Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform

Abstract

Microfluidics-based protoplast isolation, which enabled intracellular uptake of singlet oxygen sensor fluorophore and DNA oligonucleotides in Symbiodinium spp.

Characterization of the Flash-Induced Fluorescence Wave Phenomenon in the Coral Endosymbiont Algae, Symbiodiniaceae

“…However, it is possible that these inhibitors do not penetrate intact Symbiodinium cells due to the presence of a thick cell wall, as was previously suggested for antimycin A [15]. Therefore, in order to test these inhibitors in the absence of the cell wall, protoplasts were prepared by digestion of the cell wall using cellulase, applying the modified protocol of Bashir et al 2022 [37]. As the protoplast preparation is a considerable stress to the cells, the cell wall digestion procedure that was applied previously (i.e., a complete cell wall digestion with 4% cellulose) significantly affected the fluorescence relaxation, and the wave disappeared under these conditions (Supplementary Figure S2).…”

“…However, the cell wall might be impermeable for these inhibitors, therefore, their effect cannot be judged in intact cells. To avoid the hinderance of the cell wall in the uptake of antimycin A and polymyxin B, the cell wall was removed using enzymatic digestion with cellulase [37,43]. In cells with a partially digested cell wall, polymyxin B blocked the wave phenomenon and slowed down the P700 + re-reduction kinetics, whereas the inhibitor of PGR5, antimycin A, did not have considerable effect.…”

“…Protoplasts were prepared using a modified protocol according to [37] (sorbitol was avoided and the cellulase concentration was reduced to 2%, and incubation time was shortened to 2 h) to allow partial digestion of the cell wall, which better retained the properties of fluorescence relaxation in Symbiodiniaceae.…”

Monitoring the photosynthetic activity at single-cell level in Haematococcus lacustris

“…The microfluidic chamber was fabricated from PDMS (polydimethylsiloxane) and bound on the cover slip. According to the cell size, an array of U-shaped traps was created in the chamber with the dimensions of 40-µm width and 50-µm length; the traps had 10-µm wall thickness with 5-µm gap; the chamber had inlet and outlet holes (Bashir et al 2022).…”

“…However, the physiological and morphological changes along with the Chl fluorescence characteristics on individually trapped cells over the time course of the carotenogenic process (and its reverse reaction) have not been monitored so far. We aimed to monitor simultaneously the morphological and photosynthetic activity changes at the single-cell level of Haematococcus by using the combination of microfluidics and variable Chl fluorescence imaging, which had been successfully applied on unicellular algae such as the widely used model organism Chlamydomonas reinhardtii (Széles et al 2022(Széles et al , 2023 and the important coral symbiont Symbiodinium (Bashir et al 2022). Chl fluorescence kinetics gives important information about the PSII-PSI intersystem electron transfer regulation.…”