Mechanical forces directing intestinal form and function.

The vertebrate intestine experiences a range of intrinsically generated and external forces during both development and adult homeostasis. It is increasingly understood how the coordination of these forces shapes the intestine through organ-scale folding and epithelial organization into crypt-villus compartments. Moreover, accumulating evidence shows that several cell types in the adult intestine can sense and respond to forces to regulate key cellular processes underlying adult intestinal functions and self-renewal. In this way, transduction of forces may direct both intestinal homeostasis as well as adaptation to external stimuli, such as food ingestion or injury. In this review, we will discuss recent insights from complementary model systems into the force-dependent mechanisms that establish and maintain the unique architecture of the intestine, as well as its homeostatic regulation and function throughout adult life.

Liver Colonization by Colorectal Cancer Metastases Requires YAP-Controlled Plasticity at the Micrometastatic Stage.

Micrometastases of colorectal cancer can remain dormant for years prior to the formation of actively growing, clinically detectable lesions (i.e., colonization). A better understanding of this step in the metastatic cascade could help improve metastasis prevention and treatment. Here we analyzed liver specimens of patients with colorectal cancer and monitored real-time metastasis formation in mouse livers using intravital microscopy to reveal that micrometastatic lesions are devoid of cancer stem cells (CSC). However, lesions that grow into overt metastases demonstrated appearance of de novo CSCs through cellular plasticity at a multicellular stage. Clonal outgrowth of patient-derived colorectal cancer organoids phenocopied the cellular and transcriptomic changes observed during in vivo metastasis formation. First, formation of mature CSCs occurred at a multicellular stage and promoted growth. Conversely, failure of immature CSCs to generate more differentiated cells arrested growth, implying that cellular heterogeneity is required for continuous growth. Second, early-stage YAP activity was required for the survival of organoid-forming cells. However, subsequent attenuation of early-stage YAP activity was essential to allow for the formation of cell type heterogeneity, while persistent YAP signaling locked micro-organoids in a cellularly homogenous and growth-stalled state. Analysis of metastasis formation in mouse livers using single-cell RNA sequencing confirmed the transient presence of early-stage YAP activity, followed by emergence of CSC and non-CSC phenotypes, irrespective of the initial phenotype of the metastatic cell of origin. Thus, establishment of cellular heterogeneity after an initial YAP-controlled outgrowth phase marks the transition to continuously growing macrometastases. SIGNIFICANCE: Characterization of the cell type dynamics, composition, and transcriptome of early colorectal cancer liver metastases reveals that failure to establish cellular heterogeneity through YAP-controlled epithelial self-organization prohibits the outgrowth of micrometastases. See related commentary by LeBleu, p. 1870.

Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming.

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.

A Tuba/Cdc42/Par6A complex is required to ensure singularity in apical domain formation during enterocyte polarization.

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization.

The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size.

PTEN is a tumor suppressor that is frequently lost in epithelial malignancies. A part of the tumor-suppressive properties of PTEN is attributed to its function in cell polarization and consequently its role in maintaining epithelial tissue integrity. However, surprisingly little is known about the function and regulation of PTEN during epithelial cell polarization. We used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene disruption to delete PTEN in intestinal epithelial Ls174T:W4 cells, which upon differentiation form a microvillus-covered apical membrane (brush border) on a part of the cell cortex, independent of cell-cell junctions. We show that loss of PTEN results in the formation of a larger brush border that, in a fraction of the cells, even spans the entire plasma membrane, revealing that PTEN functions in the regulation of apical membrane size. Depletion of the phosphatase PTPL1 resulted in a similar defect. PTPL1 interacts with PTEN, and this interaction is necessary for apical membrane enrichment of PTEN. Importantly, phosphatase activity of PTPL1 is not required, indicating that PTPL1 functions as an anchor protein in this process. Our work thus demonstrates a novel function for PTEN during cell polarization in controlling apical membrane size and identifies PTPL1 as a critical apical membrane anchor for PTEN in this process.

A Two-Tiered Mechanism Enables Localized Cdc42 Signaling during Enterocyte Polarization.

Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.