Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

Module-based systematic construction of plasmids for episomal gene expression in fission yeast.

The fission yeast Schizosaccharomyces pombe is a powerful model organism for cell biology and molecular biology, as genetic manipulation is easily achieved. Introduction of exogenous genes cloned in episomal plasmids into yeast cells can be done through well-established transformation methods. For expression of genes in S. pombe cells, the multi-copy plasmid pREP1 and its derivatives, including pREP41 and pREP81, have been widely used as vectors. Although recent advancement of technology brought a number of useful genetic elements such as new promoters, selection marker genes and fluorescent protein tags, introduction of those elements into conventional pREP1 requires a large commitment of both time and effort because cloning procedures need to be repeated until the final products are constructed. Here, we introduce materials and methods to construct many pREP1-type plasmids easily and systematically using the Golden Gate shuffling method, which enables one-step ligation of many DNA fragments into a plasmid. These materials and methods support creation of expression plasmids employing a variety of novel genetic elements, which will further facilitate genetic studies using S. pombe.