Brush border myosin I (BBMI): a basally localized transcript in human jejunal enterocytes.

To extend our recent observation that villin mRNA, encoding an apical microvillous protein, is dichotomously localized in the basal region of human enterocytes, we examined the localization of mRNAs for brush border myosin I (BBMI) and intestinal fimbrin (I-fim). In situ hybridization indicated that BBMI mRNA localized to the basal region of human enterocytes, whereas the mRNA for I-fim distributed diffusely. To facilitate study of potential mechanisms of mRNA targeting, we cloned a full-length cDNA for BBMI including its 5'- and 3'-untranslated regions (UTRs). This cDNA shares 86% sequence identity with bovine BBMI and 85% with rat BBMI. Sequence analysis revealed no obvious similarity between the 3'-UTRs of BBMI and villin. This study provides evidence of novel sorting pathways for intestinal microvillous cytoskeletal proteins.     

Tension, free space, and cell damage in a microfluidic wound healing assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.     

Molecular evidence for the introgression between Hylobates lar and H. pileatus in the wild

Inter-specific hybrid zones for Hylobates gibbons are known in Southeast Asia. Among these, one hybrid zone between Hylobates lar and H. pileatus is located in Khao Yai National Park, Thailand. To find molecular evidence for the natural hybridization of the gibbons in this region, we studied mitochondrial DNA (mtDNA) of 68 gibbons of the H. lar phenotype living adjacent to the hybrid zone. Nucleotide sequencing of a fragment of mtDNA spanning hyper variable segment I showed that nine gibbons had an mtDNA haplotype of H. pileatus, and that seven of these nine gibbons belonged to a single maternal lineage over three generations. It is thus confirmed that introgression between H. lar and H. pileatus exists and the initial hybridization took place ages ago.     

Structure and dynamics of macrophage podosomes

Podosomes are short-lived, dynamic actin-rich adhesions classically formed in macrophages, osteoclasts and Src-transformed fibroblasts. Though related to the more commonly studied focal adhesions, sharing several structural and regulatory proteins, podosomes have distinct functional, structural, and dynamic characteristics. Here, we discuss current understanding of the function of podosomes in disparate cell types and how this relates to their structure and dynamics.     

Differential stabilization of two hydrophobic cores in the transition state of the villin 14T folding reaction

We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.     

TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.