During clathrin-mediated endocytosis in eukaryotes, actin assembly is required to overcome large membrane tension and turgor pressure. However, the molecular mechanisms by which the actin machinery adapts to varying membrane tension remain unknown. In addition, how cells reduce their membrane tension when they are challenged by hypotonic shocks remains unclear. We used quantitative microscopy to demonstrate that cells rapidly reduce their membrane tension using three parallel mechanisms. In addition to using their cell wall for mechanical protection, yeast cells disassemble eisosomes to buffer moderate changes in membrane tension on a minute time scale. Meanwhile, a temporary reduction of the rate of endocytosis for 2 to 6 minutes, and an increase in the rate of exocytosis for at least 5 minutes allow cells to add large pools of membrane to the plasma membrane. We built on these results to submit the cells to abrupt increases in membrane tension and determine that the endocytic actin machinery of fission yeast cells rapidly adapts to perform clathrin-mediated endocytosis. Our study sheds light on the tight connection between membrane tension regulation, endocytosis and exocytosis.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Julien Berro
- Julien Berro
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Christophe Lamaze, Institut Curie
© 2021, Lemière et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.
Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.