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A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum ‘ Candidatus Atribacteria’ (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov. A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, the authors isolate a member of the ubiquitous, yet-to-be-cultivated bacterial phylum ‘ Candidatus Atribacteria’ that has an intracytoplasmic membrane apparently surrounding the nucleoid.

Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3′‐end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein‐tagged full‐length cDNA library. Most of the newly identified PSPs possessed various putative RNA‐binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3′‐end polyadenylation of the NEAT1_1 isoform. An in vitro 3′‐end processing assay revealed that HNRNPK arrested binding of the CPSF6–NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. Paraspeckle formation is initiated by the long noncoding RNA isoform NEAT1_2. This study identifies 35 paraspeckle proteins, seven of which are essential for paraspeckle formation. One of these, hnRNP K, governs alternative 3′‐end processing of NEAT to generate NEAT1_2.

Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α 6 β 1 . In contrast, the binding of α 6 β 1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α 6 β 1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis. Seano, Primo and colleagues report that blood vessel branching during tumour angiogenesis is mediated by the formation of podosome rosettes that depends on VEGF-A and integrin α 6 β 1 .

Tunneling nanotubes (TNTs) and associated structures are recently recognized structures for intercellular communication. They are F-actin-containing thin protrusions of the plasma membrane of a cell and allow a direct physical connection to the plasma membranes of remote cells. TNTs and associated structures serve as mediators for intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, several pathogens have been shown to exploit these structures to spread among cells. Because of their contribution to normal cellular functions and importance in pathological conditions, studies on TNTs and related structures have accelerated over the past few years. These studies have revealed key molecules for their induction and/or formation; HIV Nef and M-Sec can induce the formation of TNTs in coordination with the remodeling of the actin cytoskeleton and vesicle trafficking.

The replication of many viruses is associated with specific intracellular compartments called virus factories or virioplasm. These are thought to provide a physical scaffold to concentrate viral components and thereby increase the efficiency of replication. The formation of virus replication sites often results in rearrangement of cellular membranes and reorganization of the cytoskeleton. Similar rearrangements are seen in cells in response to protein aggregation, where aggresomes and autophagosomes are produced to facilitate protein degradation. Here I review the evidence that some viruses induce aggresomes and autophagosomes to generate sites of replication.

Key Points Speckles are dynamic subnuclear structures that contain pre-messenger RNA splicing factors and other proteins that are involved in transcription, 3′- end RNA-processing and reversible protein phosphorylation. The formation of speckles is regulated during the cell-division cycle. Splicing factors cycle continually between speckles and the nucleoplasm. Their size and shape results from the dynamic exchange of factors into and out of speckles. A reversible protein phosphorylation mechanism can regulate the movement of speckle components between speckles and other nuclear structures. It is likely that protein–protein interactions are primarily responsible for the formation and integrity of speckles. Speckles contain little or no DNA and are not principal sites of transcription. Instead, they function as assembly/modification sites that can supply active splicing factors to sites of transcription. We propose a 'regulated-exchange' model to account for the steady-state level of proteins in speckles. This envisages that the concentration of factors that are localized in speckles results from a regulated and cell-type-specific basal exchange rate of speckle components. Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA–protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.

Magnetosomes are membranous bacterial organelles sharing many features of eukaryotic organelles. Using electron cryotomography, we found that magnetosomes are invaginations of the cell membrane flanked by a network of cytoskeletal filaments. The filaments appeared to be composed of MamK, a homolog of the bacterial actin-like protein MreB, which formed filaments in vivo. In a mamK deletion strain, the magnetosome-associated cytoskeleton was absent and individual magnetosomes were no longer organized into chains. Thus, it seems that prokaryotes can use cytoskeletal filaments to position organelles within the cell.

Patients with microsatellite instability‐high (MSI‐H) colorectal cancer (CRC) have high tumor mutation burden and tumor immunogenicity, exhibiting a higher response rate to immunotherapy and better survival. However, a portion of MSI‐H CRC patients still experience adverse disease outcomes. We aimed to identify the tumor‐autonomous regulators determining these heterogeneous clinical outcomes. The Cancer Genome Atlas (TCGA) dataset was used to identify regulators in MSI‐H CRC patients with unfavorable outcomes. Stable CRC tumor clones expressing targeted regulators were established to evaluate migratory and stemness properties, immune cell vulnerability, and cell‐in‐cell (CIC) structure formation. RNA‐sequencing (RNA‐seq) was used to identify enriched biological pathways in stable CRC tumor clones. Clinicopathological characterization of formalin‐fixed paraffin‐embedded (FFPE) MSI‐H CRC specimens was performed to explore the underlying mechanisms involved. We showed that cancer/testis antigen family 45 member A1 (CT45A1) expression was upregulated in MSI‐H CRC patients with poor survival outcomes. CT45A1‐expressing microsatellite stable (MSS) CRC cells showed enhanced migratory ability. However, CT45A1‐expressing MSI‐H CRC cells, but not MSS CRC cells, showed higher resistance to natural killer (NK) cell cytotoxicity and served as outer cells in homotypic CIC structures, preventing exogenous or therapeutic antibody access to inner CRC cells. Inactivating RHO‐ROCK/MLCK‐MLC2 signaling with small‐molecule inhibitors or short‐hairpin RNAs (shRNAs) targeting myosin light chain kinase (MYLK) abolished NK cell resistance and reduced the outer cell fate of CT45A1‐expressing MSI‐H CRC cells. In MSI‐H CRC patients, CT45A1‐positive tumors exhibited increased MLC2 phosphorylation, increased outer cell fate, and decreased survival. We demonstrated that CT45A1 potentiates the advanced progression of MSI‐H CRC, and targeting MLC2 phosphorylation may enhance immunotherapy efficacy in CT45A1‐positive MSI‐H CRC patients. CT45A1, a vital driver of the RHO‐ROCK/MLCK‐MLC2 signaling, is found to enhance cancer cell resistance to natural killer (NK) cell killing and generate a protective cell‐in‐cell (CIC) structure in microsatellite instability‐high (MSI‐H) colorectal cancer (CRC) cells. This structure shields the inner CT45A1 (Low) cancer cells from targeted antibody therapy and potentially subsequent antitumor immunity, thereby driving aggressive MSI‐H CRC outcomes.

A cell-free system has been developed to image vesicle budding and fission events. This method reveals an important role for the F-BAR protein FBP17 in regulating tubulation and clathrin-dependent budding. Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport 1 , 2 , 3 . To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) 4 , 5 , 6 , revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPγS. Triggering fission by replacing GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.