Claudin-4 polymerizes after a small extracellular claudin-3-like substitution.

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers that compartmentalize tissues and protect the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is crucial, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.

Senescence-Associated Sugar Transporter1 affects developmental master regulators and controls senescence in Arabidopsis.

Sugar transport across membranes is critical for plant development and yield. However, an analysis of the role of intracellular sugar transporters in senescence is lacking. Here, we characterized the role of Senescence-Associated Sugar Transporter1 (SAST1) during senescence in Arabidopsis (Arabidopsis thaliana). SAST1 expression was induced in leaves during senescence and after the application of abscisic acid (ABA). SAST1 is a vacuolar protein that pumps glucose out of the cytosol. sast1 mutants exhibited a stay-green phenotype during developmental senescence, after the darkening of single leaves, and after ABA feeding. To explain the stay-green phenotype of sast1 mutants, we analyzed the activity of the glucose-induced master-regulator TOR (target of rapamycin), which is responsible for maintaining a high anabolic state. TOR activity was higher in sast1 mutants during senescence compared to wild types, whereas the activity of its antagonist, SNF1-related protein kinase 1 (SnRK1), was reduced in sast1 mutants under senescent conditions. This deregulation of TOR and SnRK1 activities correlated with high cytosolic glucose levels under senescent conditions in sast1 mutants. Although sast1 mutants displayed a functional stay-green phenotype, their seed yield was reduced. These analyses place the activity of SAST1 in the last phase of a leaf's existence in the molecular program required to complete its life cycle.

Tight junctions control lumen morphology via hydrostatic pressure and junctional tension.

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.

The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome. Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.

Canonical and non-canonical integrin-based adhesions dynamically interconvert.

Adhesions are critical for anchoring cells in their environment, as signaling platforms and for cell migration. In line with these diverse functions different types of cell-matrix adhesions have been described. Best-studied are the canonical integrin-based focal adhesions. In addition, non-canonical integrin adhesions lacking focal adhesion proteins have been discovered. These include reticular adhesions also known as clathrin plaques or flat clathrin lattices, that are enriched in clathrin and other endocytic proteins, as well as extensive adhesion networks and retraction fibers. How these different adhesion types that share a common integrin backbone are related and whether they can interconvert is unknown. Here, we identify the protein stonin1 as a marker for non-canonical αVß5 integrin-based adhesions and demonstrate by live cell imaging that canonical and non-canonical adhesions can reciprocally interconvert by the selective exchange of components on a stable αVß5 integrin scaffold. Hence, non-canonical adhesions can serve as points of origin for the generation of canonical focal adhesions.

Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis toward the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

Supporting regional pandemic management by enabling self-service reporting-A case report.

BACKGROUND: The COVID-19 pandemic revealed a need for better collaboration among research, care, and management in Germany as well as globally. Initially, there was a high demand for broad data collection across Germany, but as the pandemic evolved, localized data became increasingly necessary. Customized dashboards and tools were rapidly developed to provide timely and accurate information. In Saxony, the DISPENSE project was created to predict short-term hospital bed capacity demands, and while it was successful, continuous adjustments and the initial monolithic system architecture of the application made it difficult to customize and scale. METHODS: To analyze the current state of the DISPENSE tool, we conducted an in-depth analysis of the data processing steps and identified data flows underlying users' metrics and dashboards. We also conducted a workshop to understand the different views and constraints of specific user groups, and brought together and clustered the information according to content-related service areas to determine functionality-related service groups. Based on this analysis, we developed a concept for the system architecture, modularized the main services by assigning specialized applications and integrated them into the existing system, allowing for self-service reporting and evaluation of the expert groups' needs. RESULTS: We analyzed the applications' dataflow and identified specific user groups. The functionalities of the monolithic application were divided into specific service groups for data processing, data storage, predictions, content visualization, and user management. After composition and implementation, we evaluated the new system architecture against the initial requirements by enabling self-service reporting to the users. DISCUSSION: By modularizing the monolithic application and creating a more flexible system, the challenges of rapidly changing requirements, growing need for information, and high administrative efforts were addressed. CONCLUSION: We demonstrated an improved adaptation towards the needs of various user groups, increased efficiency, and reduced burden on administrators, while also enabling self-service functionalities and specialization of single applications on individual service groups.

Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM.

The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5-15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.