Design and Validation of a Tool for Neurite Tracing and Analysis in Fluorescence Microscopy Images

Background: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. Methods: The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. Results: Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3. Conclusions: Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods

BKV Agnoprotein Interacts with α-Soluble N-Ethylmaleimide-Sensitive Fusion Attachment Protein, and Negatively Influences Transport of VSVG-EGFP

Background: The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. Principal Findings: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them a-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and a-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and a-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with a-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. Conclusions: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with a-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence

Role of 3′UTRs in the Translation of mRNAs Regulated by Oncogenic eIF4E—A Computational Inference

Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3′UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3′UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5′UTR and microRNA binding at the 3′UTR

Endosomal Trafficking Of Ampa-Type Glutamate Receptors

Many different forms of synaptic plasticity have been shown to ultimately modulate the number of AMPA-type glutamate receptors at the synapse. This trafficking involves lateral movements between synaptic and extrasynaptic sites at the neuron surface, as well as vesicular transport between the plasma membrane and intracellular compartments. Several new studies have shed light on the location and regulation of AMPA-type receptor (AMPAR) endocytosis, their intracellular sorting to divergent pathways at the level of endosomes, and the mechanism and sites of receptor recycling. This review summarizes this recent data on the trafficking along the endocytic pathway, and follows the path of internalized AMPAR from endocytosis up to sites of recycling. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved