NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.     

YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.     

Cytosolic Iron-Sulfur Cluster Assembly (CIA) System: Factors,Mechanism, and Relevance to Cellular Iron Regulation

FeS cluster biogenesis is an essential process in virtually all forms of life. Complex protein machineries that are conserved from bacteria through higher eukaryotes facilitate assembly of the FeS cofactor in proteins. In the last several years, significant strides have been made in our understanding of FeS cluster assembly and the functional overlap of this process with cellular iron homeostasis. This minireview summarizes the present understanding of the cytosolic iron-sulfur cluster assembly (CIA) system in eukaryotes, with a focus on information gained from studies in budding yeast and mammalian systems.     

A High‐Resolution Approach to Mapping Energy Flows through Water Infrastructure Systems

Using data from the water service area of the East Bay Municipal Utility District in Northern California, we develop and discuss a method for assessing, at a high resolution, the energy intensity of water treated and delivered to customers of a major metropolitan water district. This method extends previous efforts by integrating hourly data from supervisory control and data acquisition systems with calculations based on the actual structure of the engineered infrastructure to produce a detailed understanding of energy use in space and time within the territory of a large‐scale urban water provider. We found significant variations in the energy intensity of delivered potable water resulting from seasonal and topographic effects. This method enhances our understanding of the energy inputs for potable water systems and can be applied to the entire delivery and postuse water life cycle. A nuanced understanding of water's energy intensity in an urban setting enables more intelligent, targeted efforts to jointly conserve water and energy resources that take seasonal, distance, and elevation effects into account.     

Oligomerization of the chitin synthase Chs3 is monitored at the Golgi and affects its endocytic recycling

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP‐1‐dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N‐terminal cytosolic region. However, the truncated ^Δ126Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI‐ and Rer1‐dependent manner. A subset of the oligomerization‐deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N‐terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly.     

Cotranslational transport of ABP140 mRNA to the distal pole of S. cerevisiae

In budding yeast, several mRNAs are selectively transported into the daughter cell in an actin-dependent manner by a specialized myosin system, the SHE machinery. With ABP140 mRNA, we now describe the first mRNA that is transported in the opposite direction and localizes to the distal pole of the mother cell, independent of the SHE machinery. Distal pole localization is not observed in mutants devoid of actin cables and can be disrupted by latrunculin A. Furthermore, localization of ABP140 mRNA requires the N-terminal actin-binding domain of Abp140p to be expressed. By replacing the N-terminal localization motif, ABP140 mRNA can be retargeted to different subcellular structures. In addition, accumulation of the mRNA at the distal pole can be prevented by disruption of polysomes. Using the MS2 system, the mRNA was found to associate with actin cables and to follow actin cable dynamics. We therefore propose a model of translational coupling, in which ABP140 mRNA is tethered to actin cables via its nascent protein product and is transported to the distal pole by actin retrograde flow.     

Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.     

Compensating for unknown confounders in microarray data analysis using filtered permutations.

Permutation of class labels is a common approach in microarray analysis. It is assumed to produce random score distributions, which are not affected by biological differences between samples. However, hidden confounding variables like the genetic background of patients or undetected experimental artifacts leave traces in the expression data contaminating the score distributions obtained from random permutations. While the effects of known confounders can be compensated using established methodology, little is known on how to deal with unknown confounders. We discuss a computational method called permutation filtering, which aims to borrow information across genes to detect and compensate the effects of unknown confounders.