α-Spectrin regulates cell shape changes during disassembly of microtubule-driven protrusions in Drosophila wings.

The dynamics of microtubule-mediated protrusions, termed Interplanar Amida Network (IPAN) in Drosophila pupal wing, involve cell shape changes. The molecular mechanisms underlying these processes are yet to be fully understood. This study delineates the stages of cell shape alterations during the disassembly of microtubule protrusions and underscores the pivotal role of α-Spectrin in driving these changes by regulating both the microtubule and actomyosin networks. Our findings also demonstrate that α-Spectrin is required for the apical relaxation of wing epithelia during protrusion disassembly, indicating its substantial contribution to the robustness of 3D tissue morphogenesis.

Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.

Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.

STUDY QUESTION: Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells? SUMMARY ANSWER: A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted. WHAT IS KNOWN ALREADY: The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted. STUDY DESIGN SIZE DURATION: Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in the embryo implantation process were highlighted. LARGE SCALE DATA: RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929. LIMITATIONS REASONS FOR CAUTION: Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only. WIDER IMPLICATIONS OF THE FINDINGS: The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest.

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing.

PURPOSE: The purpose of this study was to develop a feasible approach for single sperm isolation and chromosome analysis by next-generation sequencing (NGS). METHODS: Single sperm cells were isolated from semen samples of normozoospermic male and an infertile reciprocal translocation (RcT) carrier with the 46,XY,t(7;13)(p12;q12.1) karyotype using the optimized fluorescence-activated cell sorting (FACS) technique. Genome profiling was performed using NGS. RESULTS: Following whole-genome amplification, NGS, and quality control, the final chromosome analysis was performed on 31 and 6 single cell samples derived from the RcT carrier and normozoospermic male, respectively. All sperm cells from normozoospermic male showed a normal haploid 23-chromosome profile. For the RcT carrier, the sequencing data revealed that 64.5% of sperm cells harbored different variants of chromosome aberrations, involving deletion of 7p or 7q, duplication of 7p, and duplication of 13q, which is concordant with the expected chromosome segregation patterns observed in balanced translocation carriers. In one sample, a duplication of 9q was also detected. CONCLUSIONS: We optimized FACS protocol for simple and efficient isolation of single human sperm cells that subsequently enabled a successful genome-wide chromosome profiling and identification of segmental aneuploidies from these individual cells, following NGS analysis. This approach may be useful for analyzing semen samples of infertile men or chromosomal aberration carriers to facilitate the reproductive risk assessment.

Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes.

Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

Translocation of cell-penetrating peptides across the plasma membrane is controlled by cholesterol and microenvironment created by membranous proteins.

Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs). GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.

Penetration without cells: membrane translocation of cell-penetrating peptides in the model giant plasma membrane vesicles.

The cellular internalization of cell-penetrating peptides (CPPs) is proposed to take place by both endocytic processes and by a direct translocation across the plasma membrane. So far only scarce data is available about what determines the choice between the two uptake routes, or the proportion of used pathways when both are active simultaneously. Furthermore, the mechanism(s) of membrane penetration by peptides is itself still a matter of debate. We have introduced the giant plasma membrane vesicles (GPMVs) to study the interaction of six well-described CPPs (fluorescently labeled nona-arginine, Tat peptide, Penetratin, MAP, Transportan and TP10) in a model system of native plasma membrane without the interference of endocytic processes. The membranes of GPMVs are shown to segregate into liquid-ordered and liquid-disordered phases at low temperatures and we demonstrate here by confocal microscopy that amphipathic CPPs preferentially associate with liquid-disordered membrane areas. Moreover, all tested CPPs accumulate into the lumen of GPMVs both at ambient and low temperature. The uncharged control peptide and dextran, in contrary, do not translocate from the medium into the lumen of vesicles. The absence of energy-dependent cellular processes and the impermeability to hydrophilic macromolecules makes the GPMVs a useful model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis.