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 themselves to the egg by regulating ciliary motility in response to a concentration gradient of attractants released from the egg and/or surrounding cells. However, the structural components of spermatozoid cilia in the land plants are largely unknown. In this study, we investigated MpCAFA (combined CAPS and FAP115; Mp1g04120) in the liverwort Marchantia polymorpha . The N - terminal and near C-terminal regions of MpCAFA show similarity to calcyphosine (CAPS), a mammalian EF-hand protein, and to FAP115, a ciliary protein of the green alga Chlamydomonas reinhardtii , respectively. Mp CAFA is expressed specifically in antheridia, and its orthologs are found in some algae, bryophytes, pteridophytes and cycads, but not in most of seed plants. Spermatozoids of mutants that lack functional MpCAFA exhibited significant decrease in swimming speed. Notably, these mutants showed no obvious morphological defect, including the 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. Introduction of Mp CAFA pro :Mp CAFA - mCitrine fully complemented the mutant phenotype and revealed MpCAFA-mCitrine was localized along the entire length of the two spermatozoid cilia. Both the CAPS-like and FAP115-like regions 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 to visualize spermatozoid ciliary movement.
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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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