The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity.     

Parentage analysis with few contributing breeders: validation and improvement

Validation of parental allocation using PAPA software (Duchesne P, Godbout MH, Bernatchez L. 2002. PAPA (package for the analysis of parental allocation): a computer program for simulated and real parental allocation. Mol Ecol Notes. 2:191-193.) was investigated under the assumption that only a small proportion of potential breeders contributed to the offspring sample. Inbreeding levels proved to have a large impact on allocation error rate. Consequently, simulations from artificial, unrelated parents may strongly underestimate allocation error, and so, whenever possible, simulations based on the actual parental genotypes should be run. An unexpected and interesting finding was that ambiguity (the highest likelihood is shared by several parental pairs) rates below 10% stood very close to exact allocation error rates (true proportions of wrong allocations). Hence, the ambiguity rate statistic may be viewed as a ready-made indicator of the resolution power of a specific parental allocation run and, if not exceeding 10%, used as an estimate of allocation error rate. It was found that the PAPA simulator, even with few contributing breeders, can be trusted to output reasonably accurate estimates of allocation error as long as those estimates do not exceed 15%. Indeed, most discrepancies between exact and estimated error then stood below 3%. Reproductive success variance had little impact on error estimate discrepancies within the same range. Finally, a (focal set) method was described to correct the estimated family sizes computed directly from parental allocations. Essentially, this method makes use of the detailed structure of the allocation probabilities associated with each parental pair with at least 1 allocated offspring. The allocation probabilities are expressed in matrix form, and the subsequent calculations are run based on standard matrix algebra. On average, this method provided better estimates of family sizes for each investigated combination of parameter values. As the size of offspring samples increased, the corrections improved until a plateau was finally reached. Typically, samples comprising 250, 500, and 1000 offspring would bring corrections in the order of 10-20%, 20-30%, and 30-40%, respectively.     

Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several μm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (～110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (μm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.     

Multivariate analysis of single quadrupole LC‐MS spectra for routine characterization and quantification of intact proteins

Modern high‐throughput proteomic platforms allow incomparable protein mixture resolution and identification. However, such sophisticated facilities are expensive and not always accessible for routine analysis of simple mixtures. In this paper, we propose a simple methodology, based on detection of intact, nondigested proteins by LC coupled to single quadrupole MS (sqLC‐MS), followed by the analysis of the resulting spectra by multivariate analysis (MA). By doing so, even large molecular weight (MW) proteins, generating complex spectra, can be characterized to a level that allows isoform discrimination, while standard algorithms, such as MS spectrum deconvolution, cannot. To demonstrate the effectiveness of the proposed approach, we have analyzed the spectra of a set of purified, intact albumins from seven different organisms (bovine, human, rabbit, rat, sheep, mouse, and pig) as a model of microheterogenous proteins, using Projection to Latent Structure Discriminant Analysis (PLS‐DA). Although these proteins are very similar (less than 1% difference in MW), sqLC‐MS/MA allowed their classification, and the identification of unknown source samples. In addition, MA allowed precise protein quantification from the same data (calibration curve R² = 0.9966). The ability to rapidly characterize and quantify proteins, together with simplicity and affordability, could make of combined sqLC‐MS/MA a routine method for the characterization of simple mixture of known proteins.     

Paget disease of bone: mapping of two loci at 5q35-qter and 5q31

Paget disease of bone is characterized by focal increases of the bone-remodeling process. It is the second most common metabolic bone disease after osteoporosis. Genetic factors play a major role in the etiology of Paget disease of bone, and two loci have been mapped for the disorder: PDB1 and PDB2. The gene(s) causing the typical form of the disorder remains to be characterized. To decipher the molecular basis of Paget disease of bone, we performed genetic linkage analysis in 24 large French Canadian families (479 individuals) in which the disorder was segregating as an autosomal dominant trait. After exclusion of PDB2, a genomewide scan was performed on the three most informative family nuclei. LOD scores >1.0 were observed at seven locations. The 24 families were then used to detect strong evidence for linkage to chromosome 5q35-qter. Under heterogeneity, a maximum LOD score of 8.58 was obtained at D5S2073, at straight theta= .1. The same characteristic haplotype was carried by all patients in eight families, suggesting a founder effect. A recombination event in a key family confined the disease region within a 6-cM interval between D5S469 and the telomere. The 16 other families, with very low conditional probability of linkage to 5q35-qter, were further used, to map a second locus at 5q31. Under heterogeneity, a maximum LOD score of 3.70 was detected at D5S500 with straight theta=.00. Recombination events refined the 5q31 region within 12.2 cM, between D5S642 and D5S1972. These observations demonstrate the mapping of two novel loci for Paget disease of bone and provide further evidence for genetic heterogeneity of this highly prevalent disorder. It is proposed that the 5q35-qter and 5q31 loci be named "PDB3" and "PDB4," respectively.     

Impact of clear-cutting and prescribed burning on microbial diversity and community structure in a Jack pine (Pinus banksiana Lamb.) clear-cut using Biolog Gram-negative microplates

The impacts of clear-cutting and clear-cutting followed by prescribed burning on soil microbial diversity and community structure were assessed using sole-carbon-source utilization. Microorganisms were washed from organic and mineral soil samples and used to inoculate Biolog Gram-negative microplates. Patterns of colour development were analysed using principal component analyses and calculation of Shannon diversity indices. No differences between treatments were detected when individual samples were used. However, both organic and mineral soil samples from the burned plot showed lower microbial diversity when pooled samples were examined.