Tracking bio‐molecules in live cells using quantum dots

Single particle tracking (SPT) techniques were developed to explore bio‐molecules dynamics in live cells at single molecule sensitivity and nanometer spatial resolution. Recent developments in quantum dots (Qdots) surface coating and bio‐conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell experiments. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Tumor necrosis factor receptor-associated factor 4 is a dynamic tight junction-related shuttle protein involved in epithelium homeostasis

Background

Despite numerous in vivo evidences that Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) plays a key biological function, how it works at the cellular and molecular level remains elusive.

Methodology/Principal Findings

In the present study, we show using immunofluorescence and immuohistochemistry that TRAF4 is a novel player at the tight junctions (TJs). TRAF4 is connected to assembled TJs in confluent epithelial cells, but accumulates in the cytoplasm and/or nucleus when TJs are open in isolated cells or EGTA-treated confluent cells. In vivo, TRAF4 is consistently found at TJs in normal human mammary epithelia as well as in well-differentiated in situ carcinomas. In contrast, TRAF4 is never localized at the plasma membrane of poorly-differentiated invasive carcinomas devoid of correct TJs, but is observed in the cytoplasm and/or nucleus of the cancer cells. Moreover, TRAF4 TJ subcellular localization is remarkably dynamic. Fluorescence recovery after photobleaching (FRAP) experiments show that TRAF4 is highly mobile and shuttles between TJs and the cytoplasm. Finally, we show that intracellular TRAF4 potentiates ERK1/2 phosphorylation in proliferating HeLa cells, an epithelial cell line known to be devoid of TJs.

Conclusions/Significance

Collectively, our data strongly support the new concept of TJs as a dynamic structure. Moreover, our results implicate TRAF4 in one of the emerging TJ-dependent signaling pathways that responds to cell polarity by regulating the cell proliferation/differentiation balance, and subsequently epithelium homeostasis. Drastic phenotypes or lethality in TRAF4-deficient mice and drosophila strongly argue in favor of such a function.     

The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.     

Molecular Evidence of Reproductive Isolation in Sympatric Sibling Species of Mouse Lemurs

Recent morphological and molecular phylogenetic studies of mouse lemurs (Microcebus) living in the western and southern regions of Madagascar have shown that specific diversity had been considerably underestimated. In large part, this underestimate was due to the lack of sufficient specimens from given localities to assess properly the level of phenotypic variation within and between populations. The accurate delineation of specific boundaries has no doubt been confounded by the diminutive size, nocturnal habits, and subtle morphological variation characteristic of mouse lemurs, which can make field identification of individuals problematic. We illustrate the use of molecular phylogenetic analysis to reveal reproductive isolation in two sympatric mouse lemur species, Microcebus murinus and M. griseorufus. Their documentation in the Berenty Private Reserve in the extreme south of Madagascar verifies the historically-broad distribution of Microcebus griseorufus, a species recently resurrected from synonomy.     

Taste,movement, and death: Varying effects of new prospero mutants during Drosophila development

The PGal4 transposon inserted upstream of the pan‐neural gene prospero (pros) causes several neural and behavioral defects in the Voila¹ strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new pros^V alleles, including the null allele pros^V17. Here, we describe the relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response. These two behaviors showed varying degrees of variation depending upon the pros allele. We also found a good relation between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 1–13, 2003     

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH 相似文献   

Increased diaphragmatic strength and tolerance to fatigue after bilateral lung transplantation: an electromyographic study.

We evaluated the diaphragmatic function of seven patients with severe chronic respiratory failure before and after a bilateral lung transplantation (BLT), with follow-up at one year of pulmonary function tests, maximal inspiratory mouth pressure (MIP) and surface diaphragmatic electromyogram (Edi). The patients were asked to sustain target inspiratory pressures at -15, -30, and -50 cmH(2)O. We measured the endurance time (Tlim) to sustain inspiratory efforts and the power spectrum density function of Edi at each inspiratory maneuver. The Edi power spectra was analysed in terms of median frequency (MF), total power (TP) and energies in high-and low-frequency bands (EL and EH). Before BLT, a defect of the diaphragmatic function was evident: MIP was 62+/-7% of the predicted value and the Tlim measured at each inspiratory effort was very short ( 13+/-1 s, 10+/-1 s and 8+/-1 s at pressures of -15, -30, and -50 cmH(2)O, respectively). One month after BLT, the Tlim began to increase at all target inspiratory pressures and at 6 months MIP recovered to normal values. One month after BLT, there was a significant decrease in TP measured at the beginning of each inspiratory efforts and also an increase in the concomitant MF value. BLT markedly accentuated the maximal variations of TP, MF and low-frequency Edi energy. Some hypotheses are raised to explain this dramatic improvement in diaphragmatic function after BLT.