Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that .70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A 2 receptor-1 (PLA 2 R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA 2 R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA 2 R by probing isolated truncated PLA 2 R extracellular domains with sera from patients with IMN that contain anti-PLA 2 R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteinerich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA 2 R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA 2 R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA 2 R. These results indicate that the immunodominant epitope in PLA 2 R is exclusively located in the CysR-FnII-CTLD1 region.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis. Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A’s genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli’s, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome’s configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.

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Most of the recent work on Web security focuses on preventing attacks that directly harm the browser's host machine and user. In this paper we attempt to quantify the threat of browsers being indirectly misused for attacking third parties. Specifically, we look at how the existing Web infrastructure (e.g., the languages, protocols, and security policies) can be exploited by malicious Web sites to remotely instruct browsers to orchestrate actions including denial of service attacks, worm propagation and reconnaissance scans. We show that, depending mostly on the popularity of a malicious Web site and user browsing patterns, attackers are able to create powerful botnet-like infrastructures that can cause significant damage. We explore the effectiveness of countermeasures including anomaly detection and more fine-grained browser security policies.

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Understanding the architecture of the nuclear periphery remains one of the biggest challenges in biology, with the nucleus presenting an impregnable frontier for structural studies. Molecular complexes and DNA that conform this region of the cell are deeply rooted in their environment, and in many cases, such as chromatin, any form of fixation, staining, and extraction is thought to alter their structure.. A variety of imaging techniques intend to reveal the architecture of the nuclear periphery, with a strong focus on chromatin. Structural biology provides deep insight into isolated complexes devoid of their natural context, with the latter often determining their conformation, composition, and function. Light microscopy has many advantages but relies on in vivo tagging but lacks detailed substructure information. Traditional electron microscopy allows detailed ultrastructural analyses but relies on fixation, embedding, and staining, often compromising structural integrity and hampering interpretation at the molecular level. Thus, a major challenge remains to combine structural analyses of chromatin with native preservation of its 3D folding and positioning within the nuclear landscape. Cryo-electron microscopy has undergone a resolution revolution due to technological advances. In its single-particle modality for structural biology, it has become a central technique capable of achieving atomic-resolution structures even flexible and transient complexes. In the modality of CET, structures can be visualized within 3-D images of cells. CET was limited by the size of the objects (< 500 nm) that can be examined in situ with molecular resolution. Using cryo-FIB milling of cells, we have eliminated this limitation ( Fig. 1)[1,2], We have demonstrated the power of cryo-FIB/CET by visualizing the nuclear periphery of HeLa cells and determining the structural dynamics of large and complex machines such as the nuclear pore complex [3] and revealing how large viruses affect the cellular machinery of bacteria during infection by creating a nucleus-like compartment that separates transcription and translation [4].Using cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (CET), we obtain highresolution 3-D images of intact (cryo-frozen) cells that have not been fixed, permeabilized, or stained. The resulting data reveal the local architecture of chromatin at the single nucleosome level, along with the structures of individual architectural complexes. To characterize these structures, we will combine CET with rigorous computational methodologies that can perform large-scale template-free discovery of structurally unknown complexes. Using cryo-FIB milling and CET to obtain 3-D molecular landscapes of the nuclear periphery of mammalian cells, we demonstrate that CET can be leveraged into the highest resolution method to (a) extract the local architecture of native chromatin at the molecular level in situ (i.e., single and stretches of nucleosomes) and (b) we will show the potential to determine the in situ st...