Cell polarity is a fundamental property of most animal cells and is critical during development and for most cell and tissue functions. Epithelial cells are organized into apical and basolateral compartments, and this intrinsic cellular asymmetry is essential for all functions that are carried out by epithelial tissue. The establishment of a polarized epithelial phenotype is orchestrated by major rearrangements of the cell cytoskeleton, polarized membrane trafficking, the formation and maturation of epithelial cell junctions, cell signaling pathways, and the generation of cortical phospholipid asymmetry. These processes need to be coordinated precisely in time and space and integrated with physical and chemical signals from the environment, failure of which leads to severe developmental disorders and various human diseases. At the heart of this regulatory network are the evolutionarily conserved polarity modules Par, Crumbs, and Scribble, whose components engage in complex cooperative and antagonistic interactions to compartmentalize and functionalize the epithelial cell cortex and to control the spatiotemporal activity of downstream polarity effectors. In this review, we will discuss recent insights into the organization and regulation of the mammalian Par and Crumbs modules and outline a hypothetical framework of how these proteins orchestrate epithelial polarity development, HIPPO signaling, and actomyosin activity at the apical–lateral border.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.

CFTR (ABCC7) is a phospho-regulated chloride channel that is found in the apical membranes of epithelial cells, is gated by ATP and the activity of the protein is crucial in the homeostasis of the extracellular liquid layer in many organs [ (2008) , 701-726; (1989) , 1066-1073]. Mutations in CFTR cause the inherited disease cystic fibrosis (CF), the most common inherited condition in humans of European descent [ (1989) , 1066-1073; (2007) , 555-567]. The structural basis of CF will be discussed in this article.

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction and molecular recognition. However, this information has often been gleaned under extremely non-physiological protein, salt, and H + concentrations. Here, we describe the development of a robust Inka1-Box (iBox)-PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semiquantitative assay described here allows the measurement of in-vivo protein-protein interactions using a novel GFP-linked reporter system which produces fluorescent readouts from protein crystals. We combined this assay with invitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between PDZ2 domain of Na + /H + exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.The catalytic domain of the serine/threonine kinase PAK4 (PAK4cat) and its endogenous inhibitor Inka1 have recently been shown to readily form rod-type crystals on co-transfection into a variety of mammalian cell types [1]. Crystals formed following transfection of a fusion construct comprised of a central 38 residue region of Inka1, termed the Inka1-Box (iBox), and PAK4cat (iBox-PAK4cat) were diffracted at the synchrotron beamline, enabling the first in-cellulo human protein structure to be determined to 2.95 Å (PDB: 4XBR). The crystal lattice revealed that iBox-PAK4cat forms a hexagonal array with channels 80 Å in diameter that run the length of the crystal. The size of this cavity allowed a guest protein (GFP) to be incorporated into the crystal lattice [1]. This, as well as the relative ease with https://www.jbc.org/cgi/