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Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii . Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo . Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.
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Exactly, as UVM4 has no cell wall, it is pretty easy to do genome editing. that is, autolysin is not needed to add before transformation. Maybe this strain is also nice to test the efficiency of gRNA on the genome editing.
For you second question, I don't think so. based on two evidences. 1) from the genome sequencing data of UVM4 (Neupert et al., 2020), we did see any mutation of this gene; 2) according our proteolytic activity test, UVM4 has a metacaspase activity while the mutants have no activity (this work).
Exactly as you say, we use this strain to have a better expression of mVenus, even though the expression efficiency, the transformants that have a high mVenus expression, is still much lower than UVM4 strain. actually, CC-4533 or CC-5325 has intact cell wall but maybe reduced thickness (Zhang et al., 2022).
It is interesting to know the survival rate of CrMCA-II KO line in WT line under heat stress. Unfortunately, we still do not have KO line in WT background.
So far, we have not check. on the other hand, there are more than three insertion of the vector fragment (by qPCR check) in this strain, it may be not a good strain for checking this phenotype.
You are right. These two strains have slight different tolerance against heat, maybe due to the strain difference. In our genome editing process, we cannot rule out the possibility of off-target, which may have some slight contribution to heat stress. So we confirm the phenotype by complemented line.
After transformation into UVM4 cells (Neupert et al., 2009), we obtained a strain overexpressing CrMCA-II with a C-terminal mVenus tag, that we named CrMCA-II-overexpressor 14-3 (OE14-3).
Are these over-expression cells more resistant to HS than WT cells?
PM localization of CrMCA-II-mVenus was confirmed using strain CC-4533, distinguished from UVM4 by the presence of both an intact cell wall and flagella (Supplemental Figure S12), suggesting that this localization is a strain-independent characteristic of Chlamydomonas.
Thank you for including this! Its nice to see such clear membrane localization in a more "normal" cell line with flagella! While this does have a cell wall, its still a cw15 mutant. I'm guessing this was necessary to get sufficient mVenus expression? I'd be super interested to see if this strain (and a fully intact cell wall strain) with MCA-II deletions are able to survive after HS
We found that the mutant strains had a higher frequency of cell death than the UVM4 strain (Figure 4A)
Very interesting and convincing phenotype. Do you think the lack of a cell wall contributes to the amount of cell death? If this experiment were done in cells with cell walls, would you miss out on this observation?
Also, why do you think ii-23 is so much more susceptible to heat than ii-9, since they're essentially the same strain, right?
A Crispr/Cas9 based targeted insertional mutagenesis approach (Picariello et al., 2020) was employed to knockout the CrMCA-II gene.
This was done on the the uvm4 strain, correct? Just confirming since the Picariello et al 2020 paper doesn't use uvm4. Have you found this Crispr/Cas9 protocol to be more effective on the uvm4 strain, compared to the strains used in Picariello et al 2020?
If so, were the CrMCA genes effected by the UV mutagenesis used to generate the uvm4 strain?