The Mp CAFA gene encodes a ciliary protein required for spermatozoid motility in the liverwort Marchantia polymorpha
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Abstract
Bryophytes, pteridophytes, and some gymnosperm species produce motile ciliated spermatozoids that navigate to the egg by regulating ciliary motility in response to a concentration gradient of attractants released from the egg and/or the surrounding cells. However, the structural components of spermatozoid cilia in land plants remain largely unknown. In this study, we investigated MpCAFA (combined calcyphosine [ CA PS] with flagellar-associated protein 115 [ FA P115]; Mp1g04120) in the liverwort Marchantia polymorpha . The N - terminal and near C-terminal regions of MpCAFA showed similarity to CAPS, a mammalian EF-hand protein, and FAP115, a ciliary protein of the green alga Chlamydomonas reinhardtii , respectively. Mp CAFA was expressed specifically in antheridia and its orthologs were found in some algae, bryophytes, pteridophytes, and some gymnosperms, but not in most seed plants. Spermatozoids from mutants lacking functional MpCAFA exhibited a significant decrease in swimming speed. Notably, these mutants showed no obvious morphological defects, including a 9 + 2 axoneme arrangement, and retained chemotactic capability and fertility, forming normal spores. This suggests that MpCAFA is required for spermatozoid motility, but not for sperm chemotaxis or subsequent reproductive processes. The introduction of Mp CAFA pro :Mp CAFA - mCitrine fully complemented the mutant phenotype and revealed that MpCAFA-mCitrine was localized along the lengths of the two spermatozoid cilia. Both the CAPS-like and FAP115-like domains were essential for MpCAFA function and subcellular localization in spermatozoid, whereas the C-terminal proline-rich region was dispensable. These findings indicate that MpCAFA is a major ciliary protein in land plants and can serve as a marker for visualizing spermatozoid ciliary movements.
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In this study, we identified MpCAFA, a protein that functions in spermatozoid motility. Our results
Very cool work on MpCAFA's role in liverwort spermatazoid motility! This is a really neat and mysterious protein. It will be very interesting to see if both CAPS and FAP115 domains are both essential in contributing the ciliary rigidity. Nice work!
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While the swimmingdirection of most wild-type spermatozoids was predominantly straight, Mpcafage spermatozoids frequentlyexhibited circular trajectories or increased swing width (Fig. 7a). We examined two aspects of spermatozoidswimming behavior: swimming speed and linearity (see Materials and Methods).
Wow! The motility phenotype is very striking! It appears the WT and mutant spermatozoids have a similar number of cells in the field of view but the m-Citrine recovery strain has a lot more cells in the background. Were the sample densities normalized before imaging? You mentioned they perform chemotaxis so I'm curious if the recovery could be influenced by cell density. Regardless, it is a very clear and convincing recovery
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Morphologicallynormal spermatozoids were released from all male Mpcafage plants upon the application of water to theirreceptacles (Fig. 6ab), and the cilia of spermatozoids from Mpcafage retained the typical 9+2 arrangement (Fig.6cd), indicating that MpCAFA is not essential for normal spermatozoid development.
I appreciate how you've shown that the mutant spermatozoids maintain normal morphology and axonemal structure despite their motility defects. I'm curious about the sample size that informed this conclusion - approximately how many spermatozoids did you analyze by phase-contrast microscopy, and how many axonemal cross-sections were examined by TEM? This information would help contextualize the robustness of the structural assessment, particularly since subtle ultrastructural differences might potentially contribute to the …
Morphologicallynormal spermatozoids were released from all male Mpcafage plants upon the application of water to theirreceptacles (Fig. 6ab), and the cilia of spermatozoids from Mpcafage retained the typical 9+2 arrangement (Fig.6cd), indicating that MpCAFA is not essential for normal spermatozoid development.
I appreciate how you've shown that the mutant spermatozoids maintain normal morphology and axonemal structure despite their motility defects. I'm curious about the sample size that informed this conclusion - approximately how many spermatozoids did you analyze by phase-contrast microscopy, and how many axonemal cross-sections were examined by TEM? This information would help contextualize the robustness of the structural assessment, particularly since subtle ultrastructural differences might potentially contribute to the observed swimming phenotype.
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