Rho-associated kinase (ROCK) is

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs, and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here, we create a culture system in which these mesoderm-like cell types and paraxial and lateral plate mesoderm-like cells are separately generated from hPSCs. We recapitulate nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which are subsequently differentiated into glomeruli, renal tubules, and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and disease and also supply cell sources for regenerative therapies.

Highlights d Induced ureteric bud (iUB) organoids have tubular lumens and repeat branching d In vitro monitoring and expansion methods for UB tip cells are established d iUB organoids can be differentiated into collecting duct progenitors d iUB organoids can be used to model multicystic dysplastic kidney (MCDK)

SummaryRegulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-a as an mDia-binding protein. The binding is mediated through the central region of Liprin-a and through the Nterminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-a competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-a fragment containing the mDiabinding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-a by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-a negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.