cells became bright first, and later fluorescence became homogeneous in the whole cells, as indicated by the red arrow in Figure 3a and Figure S41a, Supporting Information. Thus, the most likely reason is intercellular communication by secreted chemical messenger molecules.…”

Embryos assist morphogenesis of others through calcium and ATP signaling mechanisms in collective teratogen resistance

“…Studies have shown calcium and ATP acting in a range of different communication pathways, including injury response and intercellular communication 89 – 94 . Calcium waves in particular have been shown to act as communicators for damage and regeneration 93 , 95 – 97 , suggesting these as potential candidates for inter-embryo signaling mechanisms for CEMA. We made use of a loss-of-function reagent known to block ATP signaling via the P2X/P2Y receptors: the purinergic receptor type 2 antagonist suramin 98 – 101 to see whether it would prevent the CEMA effect and render large groups as susceptible to defects as small ones 93 , 94 , 102 .…”

Mesenchymal Migration on Adhesive–Nonadhesive Alternate Surfaces in Macrophages

“…Fabrication of Micropatterned Substrate: Micropatterned substrates were prepared as previously described. [48] In brief, glass substrates (𝜑 = 25 mm) were immersed in chromic acid lotion for thorough cleaning and then dried at 80 °C for 20 min. Afterward, they were put into a vacuum desiccator containing hexamethyldisilazane (HMDS, Sigma, USA) for vapor phase deposition for at least 30 min.…”