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Annexins (ANXAs), calcium-sensitive phospholipid-binding proteins, are pivotal for cellular membrane repair, which is crucial for eukaryotic cell survival under membrane stress. With their unique trimeric arrangements and crystalline arrays on the membrane surface, ANXA4 and ANXA5 induce membrane curvature and rapidly orchestrate plasma membrane resealing. However, the influence of cholesterol and anionic lipid headgroups on annexin-induced membrane curvature remains poorly understood at the molecular level. Using all-atom molecular dynamics simulations, we measured the local curvature-induced underneath ANXA4 and ANXA5 monomers and trimers when they bind to lipid bilayers of distinct lipid compositions: PSPC (20% POPS, 80% POPC), PAPC (20% POPA, 80% POPC), and PSPCCHL (14% POPS, 56% POPC, 30% cholesterol). Laser injury experiments were conducted on MCF7 cells transfected to transiently express fluorescently labeled ANXA4 or ANXA5 to facilitate the examination of protein and lipid accumulation at the damage site. Annexin trimers induce higher curvature than monomers, particularly with cholesterol present. Annexin trimers induce similar curvatures on both PAPC and PSPC membranes. Notably, among monomers, ANXA5 induces the highest curvature on PAPC, suggesting more efficient recruitment of ANXA5 compared with ANXA4 in the early stages of membrane repair near a lesion. Laser injury experiments confirm rapid coaccumulation of phosphatidic acid lipids with ANXA4 and ANXA5 at repair sites, potentially enhancing the accumulation of annexins in the early stages of membrane repair.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ebstrup, Sønder, Fogde, Heitmann, Dietrich, Dias, Jäättelä, Maeda and Nylandsted.)

Cancer/testis antigens are receiving attention as targets for cancer therapy due to their germ- and cancer cell-restricted expression. However, many of these antigens are inconsistently expressed among cancer types and individual tumors. Here, we show that members of the SSX cancer/testis antigen family comprise attractive targets in the majority of melanoma patients, as SSX is expressed in more than 90% of primary melanomas and metastases and plays a critical role in metastatic progression. Accordingly, SSX silencing in melanoma mouse xenograft models reduced tumor growth and completely abolished the formation of metastatic lesions in lungs and livers. Mechanistically, we demonstrate that silencing SSX in melanoma cells induces cell cycle S-phase stalling, leading to proliferative arrest and enhanced apoptosis, which elucidates the inhibitory effect of SSX loss on tumor growth and colonization capacity. Silencing SSX further compromised the capacity of melanoma cells to migrate and invade, influencing these cells' capability to spread and colonize. Taken together, these studies highlight SSX proteins as pivotal targets in melanoma with implications for blocking metastatic progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Traynor, Ebstrup, Gammelgaard, Mansoori, Terp, Rein, Rattenborg, Pedersen, Ditzel and Gjerstorff.)

Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension., Competing Interests: Competing interests The authors have nothing to disclose., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

The transcription factor ZBED1 is highly expressed in trophoblast cells, but its functions in the processes of trophoblast and placental biology remain elusive. Here, we characterized the role of ZBED1 in trophoblast cell differentiation using an in vitro BeWo cell model. We demonstrate that ZBED1 is enhanced in its expression early after forskolin-induced differentiation of BeWo cells and regulates many of the genes that are differentially expressed as an effect of forskolin treatment. Specifically, genes encoding markers for the differentiation of cytotrophoblast into syncytiotrophoblast and factors essential for trophoblast cell fusion and invasion were negatively regulated by ZBED1, indicating that ZBED1 might be important for maintaining a steady pool of cytotrophoblast cells. In addition, ZBED1 affected genes involved in the regulation of trophoblast cell survival and apoptosis, in agreement with the observed increase in apoptosis upon knockdown of ZBED1 in forskolin-treated BeWo cells. In addition, genes implicated in the differentiation, recruitment, and function of innate immune cells by the placenta were affected by ZBED1, further suggesting a role for this protein in the regulation of maternal immune tolerance. In conclusion, our study implicates ZBED1 in major biological processes of placental biology.

SSX proteins are normally restricted to spermatogenic cells, but ectopic expression can be observed in many types of human cancer. We recently demonstrated that SSX family members may contribute to tumorigenesis by modifying chromatin structure and, in specific settings, compromise chromatin stability. Here, we used normal and tumorigenic breast epithelial cell line models to further study the effect of ectopic expression of SSX2 on nuclear organization. We show that SSX2 induces the formation of a novel type of nucleoplasmic lamin bodies. Ectopic expression of SSX2 in various breast epithelial cell lines led to the formation of a previously undescribed type of intranuclear bodies containing both A and B type lamins but no other components of the nuclear lamina. SSX2-expressing cells contained a highly variable number of lamin bodies distributed throughout the nuclear space. SSX2-mediated establishment of intranuclear lamin bodies could not be linked to previous molecular interactions of SSX proteins, including polycomb proteins and the Mediator complex, but was, however, dependent on S-phase progression. These results reveal a novel interaction between SSX2 and lamins in the nucleoplasmic space. They further suggest that SSX2 promotes the formation of chromatin neighborhoods supporting the organization of lamins into nuclear bodies. We speculate that this may have implications for the organization and functional regulation of chromatin in cancer cells. Our study contributes to the further understanding of the biology of SSX proteins in tumorigenesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.