Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Intussusceptive Angiogenesis in Human Metastatic Malignant Melanoma.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

From cells to organs: building polarized tissue.

How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.

cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.

Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex.

Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively.

p114RhoGEF governs cell motility and lumen formation during tubulogenesis through a ROCK-myosin-II pathway.

Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis.

Scrib regulates HGF-mediated epithelial morphogenesis and is stabilized by Sgt1-HSP90.

Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. We have investigated Scrib using a three-dimensional epithelial cell culture system. We show a novel association between the leucine-rich repeat domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following hepatocyte growth factor (HGF) stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib-ßPix-PAK complex. Inhibition of any member of this complex, Scrib, ßPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for three-dimensional cell migration suggests that chaperone-mediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis.

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

Genetic control of single lumen formation in the zebrafish gut.

Most organs consist of networks of interconnected tubes that serve as conduits to transport fluid and cells and act as physiological barriers between compartments. Biological tubes are assembled through very diverse developmental processes that generate structures of different shapes and sizes. Nevertheless, all biological tubes invariably possess one single lumen. The mechanisms responsible for single lumen specification are not known. Here we show that zebrafish mutants for the MODY5 and familial GCKD gene tcf2 (also known as vhnf1) fail to specify a single lumen in their gut tube and instead develop multiple lumens. We show that Tcf2 controls single lumen formation by regulating claudin15 and Na+/K+-ATPase expression. Our in vivo and in vitro results indicate that Claudin15 functions in paracellular ion transport to specify single lumen formation. This work shows that single lumen formation is genetically controlled and appears to be driven by the accumulation of fluid.

A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.

The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.

Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b.

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.

Reduced immunoglobulin A transcytosis associated with immunoglobulin A nephropathy and nasopharyngeal carcinoma.

Polymeric IgA (pIgA) is transcytosed by the pIgA receptor (pIgR) across mucosal epithelial cells. After transcytosis to the apical surface, the extracellular, ligand-binding portion of the pIgR is proteolytically cleaved. A missense mutation in human pIgR, A580V, is associated with IgA nephropathy and nasopharyngeal carcinoma. We report that this mutation reduces the rate of transcytosis of pIgR and pIgA, and seemingly the rate of pIgR cleavage. We propose that the defects in pIgR trafficking caused by the A580V mutation may underlie the pathogenesis of both diseases.

Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism.

Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell-cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3-enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.

MDCK cystogenesis driven by cell stabilization within computational analogues.

The study of epithelial morphogenesis is fundamental to increasing our understanding of organ function and disease. Great progress has been made through study of culture systems such as Madin-Darby canine kidney (MDCK) cells, but many aspects of even simple morphogenesis remain unclear. For example, are specific cell actions tightly coupled to the characteristics of the cell's environment or are they more often cell state dependent? How does the single lumen, single cell layer cyst consistently emerge from a variety of cell actions? To improve insight, we instantiated in silico analogues that used hypothesized cell behavior mechanisms to mimic MDCK cystogenesis. We tested them through in vitro experimentation and quantitative validation. We observed novel growth patterns, including a cell behavior shift that began around day five of growth. We created agent-oriented analogues that used the cellular Potts model along with an Iterative Refinement protocol. Following several refinements, we achieved a degree of validation for two separate mechanisms. Both survived falsification and achieved prespecified measures of similarity to cell culture properties. In silico components and mechanisms mapped to in vitro counterparts. In silico, the axis of cell division significantly affects lumen number without changing cell number or cyst size. Reducing the amount of in silico luminal cell death had limited effect on cystogenesis. Simulations provide an observable theory for cystogenesis based on hypothesized, cell-level operating principles.

Identification of a cytoplasmic signal for apical transcytosis.

Polarized epithelial cells contain apical and basolateral surfaces with distinct protein compositions. To establish and maintain this asymmetry, newly made plasma membrane proteins are sorted in the trans Golgi network for delivery to apical or basolateral surfaces. Signals for basolateral sorting are generally located in the cytoplasmic domain of the protein, whereas signals for apical sorting can be in any part of the protein and can depend on N-linked glycosylation of the protein. Signals for constitutive transcytosis to the apical surface have not been reported. In this study, we used the polymeric immunoglobulin receptor (pIgR), which is biosynthetically delivered to the basolateral surface. There the pIgR can bind a ligand and, with or without bound ligand, the pIgR can then be transcytosed to the apical surface. We found that the glycosylation of the pIgR did not affect the biosynthetic transport of the pIgR. However, glycosylation had an effect on pIgR apical transcytosis. Importantly, analysis of the cytoplasmic tail of the pIgR suggested that a short peptide segment was sufficient to transcytose the pIgR or a neutral reporter from the basolateral to the apical surface. This apical transcytosis sorting signal was not involved in polarized biosynthetic traffic of the pIgR.