Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.     

Small Heat-Shock Proteins,IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox^-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox^-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains.     

Mathematical Models for Cell Migration with Real-Time Cell Cycle Dynamics

The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

A case study for late Archean and Proterozoic biogeochemical iron‐ and sulphur cycling in a modern habitat—the Arvadi Spring

As a consequence of Earth's surface oxygenation, ocean geochemistry changed from ferruginous (iron(II)‐rich) into more complex ferro‐euxinic (iron(II)‐sulphide‐rich) conditions during the Paleoproterozoic. This transition must have had profound implications for the Proterozoic microbial community that existed within the ocean water and bottom sediment; in particular, iron‐oxidizing bacteria likely had to compete with emerging sulphur‐metabolizers. However, the nature of their coexistence and interaction remains speculative. Here, we present geochemical and microbiological data from the Arvadi Spring in the eastern Swiss Alps, a modern model habitat for ferro‐euxinic transition zones in late Archean and Proterozoic oceans during high‐oxygen intervals, which enables us to reconstruct the microbial community structure in respective settings for this geological era. The spring water is oxygen‐saturated but still contains relatively elevated concentrations of dissolved iron(II) (17.2 ± 2.8 μM) and sulphide (2.5 ± 0.2 μM) with simultaneously high concentrations of sulphate (8.3 ± 0.04 mM). Solids consisting of quartz, calcite, dolomite and iron(III) oxyhydroxide minerals as well as sulphur‐containing particles, presumably elemental S⁰, cover the spring sediment. Cultivation‐based most probable number counts revealed microaerophilic iron(II)‐oxidizers and sulphide‐oxidizers to represent the largest fraction of iron‐ and sulphur‐metabolizers in the spring, coexisting with less abundant iron(III)‐reducers, sulphate‐reducers and phototrophic and nitrate‐reducing iron(II)‐oxidizers. 16S rRNA gene 454 pyrosequencing showed sulphide‐oxidizing Thiothrix species to be the dominating genus, supporting the results from our cultivation‐based assessment. Collectively, our results suggest that anaerobic and microaerophilic iron‐ and sulphur‐metabolizers could have coexisted in oxygenated ferro‐sulphidic transition zones of late Archean and Proterozoic oceans, where they would have sustained continuous cycling of iron and sulphur compounds.     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

A striking organization of a large family of human neural cadherin-like cell adhesion genes.

We have identified 52 novel human cadherin-like genes organized into three closely linked clusters. Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.