A Size Filter Regulates Apical Protein Sorting.

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. We investigated whether a size barrier at the trans-Golgi network (TGN) might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3 and Ace2 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum (ER) until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, departure from the Golgi could be significantly delayed simply by increasing cytoplasmic bulk. Moreover, large and small Crb3 segregated into spatially distinct Golgi regions as detected by super resolution imaging. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the ER and Golgi, we found that Pals1 disassociates prior to Crb3 departure. Notably, a non-dissociable mutant Pals1 hampers the exit of Crb3. We conclude that an unexpected mechanism involving a size filter at the TGN facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for the normal kinetics of Crb3 sorting.

A junction-dependent mechanism drives murine mammary cell intercalation for ductal elongation.

The luminal epithelium of the mammary gland is organized into monolayers; however, it originates from multilayered terminal end buds (TEBs) during development. Although apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it doesn't account for ductal elongation behind TEBs. Spatial calculations in mice suggest that most TEB cells integrate into the outermost luminal layer to generate elongation. We developed a quantitative cell culture assay that models intercalation into epithelial monolayers. We found that tight junction proteins play a key role in this process. ZO-1 puncta form at the new cellular interface and resolve into a new boundary as intercalation proceeds. Deleting ZO-1 suppresses intercalation both in culture and in cells transplanted into mammary glands via intraductal injection. Cytoskeletal rearrangements at the interface are critical for intercalation. These data identify luminal cell rearrangements necessary for mammary development and suggest a mechanism for integration of cells into an existing monolayer.

Temporal recording of mammalian development and precancer.

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo , while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells.

Pluripotent stem cells can be driven by manipulation of Wnt signalling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic-mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells triggers widespread apoptosis, followed by a synchronous epithelial-mesenchymal transition (EMT). Apoptosis is caused by the absence of bFGF in the differentiation medium. EMT requires induction of the transcription factors SNAI1 and SNAI2 downstream of MESP1 expression, and double knockout of SNAI1 and SNAI2 or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis, either chemically or by ablation of pro-apoptotic genes, also completely prevents EMT, suppressing even the earliest events in mesoderm conversion, including T/BRA, TBX6 and MESP1 induction. Conditioned medium from WNT-activated wild-type iPSCs overcomes the block to EMT by cells incapable of apoptosis, suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, whereas treatment with a purinergic P2-receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signalling. ATP and/or UTP was sufficient to induce a partial EMT in apoptosis-incapable cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that in addition to acting as chemo-attractants for clearance of apoptotic cells, nucleotides can function as essential paracrine signals that, with WNT signalling, create a logical AND gate for mesoderm specification.

Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation.

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.

DNA Damage Promotes Epithelial Hyperplasia and Fate Mis-specification via Fibroblast Inflammasome Activation.

DNA crosslinking agents are commonly used in cancer chemotherapy; however, responses of normal tissues to these agents have not been widely investigated. We reveal in mouse interfollicular epidermal, mammary and hair follicle epithelia that genotoxicity does not promote apoptosis but paradoxically induces hyperplasia and fate specification defects in quiescent stem cells. DNA damage to skin causes epithelial and dermal hyperplasia, tissue expansion, and proliferation-independent formation of abnormal K14/K10 dual-positive suprabasal cells. Unexpectedly, this behavior is epithelial cell non-autonomous and independent of an intact immune system. Instead, dermal fibroblasts are both necessary and sufficient to induce the epithelial response, which is mediated by activation of a fibroblast-specific NLRP3 inflammasome and subsequent IL-1ß production. Thus, genotoxic agents that are used chemotherapeutically to promote cancer cell death can have the opposite effect on wild-type epithelia by inducing, via a non-autonomous IL-1ß-driven mechanism, both hyperplasia and stem cell lineage defects.

Mutations in the exocyst component EXOC2 cause severe defects in human brain development.

The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients' neurological disorders.

Polarity proteins in oncogenesis.

Most human cancers arise from epithelial tissues, which are apical-basally polarized and possess intercellular adhesive junctions. Epithelial cells grow to characteristic densities, often from proliferative progenitors, which arrest as they mature. Homeostatic mechanisms can maintain this characteristic density if it is exceeded (crowding) or is too low (e.g. in response to wounding). During tumor initiation and progression this homeostatic mechanism is lost. Some aspects of cell polarity are also lost, although many carcinomas retain intercellular junctions and even apical domains. In other cases, and particularly in recurrent tumors, however, the cells become predominantly mesenchymal. A major question, still only incompletely answered, is whether the proteins that determine cell polarity function as tumor suppressors or tumor promoters. Here we discuss recent advances in understanding the role of polarity proteins and homeostasis in cancer.

Publisher Correction: Exocyst dynamics during vesicle tethering and fusion.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Biochemistry and Molecular Genetics and Breast Surgery, Ehime University Graduate School of Medicine, 7910295, Japan' and affiliation 3 incorrectly read 'Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Matsuyama 7910295, Japan.' These errors have now been corrected in both the PDF and HTML versions of the Article.

Exocyst dynamics during vesicle tethering and fusion.

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

A senataxin-associated exonuclease SAN1 is required for resistance to DNA interstrand cross-links.

Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. However, FA-independent repair mechanisms of ICLs remain poorly understood. Here we report a previously uncharacterized protein, SAN1, as a 5' exonuclease that acts independently of the FA pathway in response to ICLs. Deletion of SAN1 in HeLa cells and mouse embryonic fibroblasts causes sensitivity to ICLs, which is prevented by re-expression of wild type but not nuclease-dead SAN1. SAN1 deletion causes DNA damage and radial chromosome formation following treatment with Mitomycin C, phenocopying defects in the FA pathway. However, SAN1 deletion is not epistatic with FANCD2, a core FA pathway component. Unexpectedly, SAN1 binds to Senataxin (SETX), an RNA/DNA helicase that resolves R-loops. SAN1-SETX binding is increased by ICLs, and is required to prevent cross-link sensitivity. We propose that SAN1 functions with SETX in a pathway necessary for resistance to ICLs.

