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Human epithelial organoids—3D spheroids derived from adult tissue stem cells—enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24–48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology—specifically the impact of host–microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research. The polarity of gastrointestinal organoids is reversed to study epithelial biology and host–microbe interactions. Access to the apical surface of the epithelium is increased while preserving epithelial integrity and secretory and absorptive functions.

Key Points Planar cell polarity (PCP) is a polarity axis that organizes cells in the plane of the tissue. PCP is conserved in metazoans and is essential for proper development and tissue homeostasis. Asymmetric and mutually exclusive subcellular enrichment of key PCP proteins patterns cells in planar-polarized tissues. PCP proteins also coordinate planar polarity between cells and control polarized behaviours by modulating the cytoskeleton. PCP patterns develop gradually from an initially disordered state through dynamic trafficking and various feedback interactions that can influence protein localization and stability. PCP patterns seem to be globally oriented along a pre-defined axis in a given tissue. Notably, multiple mechanistic inputs may have differential influences on PCP patterning depending on developmental timing and tissue context, and may only partially overlap in different contexts. The morphogenetic events governed by PCP signalling are best understood in Drosophila melanogaster , in which the particular orientation of hairs and bristles on the fly body has served to unravel basic principles of PCP-dependent processes. Information obtained from this model has helped to better understand equivalent mechanisms in vertebrates, particularly in the context of the orientation of fluid flow mediated by multiciliated cells and cell rearrangements during convergent extension. Mutations in PCP genes have been implicated in diverse human pathologies, and the body of evidence supporting the involvement of PCP aberrations in human birth defects continues to grow rapidly. Planar cell polarity — the asymmetric distribution of proteins in the plane of a cell sheet — dictates the orientation of various subcellular structures and drives collective cell rearrangements. Better understanding of this conserved axis of polarity can shed light on the mechanisms of morphogenetic processes and explain the underlying causes of human birth defects. Planar cell polarity (PCP) is an essential feature of animal tissues, whereby distinct polarity is established within the plane of a cell sheet. Tissue-wide establishment of PCP is driven by multiple global cues, including gradients of gene expression, gradients of secreted WNT ligands and anisotropic tissue strain. These cues guide the dynamic, subcellular enrichment of PCP proteins, which can self-assemble into mutually exclusive complexes at opposite sides of a cell. Endocytosis, endosomal trafficking and degradation dynamics of PCP components further regulate planar tissue patterning. This polarization propagates throughout the whole tissue, providing a polarity axis that governs collective morphogenetic events such as the orientation of subcellular structures and cell rearrangements. Reflecting the necessity of polarized cellular behaviours for proper development and function of diverse organs, defects in PCP have been implicated in human pathologies, most notably in severe birth defects.

Planar cell polarity (PCP) is an important patterning mechanism in both vertebrates and invertebrates by which cells coherently polarize along the apical surface of the epithelium. This patterning mechanism acts upstream of many developmental processes, such as oriented growth, division, cell movements and orientation of body hairs. While various models have been proposed to explain PCP patterning, all rely on persistent global cues/gradients to obtain global orientation of large tissues. However, recent experimental work has shown that this process can happen independently of such global cues, challenging the current paradigm. In this work, we developed a new model for PCP using the Cellular Potts modelling framework to investigate the conditions under which global tissue orientation can be achieved without a tissue spanning morphogen. We found that a combination of a local boundary signal, a small initial tissue size and uniform proliferation can effectively establish long-range polarity without the need for global cues. We also investigated the impact of cell division planes and growth rates on final patterning. Finally, we compared the cell-autonomous and cell non-autonomous versions of our PCP model, as found in flies and mice, and found that the latter offers more robust patterning outcomes in the absence of gradients.

Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to IFNs, Toll-like receptor engagement, or IL-4/IL-13 signaling, macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a universe of activation states. Progress has now been made in defining the signaling pathways, transcriptional networks, and epigenetic mechanisms underlying M1-M2 or M2-like polarized activation. Functional skewing of mononuclear phagocytes occurs in vivo under physiological conditions (e.g., ontogenesis and pregnancy) and in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer). However, in selected preclinical and clinical conditions, coexistence of cells in different activation states and unique or mixed phenotypes have been observed, a reflection of dynamic changes and complex tissue-derived signals. The identification of mechanisms and molecules associated with macrophage plasticity and polarized activation provides a basis for macrophage-centered diagnostic and therapeutic strategies.

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.

