Summary
Transcription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long ...slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono‐allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady‐state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run‐on transcription analysis indicated that RNA polymerase was still fully associated with the promoter‐proximal half of the ‘active’ ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the ‘active’ ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.
Trypanosoma brucei brucei infects a wide range of mammals, but is unable to infect humans because this subspecies is lysed by normal human serum (NHS). The phenotype of cellular lysis is debated. For ...some authors the lysosome undergoes osmotic swelling due to massive influx of chloride ions from the cytoplasmic compartment, but others describe multiple small cytoplasmic vacuoles and general swelling of the cellular body. Using population-level imaging of live immobilized trypanosomes throughout the lysis process, we report that specific swelling of the lysosome is a genuine and major characteristic of NHS-mediated lysis and that this phenotype is independent of the strain of trypanosomes and of NHS aging or damaging. Thus, irrespective of experimental conditions NHS reproducibly induced the swelling of the parasite lysosome.
We have previously identified TGSGP as a gene specific to the Trypanosoma brucei gambiense subspecies. TGSGP is a truncated VSG-like telomeric gene transcribed by RNA polymerase II. The TGSGP protein ...localizes to the flagellar pocket, and exhibits features compatible with a role as surface receptor. Here we show that TGSGP is physically linked to a truncation of a gene homologous to yeast AUT1 (APG3), a gene involved in internal vesicular formation. Further analysis indicated that T. b. gambiense is heterozygous for AUT1 (AUT1/aut1), with each allele located on independent chromosome II homologues. In 18 T. b. gambiense isolates from distinct geographical origins and different hosts, this genomic rearrangement was conserved. The size of the intergenic region between TGSGP and truncated aut1 varied among isolates but was similar in isolates of the same geographical area, and this observation may be used in epidemiology to trace the geographical origin of T. b. gambiense isolates.
It has been observed in vitro that complete clot lysis is generally preceded by a slow phase of lysis during which the degradation seems to be inefficient. However, this slow regime was merely ...noticed, but not yet quantitatively discussed. In our experiments, we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average, the slow regime lasts longer than the fast one, meaning that during most of the process, the lysis is ineffective. We proposed a computational model in which the properties of the binding of the proteins change during the lysis: first, the biochemical reactions take place at the surface of the fibrin fibers, then in the bulk, resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally.
Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. ...Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.
Display omitted
•PI3K-C2α specifically produces PtdIns3P at the PRE•PI3K-C2α-dependent PtdIns3P modulates localization and activation of Rab11•PI3K-C2α controls the Rab11/Rab8 axis at the primary cilium base•PI3K-C2α is required for Smo ciliary targeting and Shh signaling
Multiple PI3Ks produce PtdIns3P, but whether enzyme specialization occurs in defined subcellular locations is unclear. Franco et al. report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) around the primary cilium base and regulates PtdIns3P-dependent membrane traffic required for Rab11 localization/activation, Smo ciliary targeting, and Shh signaling.
Vertebrate organs require locally adapted blood vessels
. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. ...Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals
. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.
Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and ...signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.
A novel way has been proposed to follow the formation of nanocrystalline magnetite. Iron oxide nanoparticles were synthesized by the thermal decomposition of Fe(acac)(3) in the presence of oleic acid ...and oleylamine surfactants at high temperature. The species produced during the synthetic process are characterized through their effects on the proton nuclear magnetic relaxation of the reaction medium and their sizes. As shown by transmission electron microscopy, photon correlation spectroscopy and x-ray diffraction, the diameter of nano-objects increases when the time synthesis is longer. Magnetic properties evaluated by nuclear magnetic resonance (NMRD profiles, T(1) and T(2) measurements) were correlated with the size parameters.
Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that
this protein contains a membrane pore-forming domain functionally similar to that
of bacterial colicins, flanked by a ...membrane-addressing domain. In lipid bilayer
membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei,
apolipoprotein L-I was targeted to the lysosomal membrane and triggered
depolarization of this membrane, continuous influx of chloride, and subsequent
osmotic swelling of the lysosome until the trypanosome lysed.
Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The ...underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about STAT3 in β-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese subjects with diabetes. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high-fat diet before analysis. Interestingly, β-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis with RNA sequencing showed that reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-β1H cells, confirmed in FACS-purified β-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-β1H cells and human islets, suggesting a mechanism for STAT3-modulated β-cell function. Our study postulates STAT3 as a novel regulator of β-cell function in obesity.
PDF
