Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.     

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.     

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.     

Studies on the mechanism of crown-ether-induced activation of enzymes in non-aqueous media.

Studies on the mechanism of crown-ether-induced activation are described in this paper. Michaelis Menten kinetics of -chymotrypsin in toluene in the presence and absence of 18-crown-6 showed that only V_max is increased upon crown ether treatment. Parallel Lineweaver–Burk plots indicate that crown ethers do not activate the enzyme by specific interactions in the active site, such as transition state stabilization or facilitated transport of water molecules. Increased V_max values of crown-ether-treated enzyme most probably originate from conformational changes, which alter k_cat as well as the amount of catalytically active enzyme.     

Essential fatty acid status of laboratory-reared mosquitos improved by supplementing crude larval foods with fish oils

Labotatory mosquitos are commonly maintained using larval media based on crude food materials such as powdered animal chows, dried yeast, liver powder, etc. Males of Culex tarsalis so reared for use in studies of sterile male control methods proved unsuccessful in competition for mates against wild males in the field, even if tested after one generation of laboratory rearing. Such laboratory-reared adults had much lower levels of an essential fatty acid, eicosapentaenoic acid (EPA) than wild C. tarsalis adults from the sterile-male-control experimental sites. Hypothesizing that suboptimal essential fatty acid status might reduce adult vigor, we attempted to raise levels of EPA in tissues of laboratory-reared C. tarsalis by supplementing the larval food mixture with fish oils, which are rich sources of EPA and related n3 polyunsaturated fatty acids. Supplementation with codliver oil doubled tissue phospholipid EPA levels compared with unsupplemented controls, and a proprietary fish oil, Walgreen EPA 500, trebled the level, as high as for wild adults. Oil-supplemented diets improved growth, as judged by bigger pupae and increased female adult longevity, and flight mill studies provided some evidence that improved EPA status improved flight. Although the phenotypic contribution to mating inferiority of laboratory-reared mosquitos remains unclear, this work illustrates how an apparently satisfactory stock rearing regimen may be cryptically suboptimal with respect to an essential nutrient.

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Résumé Les souches de laboratoire de moustiques sont généralement conservées en utilisant un régime larvaire brut tel que pâtée pour animaux domestiques, levure desséchée, poudre de foie, etc. Les mâles de C. tarsalis élevés de la sorte se sont révélés défavorisés dans la compétition sexuelle avec des mâles sauvages dans la nature, lors d'expériences sur l'utilisation de mâles stériles dans la lutte contre les moustiques, même si la compétition a eu lieu après un seul cycle larvaire en élevage au laboratoire. Les adultes provenant de ces élevages avaient une teneur en acide eicosapentaenoique (EPA),-acide gras essentiel-, beaucoup plus faible que les adultes provenant de la nature. En envisageant qu'une teneur suboptimale en acides gras essentiels pouvait diminuer la vigueur des adultes, nous avons tenté d'augmenter la teneur en EPA des tissus de C. tarsalis élevés au laboratoire en complétant le régime avec des huiles de poisson qui sont riches en EPA et en acides gras polyinsaturés n3 apparentés. L'addition d'huile de foie de moure double la teneur des tissus en phospholipides EPA par rapport aux témoins, et la spécialité Walgreen EPA 500, triple la teneur en EPA, la mettant au niveau des adultes de la nature. Les régimes complétés en huile ont amélioré la croissance (pupes plus grosses et longévité des femelles accrue), et quelques études en moulin à vol ont montré que l'amélioration de la teneur en EPA modifiait les caractéristiques du vol. Bien que ce travail n'ait pas permis de clarifier la part phénotypique de l'infériorité des mâles des moustiques élevés au laboratoire, son intérêt général a été de montrer comment un régime apparemment satisfaisant peut être en réalité suboptimal en ce qui concerne un aliment essentiel.

     

Cytokinesis in bacteria.

Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.