Reconstitution of Mdm2-Dependent Post-Translational Modifications of p53 in Yeast

p53 mediates cell cycle arrest or apoptosis in response to DNA damage. Its activity is subject to a tight regulation involving a multitude of post-translational modifications. The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network. To circumvent this complexity, we studied here the minimal requirements for functionally relevant p53 post-translational modifications by expressing human p53 together with its best characterized modifier Mdm2 in budding yeast. We find that expression of the human p53-Mdm2 module in yeast is sufficient to faithfully recapitulate key aspects of p53 regulation in higher eukaryotes, such as Mdm2-dependent targeting of p53 for degradation, sumoylation at lysine 386 and further regulation of this process by p14ARF. Interestingly, sumoylation is necessary for the recruitment of p53-Mdm2 complexes to yeast nuclear bodies morphologically akin to human PML bodies. These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies. The reductionist yeast model that was established and validated in this study will now allow to incrementally study simplified parts of the intricate p53 network, thus helping elucidate the core mechanisms of p53 regulation as well as test novel strategies to counteract p53 malfunctions

Biomaterial-Assisted Anastomotic Healing: Serosal Adhesion of Pectin Films

Anastomotic leakage is a frequent complication of intestinal surgery and a major source of surgical morbidity. The timing of anastomotic failures suggests that leaks are the result of inadequate mechanical support during the vulnerable phase of wound healing. To identify a biomaterial with physical and mechanical properties appropriate for assisted anastomotic healing, we studied the adhesive properties of the plant-derived structural heteropolysaccharide called pectin. Specifically, we examined high methoxyl citrus pectin films at water contents between 17–24% for their adhesivity to ex vivo porcine small bowel serosa. In assays of tensile adhesion strength, pectin demonstrated significantly greater adhesivity to the serosa than either nanocellulose fiber (NCF) films or pressure sensitive adhesives (PSA) (p < 0.001). Similarly, in assays of shear resistance, pectin demonstrated significantly greater adhesivity to the serosa than either NCF films or PSA (p < 0.001). Finally, the pectin films were capable of effectively sealing linear enterotomies in a bowel simulacrum as well as an ex vivo bowel segment. We conclude that pectin is a biomaterial with physical and adhesive properties capable of facilitating anastomotic healing after intestinal surgery

The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER-LD contact sites

Daughter-cell-specific modulation of nuclear pore complexes controls cell cycle entry during asymmetric division

International audienceThe acquisition of cellular identity is coupled to changes in the nuclear periphery and nuclear pore complexes (NPCs). Whether and how these changes determine cell fate remain unclear. We have uncovered a mechanism that regulates NPC acetylation to direct cell fate after asymmetric division in budding yeast. The lysine deacetylase Hos3 associates specifically with daughter cell NPCs during mitosis to delay cell cycle entry (Start). Hos3-dependent deacetylation of nuclear basket and central channel nucleoporins establishes daughter-cell-specific nuclear accumulation of the transcriptional repressor Whi5 during anaphase and perinuclear silencing of the G1/S cyclin gene CLN2 in the following G1 phase. Hos3-dependent coordination of both events restrains Start in daughter, but not in mother, cells. We propose that deacetylation modulates transport-dependent and transport-independent functions of NPCs, leading to differential cell cycle progression in mother and daughter cells. Similar mechanisms might regulate NPC functions in specific cell types and/or cell cycle stages in multicellular organisms

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission

International audienceDuring transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis.

Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.This research was supported by ‘La Caixa’ fellowships to N.A., G.N. and M.Maier, and grants from the Spanish Ministry of Economy and Competitivity (BFU2011-30185 and CDS2009-00016 to M.-I.G.; BFU2015-71308 and BFU2013-50245-EXP to J.T.-R.; and BFU2009-08213 and BFU2012-37162/nto M.Mendoza), and from the European Research Council (ERC Starting Grant 260965 to M.Mendoza). We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-020