Here we characterized an information measure for cell polarity that applies to non-motile cells responding to a chemical gradient. The central idea is that polarization represents information about the direction of the gradient. We applied a theory of optimal gradient sensing and response in the presence of external noise based on the information capacity of a Gaussian channel. First, we formulated an information framework that describes spatial gradient sensing and polarization response. As part of this section, we modeled ligand diffusion and receptor-binding dynamics as a mixed Poisson distribution, confirming the single receptor accuracy limits derived by ten Wolde and colleagues. Second, we performed numerical calculations of stochastic ligand levels at the cell surface to estimate the information provided about the directional component of the gradient vector, which was close to the Gaussian channel bound for low signal-to-noise ratios. Third, we used the information framework to evaluate the noise-robustness of three generic models of cell polarity, demonstrating that a filter-amplifier architecture and time integration can attenuate the detrimental impact of noise on polarity so that the model can approach the theoretical limits. Fourth, we compared the theory to published experimental data on yeast mating projection growth in a pheromone gradient, identifying the ligand association rate and integration time as two key parameters affecting directional accuracy. By varying these parameters, we showed that for certain ranges the theory is roughly in agreement with the data, and that the slow binding rate constant is a key limiting factor. We concluded that temporal averaging can help overcome the slow binding rate to achieve greater accuracy, but with the drawback of a slow mating response.

Tissue disorganisation is one of the main hallmarks of cancer. Polarity proteins are responsible for the arrangement of cells within epithelial tissues through the asymmetric organisation of cellular components. Partition defective 3 (PARD3) is a master regulator of the Par polarity complex primarily due to its ability to form large complexes via its self-homologous binding domain. In addition to its role in polarity, PARD3 is a scaffolding protein that binds to intracellular signalling molecules, many of which are frequently deregulated in cancer. The role of PARD3 has been implicated in multiple solid cancers as either a tumour suppressor or promoter. This dual functionality is both physiologically and cell context dependent. In this review, we will discuss PARD3’s role in tumourigenesis in both laboratory and clinical settings. We will also review several of the mechanisms underpinning PARD3’s function including its association with intracellular signalling pathways and its role in the regulation of asymmetric cell division.

Differentiation of CD4.sup.+ T cells into either follicular helper T (T.sub.FH) or type 1 helper T (T.sub.H1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4.sup.+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove T.sub.FH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into T.sub.H1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4.sup.+ T cell differentiation and might instruct vaccine design strategies.

The Epithelial Polarity Program (EPP) adapts and integrates three ancient cellular machineries to construct an epithelial cell. The polarized trafficking machinery adapts the cytoskeleton and ancestral secretory and endocytic machineries to the task of sorting and delivering different plasma membrane (PM) proteins to apical and basolateral surface domains. The domain-identity machinery builds a tight junctional fence (TJ) between apical and basolateral PM domains and adapts ancient polarity proteins and polarity lipids on the cytoplasmic side of the PM, which have evolved to perform a diversity of polarity tasks across cells and species, to provide ‘identity’ to each epithelial PM domain. The 3D organization machinery utilizes adhesion molecules as positional sensors of other epithelial cells and the basement membrane and small GTPases as integrators of positional information with the activities of the domain-identity and polarized trafficking machineries. Cancer is a disease mainly of epithelial cells (90% of human cancers are carcinomas that derive from epithelial cells) that hijacks the EPP machineries, resulting in loss of epithelial polarity, which often correlates in extent with the aggressiveness of the tumor. Here, we review how the EPP integrates its three machineries and the strategies used by cancer to hijack them.

Key Points Actin and microtubules are dynamic polar polymers that are well suited for providing the structural basis for cell polarity. Nucleation and plus-end regulation are key mechanisms for the assembly of orientated actin and microtubule arrays, respectively. Actin and actin-based motor proteins drive symmetry breaking in several well-studied, polarized cell types. Microtubules promote symmetry breaking in the establishment of neuronal polarity. Microtubules have key roles in the maintenance of cell polarity in many mammalian cell types. Crosstalk coordinates the function of actin and microtubules in cell polarity. Actin and microtubules are dynamic polar polymers that are well suited for providing the structural basis for cell polarity. The actin cytoskeleton generally drives symmetry breaking, whereas microtubules control polarity maintenance. These events are coordinated by bidirectional crosstalk between actin and microtubules. Cell polarity relies on the asymmetric organization of cellular components and structures. Actin and microtubules are well suited to provide the structural basis for cell polarization because of their inherent structural polarity along the polymer lattices and intrinsic dynamics that allow them to respond rapidly to polarity cues. In general, the actin cytoskeleton drives the symmetry-breaking process that enables the establishment of a polarized distribution of regulatory molecules, whereas microtubules build on this asymmetry and maintain the stability of the polarized organization. Crosstalk coordinates the functions of the two cytoskeletal systems.