A cation counterflux supports lysosomal acidification

The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl⁻ with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.     

Induction,rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed‐propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC‐AT transition mutations were recovered, similar to that reported in seed‐propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation‐based functional genomics and improvement of vegetatively propagated crops.     

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.     

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus–insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl + P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell–baculovirus systems for bioprocess optimization.     

Dynamic localization of HmpF regulates type IV pilus activity and directional motility in the filamentous cyanobacterium Nostoc punctiforme

Many cyanobacteria exhibit surface motility powered by type 4 pili (T4P). In the model filamentous cyanobacterium Nostoc punctiforme, the T4P systems are arrayed in static, bipolar rings in each cell. The chemotaxis‐like Hmp system is essential for motility and the coordinated polar accumulation of PilA on cells in motile filaments, while the Ptx system controls positive phototaxis. Using transposon mutagenesis, a gene, designated hmpF, was identified as involved in motility. Synteny among filamentous cyanobacteria and the similar expression patterns for hmpF and hmpD imply that HmpF is part of the Hmp system. Deletion of hmpF produced a phenotype distinct from other hmp genes, but indistinguishable from pilB or pilQ. Both an HmpF‐GFPuv fusion protein, and PilA, as assessed by in situ immunofluorescence, displayed coordinated, unipolar localization at the leading pole of each cell. Reversals were modulated by changes in light intensity and preceded by the migration of HmpF‐GFPuv to the lagging cell poles. These results are consistent with a model where direct interaction between HmpF and the T4P system activates pilus extension, the Hmp system facilitates coordinated polarity of HmpF to establish motility, and the Ptx system modulates HmpF localization to initiate reversals in response to changes in light intensity.     

Free-energy simulations reveal that both hydrophobic and polar interactions are important for influenza hemagglutinin antibody binding

Antibodies binding to conserved epitopes can provide a broad range of neutralization to existing influenza subtypes and may also prevent the propagation of potential pandemic viruses by fighting against emerging strands. Here we propose a computational framework to study structural binding patterns and detailed molecular mechanisms of viral surface glycoprotein hemagglutinin (HA) binding with a broad spectrum of neutralizing monoclonal antibody fragments (Fab). We used rigorous free-energy perturbation (FEP) methods to calculate the antigen-antibody binding affinities, with an aggregate underlying molecular-dynamics simulation time of several microseconds (～2 μs) using all-atom, explicit-solvent models. We achieved a high accuracy in the validation of our FEP protocol against a series of known binding affinities for this complex system, with <0.5 kcal/mol errors on average. We then introduced what to our knowledge are novel mutations into the interfacial region to further study the binding mechanism. We found that the stacking interaction between Trp-21 in HA2 and Phe-55 in the CDR-H2 of Fab is crucial to the antibody-antigen association. A single mutation of either W21A or F55A can cause a binding affinity decrease of ΔΔG > 4.0 kcal/mol (equivalent to an ～1000-fold increase in the dissociation constant K_d). Moreover, for group 1 HA subtypes (which include both the H1N1 swine flu and the H5N1 bird flu), the relative binding affinities change only slightly (< ±1 kcal/mol) when nonpolar residues at the αA helix of HA mutate to conservative amino acids of similar size, which explains the broad neutralization capability of antibodies such as F10 and CR6261. Finally, we found that the hydrogen-bonding network between His-38 (in HA1) and Ser-30/Gln-64 (in Fab) is important for preserving the strong binding of Fab against group 1 HAs, whereas the lack of such hydrogen bonds with Asn-38 in most group 2 HAs may be responsible for the escape of antibody neutralization. These large-scale simulations may provide new insight into the antigen-antibody binding mechanism at the atomic level, which could be essential for designing more-effective vaccines for influenza.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.