Regulated by pH, membrane-anchored proteins E and M function during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo-electron microscopy at 3.5-Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino-acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch is fastened at an earlier stage, during maturation at acidic pH in the trans-Golgi network. At a later stage, to initiate infection in response to acidic pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.
The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up- or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm-induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.
Pancreatic islets of Langerhans display characteristic spatial architecture of their endocrine cell types. This architecture is critical for cell-cell communication and coordinated hormone secretion. Islet architecture is disrupted in type-2 diabetes. Moreover, the generation of architecturally correct islets in vitro remains a challenge in regenerative approaches to type-1 diabetes. Although the characteristic islet architecture is well documented, the mechanisms controlling its formation remain obscure. Here, we report that correct endocrine cell type sorting and the formation of mature islet architecture require the expression of Roundabout (Robo) receptors in β cells. Mice with whole-body deletion of Robo1 and conditional deletion of Robo2 either in all endocrine cells or selectively in β cells show complete loss of endocrine cell type sorting, highlighting the importance of β cells as the primary organizer of islet architecture. Conditional deletion of Robo in mature β cells subsequent to islet formation results in a similar phenotype. Finally, we provide evidence to suggest that the loss of islet architecture in Robo KO mice is not due to β cell transdifferentiation, cell death or loss of β cell differentiation or maturation.
The islets of Langerhans display typical, species-specific architecture, with distinct spatial organization of their various endocrine cell types1,2,3,4,5. In the mouse, the core of the islet is composed mostly of insulin-secreting β cells, while glucagon-secreting α cells, somatostatin-secreting δ cells and pancreatic polypeptide-secreting PP cells are located at the islet periphery3. In humans and other primates, islet architecture is more complex, but still conforms to the overall structure of several β cell lobules surrounded by mantles of α, δ and other endocrine cells types4,5. Correct islet architecture facilitates the mature pattern of hormone release, directionality of intra-islet paracrine signaling, and connection with the microvasculature6,7. The typical islet architecture is disrupted in obesity, insulin resistance, and diabetes in both humans and rodents8,9,10,11,12,13,14. Structural islet integrity and architecture are also disrupted in cadaver islets during isolation and culture prior to islet transplantation, as well as after infusion into the portal vein15,16,17,18. Moreover, the generation of bona fide islets of Langerhans from human pluripotent stem cells, in which the three-dimensional islet architecture is recapitulated, remains a pressing challenge in regenerative medicine approaches to diabetes19,20.
The formation of the islets of Langerhans in the mouse starts with the delamination of individual pro-endocrine cells from the pancreatic duct, beginning at embryonic day (E) 13.521. These cells then migrate into the mesenchyme, aggregate to form proto-islet clusters, and subsequently rearrange into the typical mantle/core architecture of the mature islets of Langerhans22. Interestingly, dissociated rat islets re-aggregate spontaneously in culture, recapitulating the original mantle-core islet architecture, suggesting that the signals and forces controlling islet architecture are islet-autonomous23. Despite the four decades that have passed since the typical islet architecture was first described24,25, the mechanisms controlling the formation of mature islet architecture during development and its maintenance in the adult remain largely unresolved22,26.
Here, we show that expression of Robo receptors in β cells is required for endocrine cell type sorting and mature islet architecture. Mice lacking Robo1 and Robo2 in all endocrine cells or selectively in β cells show complete loss of endocrine cell type sorting in the islets. Moreover, deletion of Robo receptors in mature β cells after islet formation has been completed also results in intermixing of endocrine cell types and loss of islet architecture. Finally, lineage-tracing experiments in β cell-selective Robo knockouts (Robo KO) provide evidence suggesting that disruption of islet architecture in Robo KO mice is not due to transdifferentiation, β cell death, or insufficient β cell differentiation or maturation.
Current understanding of the formation of the mature architecture of the islets of Langerhans during development suggests that, beginning at E13.5, individual endocrine progenitors within the pancreatic duct independently turn on the transcription factor Neurogenin3 (Neurog3), and delaminate from the duct into the surrounding mesenchyme as single cells. These delaminated cells then migrate away from the duct and coalesce to form the mature islet architecture35,37,44,45. To test our hypothesis that Robo receptors are involved in the organogenesis of the islets of Langerhans, we generated an early endocrine progenitor knockout of Robo by crossing Robo1Δ/Δ,2flx/flx mice34 with Neurog3-Cre mice46. Robo1Δ/Δ,2flx/flx mice harbor a linked Robo1 deletion allele (Robo1Δ) and conditional Robo2 deletion allele (Robo2flx). Neurog3 is expressed in all endocrine progenitors during their delamination from the duct, and its expression is subsequently turned off prior to endocrine cell aggregation to form proto-islet clusters47,48. The resulting Neurog3-Cre;Robo1Δ/Δ,2flx/flx mice carry a whole-body deletion of Robo1, and a pancreatic endocrine-selective deletion of Robo2. This strategy was chosen to eliminate redundant Robo signaling and to avoid the homozygous lethality of Robo2 whole-body deletion49. Neurog3-Cre;Robo1Δ/Δ,2flx/flx progeny are viable and appear normal.
Robo receptors are required for endocrine cell type sorting and mature architecture of the islets of Langerhans. (A) Immunofluorescence staining for β cells (Insulin, red) and α cells (glucagon, green) of control (Robo1+/Δ,2+/flx and Neurog3-Cre;Robo1+/+,2+/+) and Neurog3-Cre;Robo1Δ/Δ,2flx/flx islets from 2 month old mice. (B) Average Circularity Index of control (Robo1+/Δ,2+/flx and Neurog3-Cre;Robo1+/+,2+/+) and Neurog3-Cre;Robo1Δ/Δ,2flx/flx islets. (C) Percentage of total α cells found in periphery of the islet in control (Robo1+/Δ,2+/flx and Neurog3-Cre;Robo1+/+,2+/+) vs. Neurog3-Cre;Robo1Δ/Δ,2flx/flx mice. (D) Average size of control (Robo1+/Δ,2+/flx and Neurog3-Cre;Robo1+/+,2+/+) and Neurog3-Cre;Robo1Δ/Δ,2flx/flx. (E) Percent of α cells out of total cells in control (Robo1+/Δ,2+/flx and Neurog3-Cre;Robo1+/+,2+/+) and Neurog3-Cre;Robo1Δ/Δ,2flx/flx.
The disrupted islet architecture and increase in α cell ratio in Ucn3-Cre;Robo1Δ/Δ,2flx/flx islets is not due to transdifferentiation of β to α cells. (A) Schematic of constructs used for lineage tracing Ucn3-Cre expressing cells. (B) Lineage tracing of mature β cells in control Ucn3-Cre;Robo1+/+,2+/+ and Ucn3-Cre;Robo1Δ/Δ,2flx/flx islets. Lineage traced β cells express nuclear mCherry (white), and counterstained for Insulin (red), Glucagon (green), and DAPI (blue), showing no transdifferentiation of α to β cells. 041b061a72