A potential new, stable state of the E-cadherin strand-swapped dimer in solution

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution

Identifying the mechanism for superdiffusivity in mouse fibroblast motility.

We seek to characterize the motility of mouse fibroblasts on 2D substrates. Utilizing automated tracking techniques, we find that cell trajectories are super-diffusive, where displacements scale faster than t1/2 in all directions. Two mechanisms have been proposed to explain such statistics in other cell types: run and tumble behavior with Lévy-distributed run times, and ensembles of cells with heterogeneous speed and rotational noise. We develop an automated toolkit that directly compares cell trajectories to the predictions of each model and demonstrate that ensemble-averaged quantities such as the mean-squared displacements and velocity autocorrelation functions are equally well-fit by either model. However, neither model correctly captures the short-timescale behavior quantified by the displacement probability distribution or the turning angle distribution. We develop a hybrid model that includes both run and tumble behavior and heterogeneous noise during the runs, which correctly matches the short-timescale behaviors and indicates that the run times are not Lévy distributed. The analysis tools developed here should be broadly useful for distinguishing between mechanisms for superdiffusivity in other cells types and environments

Nuclear position relative to the Golgi body and nuclear orientation are differentially responsive indicators of cell polarized motility.

Cell motility is critical to biological processes from wound healing to cancer metastasis to embryonic development. The involvement of organelles in cell motility is well established, but the role of organelle positional reorganization in cell motility remains poorly understood. Here we present an automated image analysis technique for tracking the shape and motion of Golgi bodies and cell nuclei. We quantify the relationship between nuclear orientation and the orientation of the Golgi body relative to the nucleus before, during, and after exposure of mouse fibroblasts to a controlled change in cell substrate topography, from flat to wrinkles, designed to trigger polarized motility. We find that the cells alter their mean nuclei orientation, in terms of the nuclear major axis, to increasingly align with the wrinkle direction once the wrinkles form on the substrate surface. This change in alignment occurs within 8 hours of completion of the topographical transition. In contrast, the position of the Golgi body relative to the nucleus remains aligned with the pre-programmed wrinkle direction, regardless of whether it has been fully established. These findings indicate that intracellular positioning of the Golgi body precedes nuclear reorientation during mouse fibroblast directed migration on patterned substrates. We further show that both processes are Rho-associated kinase (ROCK) mediated as they are abolished by pharmacologic ROCK inhibition whereas mouse fibroblast motility is unaffected. The automated image analysis technique introduced could be broadly employed in the study of polarization and other cellular processes in diverse cell types and micro-environments. In addition, having found that the nuclei Golgi vector may be a more sensitive indicator of substrate features than the nuclei orientation, we anticipate the nuclei Golgi vector to be a useful metric for researchers studying the dynamics of cell polarity in response to different micro-environments

How Bacteria Respond to Material Stiffness during Attachment: A Role of <i>Escherichia coli</i> Flagellar Motility

Material stiffness has been shown to have potent effects on bacterial attachment and biofilm formation, but the mechanism is still unknown. In this study, response to material stiffness by <i>Escherichia coli</i> during attachment was investigated with biofilm assays and cell tracking using the Automated Contour-base Tracking for in Vitro Environments (ACT<i>IV</i>E) computational algorithm. By comparing the movement of <i>E. coli</i> cells attached on poly­(dimethylsiloxane) (PDMS) surfaces of different Young’s moduli (0.1 and 2.6 MPa, prepared by controlling the degree of cross-linking) using ACT<i>IV</i>E, attached cells on stiff surfaces were found more motile during early stage biofilm formation than those on soft surfaces. To investigate if motility is important to bacterial response to material stiffness, we compared <i>E. coli</i> RP437 and its isogenic mutants of flagellar motor (<i>motB</i>) and synthesis of flagella (<i>fliC</i>) and type I fimbriae (<i>fimA</i>) for attachment on 0.1 and 2.6 MPa PDMS surfaces. The <i>motB</i> mutant exhibited defects in response to PDMS stiffness (based on cell counting and tracking with ACT<i>IV</i>E), which was recovered by complementing the <i>motB</i> gene. Unlike <i>motB</i> results, mutants of <i>fliC</i> and <i>fimA</i> did not show significant defects on both face-up and face-down surfaces. Collectively, these findings suggest that <i>E. coli</i> cells can actively respond to material stiffness during biofilm formation, and <i>motB</i> is involved in this response