Epithelial plasticity in the mammary gland.

Many epithelial tissues rely on multipotent stem cells for the proper development and maintenance of their diverse cell lineages. Nevertheless, the identification of multipotent stem cell populations within the mammary gland has been a point of contention over the past decade. In this review, we provide a critical overview of the various lineage-tracing studies performed to address this issue and conclude that although multipotent stem cells exist in the embryonic mammary placode, the postnatal mammary gland instead contains distinct unipotent progenitor populations that contribute to stage-specific development and homeostasis. This begs the question of why differentiated mammary epithelial cells can exhibit stem cell behavior in culture. We speculate that such reprogramming potential is repressed in situ under normal conditions but revealed in vitro and might drive breast cancer development.

The Par3 polarity protein is an exocyst receptor essential for mammary cell survival.

The exocyst is an essential component of the secretory pathway required for delivery of basolateral proteins to the plasma membranes of epithelial cells. Delivery occurs adjacent to tight junctions (TJ), suggesting that it recognizes a receptor at this location. However, no such receptor has been identified. The Par3 polarity protein associates with TJs but has no known function in membrane traffic. We now show that, unexpectedly, Par3 is essential for mammary cell survival. Par3 silencing causes apoptosis, triggered by phosphoinositide trisphosphate depletion and decreased Akt phosphorylation, resulting from failure of the exocyst to deliver basolateral proteins to the cortex. A small region of PAR3 binds directly to Exo70 and is sufficient for exocyst docking, membrane-protein delivery and cell survival. PAR3 lacking this domain can associate with the cortex but cannot support exocyst function. We conclude that Par3 is the long-sought exocyst receptor required for targeted membrane-protein delivery.

An In Vivo Gain-of-Function Screen Identifies the Williams-Beuren Syndrome Gene GTF2IRD1 as a Mammary Tumor Promoter.

The broad implementation of precision medicine in cancer is impeded by the lack of a complete inventory of the genes involved in tumorigenesis. We performed in vivo screening of ∼1,000 genes that are associated with signaling for positive roles in breast cancer, using lentiviral expression vectors in primary MMTV-ErbB2 mammary tissue. Gain of function of five genes, including RET, GTF2IRD1, ADORA1, LARS2, and DPP8, significantly promoted mammary tumor growth. We further studied one tumor-promoting gene, the transcription factor GTF2IRD1. The mis-regulation of genes downstream of GTF2IRD1, including TßR2 and BMPR1b, also individually promoted mammary cancer development, and silencing of TßR2 suppressed GTF2IRD1-driven tumor promotion. In addition, GTF2IRD1 is highly expressed in human breast tumors, correlating with high tumor grades and poor prognosis. Our in vivo approach is readily expandable to whole-genome annotation of tumor-promoting genes.

Mechanisms of polarity protein expression control.

Polarity is a universal feature of cells during division and often at other stages of the cell cycle or after post-mitotic differentiation. A conserved machinery, present in all animals, initiates and maintains polarity. Multi-cellular animals organize themselves with respect to the axes of symmetry of the organism through the process of planar cell polarity, but many tissues also express a cell-intrinsic form of polarity, for instance to segregate the apical and basolateral membranes of epithelial cells. Although the genes and proteins involved in apical-basal polarity have been known for many years, the regulation of their expression remains ill-defined. Maintenance of the correct expression levels is essential for normal cell lineage allocation, tissue morphogenesis and cell survival. Here we summarize what is known about the transcriptional and post-transcriptional regulation of polarity protein expression, and discuss areas that remain to be understood.

Detection of RNA-Protein Interactions Using Tethered RNA Affinity Capture.

Recent progress in large-scale nucleic acid analysis technology has revealed the presence of vast numbers of RNA species in cells, and extensive processing. To investigate the functions of these transcripts highly efficient methods are needed to analyze their interactions with RNA-binding proteins (RNBPs), and to understand the binding mechanisms. Many methods have been described to identify RNBPs, but none are wholly satisfactory, in part because RNAs are flexible macromolecules that adopt multiple conformations only some of which might bind to specific proteins. Here we describe a novel in vitro RNA-pull-down assay using tRNA scaffolded Streptavidin Aptamer (tRSA), to identify transcript specific RNA binding protein from mammalian cell lysates. The tRNA scaffold functions to stabilize the structure of the aptamer and the attached RNA, increasing the efficiency of the affinity purification.

Loss of the polarity protein PAR3 activates STAT3 signaling via an atypical protein kinase C (aPKC)/NF-κB/interleukin-6 (IL-6) axis in mouse mammary cells.

PAR3 suppresses tumor growth and metastasis in vivo and cell invasion through matrix in vitro. We propose that PAR3 organizes and limits multiple signaling pathways and that inappropriate activation of these pathways occurs without PAR3. Silencing Pard3 in conjunction with oncogenic activation promotes invasion and metastasis via constitutive STAT3 activity in mouse models, but the mechanism for this is unknown. We now show that loss of PAR3 triggers increased production of interleukin-6, which induces STAT3 signaling in an autocrine manner. Activation of atypical protein kinase C ι/λ (aPKCι/λ) mediates this effect by stimulating NF-κB signaling and IL-6 expression. Our results suggest that PAR3 restrains aPKCι/λ activity and thus prevents aPKCι/λ from activating an oncogenic signaling network.

Does FACS perturb gene expression?

Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub-populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short-term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNA's and rapid decreases in their predicted target transcripts. © 2015 International Society for Advancement of Cytometry.