Claudin-3 in the non-neural ectoderm is essential for neural fold fusion in chicken embryos.

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.

NoLogo: a new statistical model highlights the diversity and suggests new classes of Crm1-dependent nuclear export signals.

BACKGROUND: Crm1-dependent Nuclear Export Signals (NESs) are clusters of alternating hydrophobic and non-hydrophobic amino acid residues between 10 to 15 amino acids in length. NESs were largely thought to follow simple consensus patterns, based on which they were categorized into 6-10 classes. However, newly discovered NESs often deviate from the established consensus patterns. Thus, identifying NESs within protein sequences remains a bioinformatics challenge. RESULTS: We describe a probabilistic representation of NESs using a new generative model we call NoLogo that can account for a large diversity of NESs. Using this model to predict NESs, we demonstrate improved performance over PSSM and GLAM2 models, but do not achieve the performance of the state-of-the-art NES predictor LocNES. Our findings illustrate that over 30% of NESs are best described by novel NES classes rather than the 6-10 classes proposed by current/existing models. Finally, many NESs have additional hydrophobic residues either upstream or downstream of the canonical four residues, suggesting possible functionality. CONCLUSION: Applying the NoLogo model highlights the observation that NESs are more diverse than previously appreciated. Our work questions the practice of assigning each NES to one of several predefined NES classes. Finally, our analysis suggests a novel and testable biophysical perspective on interaction between Crm1 receptor and Crm1-dependent NESs.

Parallel reorganization of protein function in the spindle checkpoint pathway through evolutionary paths in the fitness landscape that appear neutral in laboratory experiments.

Regulatory networks often increase in complexity during evolution through gene duplication and divergence of component proteins. Two models that explain this increase in complexity are: 1) adaptive changes after gene duplication, such as resolution of adaptive conflicts, and 2) non-adaptive processes such as duplication, degeneration and complementation. Both of these models predict complementary changes in the retained duplicates, but they can be distinguished by direct fitness measurements in organisms with short generation times. Previously, it has been observed that repeated duplication of an essential protein in the spindle checkpoint pathway has occurred multiple times over the eukaryotic tree of life, leading to convergent protein domain organization in its duplicates. Here, we replace the paralog pair in S. cerevisiae with a single-copy protein from a species that did not undergo gene duplication. Surprisingly, using quantitative fitness measurements in laboratory conditions stressful for the spindle-checkpoint pathway, we find no evidence that reorganization of protein function after gene duplication is beneficial. We then reconstruct several evolutionary intermediates from the inferred ancestral network to the extant one, and find that, at the resolution of our assay, there exist stepwise mutational paths from the single protein to the divergent pair of extant proteins with no apparent fitness defects. Parallel evolution has been taken as strong evidence for natural selection, but our results suggest that even in these cases, reorganization of protein function after gene duplication may be explained by neutral processes.

Recent Progress in CFTR Interactome Mapping and Its Importance for Cystic Fibrosis.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel found in secretory epithelia with a plethora of known interacting proteins. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease that leads to progressive respiratory illness and other complications of phenotypic variance resulting from perturbations of this protein interaction network. Studying the collection of CFTR interacting proteins and the differences between the interactomes of mutant and wild type CFTR provides insight into the molecular machinery of the disease and highlights possible therapeutic targets. This mini review focuses on functional genomics and proteomics approaches used for systematic, high-throughput identification of CFTR-interacting proteins to provide comprehensive insight into CFTR regulation and function.