Transcriptional Activator-Like Effector (TALE) proteins have become focused as designable DNA-targeting tool. TALEs comprise of a tandem array of TAL-repeats, each of which repeat recognizes a specific single base-pair. TALE is, thus, designed to bind at any predefined genomic site by crafting an array of TAL-repeats to recognize DNA sequence typically in 17 base-pair long. Fusion of TALE to a protein module produces an orthogonal gene-targeting modifier, including TALE-nuclease (TALEN) in genome editing. In spite of prevailing TALEs applications, it still remains elusive how TALE proteins recognize DNA sequence. Here, we review the biophysical aspects of TALE-DNA interactions.
How do organs reach their reproducible shapes? This question remains open because it has been difficult to connect the microscopic dynamics/stochasticity to the macroscopic regulation/reproducibility at the molecular-cellular-organ level. We challenged by data science to quantify the heterogeneity of microtubule alignments to connect the molecular dynamics to the regulation of cell shape, and to quantify the variability of cell growth to connect the cellular stochasticity to the reproducible organ shape. Our key and counterintuitive result was “cellular randomness is required for reaching the correct organ size and shape” meaning that the order can be emerged from disorder.
Well-designed microscopic elasticity gradients on cell culture substrate can be an useful platform for investigating the mechanics of cell movements. To understand mechanical aspect of cell movement, we have focused on durotaxis which is a form of cell migration guided by elasticity gradient on extracellular millieu. To systematically investigate the effect of the elasticity gradient on emergence of cell polarity that determines direction of cell movement, we have originally developed microelastically-patterned hydrogel substrate, which enable manipulating the cell durotaxis. Width, strength, and curvature of the elasticity gradient around the elasticity transition region could be successfully designed by employing the mask-free photolithographic microelasticity patterning system. In this mini-review, the following three issues are discussed: 1) determining a threshold strength of elasticity gradient that induces durotaxis, 2) designing an asymmetric elastic gradient that rectifies durotactic cells, and 3) investigating how the durotaxis depends on curvatures on the elasticity boundaries.
Based on the structure of invertebrate-type lyzozyme, Venerupis philippinarum lysozyme (VpL), we designed Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEWL) mutants. Both lysozyme mutants possessed lysozyme acitivity like VpL and HEWL. Although Asn46Glu/Asp52Ser mutant formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer, however, the turnover rate of estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. From these results, we discussed the catalytic mechanism of lysozymes.
In living cells, the 3D architecture of molecular assemblies such as chromosomes, lipid bilayers, and the cytoskeleton is regulated through the interaction among their component molecules. Monitoring the position and orientation of constituent molecules is important for understanding the mechanisms that govern the structure and function of these assemblies. We have developed an instantaneous fluorescence polarization microscope to track the position and orientation of fluorescently labeled particles, including single molecules, which form micron-scale macromolecular assemblies in living cells. Our imaging approach is broadly applicable to the study of dynamic molecular interactions that underpin the function of micron-scale assemblies in living cells.
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