BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.     

Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism''s niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).     

Non-ideal effects in indentation testing of soft tissues

Indentation has several advantages as a loading mode for determining constitutive behavior of soft, biological tissues. However, indentation induces a complex, spatially heterogeneous deformation field that creates analytical challenges for the calculation of constitutive parameters. As a result, investigators commonly assume small indentation depths and large sample thicknesses to simplify analysis and then restrict indentation depth and sample geometry to satisfy these assumptions. These restrictions limit experimental resolution in some fields, such as brain biomechanics. However, recent experimental evidence suggests that conventionally applied limits are in fact excessively conservative. We conducted a parametric study of indentation loading with various indenter geometries, surface interface conditions, sample compressibility, sample geometry and indentation depth to quantitatively describe the deviation from previous treatments that results from violation of the assumptions of small indentation depth and large sample thickness. We found that the classical solution was surprisingly robust to violation of the assumption of small strain but highly sensitive to violation of the assumption of large sample thickness, particularly if the indenter was cylindrical. The ramifications of these findings for design of indentation experiments are discussed and correction factors are presented to allow future investigators to account for these effects without recreating our finite element models.     

Trpm4 Gene Invalidation Leads to Cardiac Hypertrophy and Electrophysiological Alterations

Rationale

TRPM4 is a non-selective Ca²⁺-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.

Objectives

We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 ^-/-) model.

Methods and Results

Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 ^-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4^+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4^-/- mice were smaller than Trpm4^+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4^-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 ^-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4^-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 ^-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca²⁺ or K⁺ currents involved in the repolarizing phase.

Conclusions

TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.     

Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

Osmotic stress is a potent regulator of biological function in many cell types, but its mechanism of action is only partially understood. In this study, we examined whether changes in extracellular osmolality can alter chromatin condensation and the rate of nucleocytoplasmic transport, as potential mechanisms by which osmotic stress can act. Transport of 10 kDa dextran was measured both within and between the nucleus and the cytoplasm using two different photobleaching methods. A mathematical model was developed to describe fluorescence recovery via nucleocytoplasmic transport. As osmolality increased, the diffusion coefficient of dextran decreased in the cytoplasm, but not the nucleus. Hyper-osmotic stress decreased nuclear size and increased nuclear lacunarity, indicating that while the nucleus was getting smaller, the pores and channels interdigitating the chromatin had expanded. The rate of nucleocytoplasmic transport was increased under hyper-osmotic stress but was insensitive to hypo-osmotic stress, consistent with the nonlinear osmotic properties of the nucleus. The mechanism of this osmotic sensitivity appears to be a change in the size and geometry of the nucleus, resulting in a shorter effective diffusion distance for the nucleus. These results may explain physical mechanisms by which osmotic stress can influence intracellular signaling pathways that rely on nucleocytoplasmic transport.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.     

The depletion attraction: an underappreciated force driving cellular organization

Cellular structures are shaped by hydrogen and ionic bonds, plus van der Waals and hydrophobic forces. In cells crowded with macromolecules, a little-known and distinct force-the "depletion attraction"-also acts. We review evidence that this force assists in the assembly of a wide range of cellular structures, ranging from the cytoskeleton to chromatin loops and whole chromosomes.     

Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.     

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathione S-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.