The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.

Single domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, as well as therapeutic and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research - i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat IgG. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

Adipokines measured during pregnancy and at birth are associated with infant negative affect.

BACKGROUND: Increased adiposity during pregnancy may be related to offspring risk for mental health disorders, although the biological mechanisms are poorly understood. One promising hypothesis is that factors secreted from adipocytes such as leptin and adiponectin may explain this association. The current study examined whether pregnancy or umbilical cord blood concentrations of leptin and/or adiponectin a) predict elevated infant negative affect at 6 months (an early life marker of risk for psychopathology); and b) help explain the association between pregnancy adiposity and increased infant negative affect. METHODS: Data came from a prospective cohort (N = 305) of pregnant individuals and their offspring. Second trimester adiposity was assessed using air displacement plethysmography. Concentrations of leptin and adiponectin were measured in second trimester plasma and umbilical cord plasma. Infant negative affect was assessed by standardized observation at 6 months. Second trimester inflammation was assessed using a comprehensive panel of cytokines. RESULTS: Lower second trimester adiponectin was associated with elevated infant negative affect, and mediated the effect of pregnancy adiposity on infant negative affect. This association was independent of the effect of second trimester inflammation. Umbilical cord leptin also predicted higher infant negative affect and mediated the association between pregnancy adiposity and infant negative affect. CONCLUSIONS: This is the first study to link pregnancy adiponectin or cord blood leptin to infant markers of risk for psychopathology, and the first to demonstrate that these adipokines mediate the association between pregnancy adiposity and offspring behavioral outcomes, suggesting novel markers of risk and potential mechanisms of effect.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis.

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

The Molecular Architecture of the Nuclear Basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of Nups in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.