MinC spatially controls bacterial cytokinesis by antagonizing the scaffolding function of FtsZ

BACKGROUND: Cytokinesis in bacteria is mediated by a cytokinetic ring, termed the Z ring, which forms a scaffold for recruitment of other cell-division proteins. The Z ring is composed of FtsZ filaments, but their organization in the Z ring is poorly understood. In Escherichia coli, the Min system contributes to the spatial regulation of cytokinesis by preventing the assembly of the Z ring away from midcell. The effector of the Min system, MinC, inhibits Z ring assembly by a mechanism that is not clear. RESULTS: Here, we report that MinC controls the scaffolding function of FtsZ by antagonizing the mechanical integrity of FtsZ structures. Specifically, MinC antagonizes the ability of FtsZ filaments to be in a solid-like gel state. MinC is a modular protein whose two domains (MinC(C) and MinC(N)) synergize to inhibit FtsZ function. MinC(C) interacts directly with FtsZ polymers to target MinC to Z rings. MinC(C) also prevents lateral interactions between FtsZ filaments, an activity that seems to be unique among cytoskeletal proteins. Because MinC(C) is inhibitory in vivo, it suggests that lateral interactions between FtsZ filaments are important for the structural integrity of the Z ring. MinC(N) contributes to MinC activity by weakening the longitudinal bonds between FtsZ molecules in a filament leading to a loss of polymer rigidity and consequent polymer shortening. On the basis of our results, we develop the first computational model of the Z ring and study the effects of MinC. CONCLUSIONS: Control over the scaffolding activity of FtsZ probably represents a universal regulatory mechanism of bacterial cytokinesis.     

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.     

Multiple loci are associated with white blood cell phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.     

Vesicle-associated membrane protein-2 (VAMP2) mediates cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ~50% and impaired cAMP-stimulated exocytosis by ~50%, as monitored with FM1-43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ~50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.     

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.     

Regulation of bidirectional melanosome transport by organelle bound MAP kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.     

Soil and greenhouse gas responses to biochar additions in a temperate hardwood forest

Biochar additions can improve soil fertility and sequester carbon, but biochar effects have been investigated primarily in agricultural systems. Biochar from spruce and maple sawdust feedstocks (with and without inorganic phosphorus in a factorial design) were added to plots in a commercially managed temperate hardwood forest stand in central Ontario, Canada; treatments were applied as a top‐dressing immediately prior to fall leaf abscission in September 2011. Forests in this region have acidic, sandy soils, and due to nitrogen deposition may exhibit phosphorus, calcium, and magnesium limitation. To investigate short‐term impacts of biochar application on soil nutrient supply and greenhouse gas fluxes as compared to phosphorus fertilization, data were collected over the first year after treatment application; linear mixed models were used to analyze data. Two to six weeks after treatment application, there were higher concentrations of potassium in spruce and maple biochar plots, and phosphorus in spruce biochar plots, as compared to the control treatment. There were higher concentrations of calcium, magnesium, and phosphorus in the phosphorus plots. In the following spring and summer (9–12 months after treatment application), there were higher soil calcium concentrations in maple biochar plots, and phosphorus plots still had higher soil phosphorus concentrations than control plots. No treatment effects on fluxes of carbon dioxide, methane, or nitrous oxide were detected in the field; however, laboratory incubations after 12 months showed higher microbial respiration in soils from maple biochar plots as compared to spruce biochar, despite no effect on microbial biomass. The results suggest that the most important short‐term impact of biochar additions in this system is the increased supply of the limiting plant nutrients phosphorus and calcium. We expect that larger changes in mineral soil physical and chemical properties will occur when the surface‐applied biochar becomes incorporated into the soil after a few years.     

Tree species effects on ecosystem water-use efficiency in a high-elevation, subalpine forest

Ecosystem water-use efficiency (eWUE; the ratio of net ecosystem productivity to evapotranspiration rate) is a complex landscape-scale parameter controlled by both physical and biological processes occurring in soil and plants. Leaf WUE (lWUE; the ratio of leaf CO₂ assimilation rate to transpiration rate) is controlled at short time scales principally by leaf stomatal dynamics and this control varies among plant species. Little is known about how leaf-scale variation in lWUE influences landscape-scale variation in eWUE. We analyzed approximately seven thousand 30-min averaged eddy covariance observations distributed across 9 years in order to assess eWUE in two neighboring forest communities. Mean eWUE was 19% lower for the community in which Engelmann spruce and subalpine fir were dominant, compared to the community in which lodgepole pine was dominant. Of that 19% difference, 8% was attributed to residual bias in the analysis that favored periods with slightly drier winds for the spruce-fir community. In an effort to explain the remaining 11% difference, we assessed patterns in lWUE using C isotope ratios. When we focused on bulk tissue from older needles we detected significant differences in lWUE among tree species and between upper and lower canopy needles. However, when these differences were scaled to reflect vertical and horizontal leaf area distributions within the two communities, they provided no power to explain differences in eWUE that we observed in the eddy covariance data. When we focused only on bulk needle tissue of current-year needles for 3 of the 9 years, we also observed differences in lWUE among species and in needles from upper and lower parts of the canopy. When these differences in lWUE were scaled to reflect leaf area distributions within the two communities, we were able to explain 6.3% of the differences in eWUE in 1 year (2006), but there was no power to explain differences in the other 2 years (2003 and 2007). When we examined sugars extracted from needles at 3 different times during the growing season of 2007, we could explain 3.8–6.0% of the differences in eWUE between the two communities, but the difference in eWUE obtained from the eddy covariance record, and averaged over the growing season for this single year, was 32%. Thus, overall, after accounting for species effects on lWUE, we could explain little of the difference in eWUE between the two forest communities observed in the eddy covariance record. It is likely that water and C fluxes from soil, understory plants, and non-needle tissues, account for most of the differences observed in the eddy covariance data. For those cases where we could explain some of the difference in eWUE on the basis of species effects, we partitioned the scaled patterns in lWUE into two components: a component that is independent of canopy leaf area distribution, and therefore only dependent on species-specific differences in needle physiology; and a component that is independent of species differences in needle physiology, and only dependent on species-specific influences on canopy leaf area distribution. Only the component that is dependent on species influences on canopy leaf area distribution, and independent of inherent species differences in needle physiology, had potential to explain differences in eWUE between the two communities. Thus, when tree species effects are important, canopy structure, rather than species-specific needle physiology, has more potential to explain patterns in eWUE.     

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.     

Revising Berg-Purcell for finite receptor kinetics

From nutrient uptake to chemoreception to synaptic transmission, many systems in cell biology depend on molecules diffusing and binding to membrane receptors. Mathematical analysis of such systems often neglects the fact that receptors process molecules at finite kinetic rates. A key example is the celebrated formula of Berg and Purcell for the rate that cell surface receptors capture extracellular molecules. Indeed, this influential result is only valid if receptors transport molecules through the cell wall at a rate much faster than molecules arrive at receptors. From a mathematical perspective, ignoring receptor kinetics is convenient because it makes the diffusing molecules independent. In contrast, including receptor kinetics introduces correlations between the diffusing molecules because, for example, bound receptors may be temporarily blocked from binding additional molecules. In this work, we present a modeling framework for coupling bulk diffusion to surface receptors with finite kinetic rates. The framework uses boundary homogenization to couple the diffusion equation to nonlinear ordinary differential equations on the boundary. We use this framework to derive an explicit formula for the cellular uptake rate and show that the analysis of Berg and Purcell significantly overestimates uptake in some typical biophysical scenarios. We confirm our analysis by numerical simulations of a many-particle stochastic system.