Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P(TF) to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P(NSE)) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed

Control of smooth muscle cell proliferation by ferrous iron.

This study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and patho-genic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requiresHOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPSbinds small membrane- associated GTPases and assembles SNAREs for fusion, but how the complexfulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the struc-ture of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities,where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore,ideally positioned between the membranes to catalyze fusion. Our data suggest a model for howHOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part ofthe membrane fusion machinery