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Chia I Grote D Marcotte M Batourina E Mendelsohn C Bouchard M 《Development (Cambridge, England)》2011,138(10):2089-2097
Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility. 相似文献
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Introduction
The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis. 相似文献24.
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An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA(Ser(CUA)) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein. 相似文献
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This protocol describes how to perform comparative measurements of the permeability of the nuclear envelope in adherent cells. The plasma membrane is permeabilized at low digitonin concentrations, leaving the nuclear membrane intact. These semi-permeabilized cells are incubated with cytosolic extracts prepared in advance and with a fluorescent reporter molecule whose molecular weight exceeds the size-exclusion limit of the nuclear envelope. Images are taken with a confocal microscope and subsequently analyzed using a custom-made software program that recognizes the nuclei automatically and calculates the mean nuclear fluorescence signal. Here, we measure the increase in nuclear permeability triggered by cytosolic extracts from cells dying by apoptosis. This method can be employed for the study of processes that affect the nucleocytoplasmic distribution of fluorescent molecules in cell populations. The large size of the samples means that subtle fluctuations in nuclear fluorescence can be detected with a high confidence level. Isolation of cell extracts takes 5-6 h, and the preparation and imaging of 15 or so specimens takes 4-5 h. 相似文献
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Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion. 相似文献
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Jens Grote Simone König Doreen Ackermann Claudia Sopalla Malgorzata Benedyk Marek Los Claus Kerkhoff 《BMC molecular biology》2006,7(1):48-10