Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer

Quantification of Nematic Cell Polarity in Three-dimensional Tissues

How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarette et al., eLife 8:e44860, 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues or in-vitro organoids.Comment: 27 pages, 9 color figure

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion

Evolution of late steps in exocytosis:conservation and specialization of the exocyst complex

Background: The eukaryotic endomembrane system most likely arose via paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist Trypanosoma brucei, and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. Methods: We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. Results: The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. Conclusions: These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms. </p

Rab11 regulates recycling through the pericentriolar recycling endosome

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive. recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo

Resilience of three-dimensional sinusoidal networks in liver tissue

Can three-dimensional, microvasculature networks still ensure blood supply if individual links fail? We address this question in the sinusoidal network, a plexus-like microvasculature network, which transports nutrient-rich blood to every hepatocyte in liver tissue, by building on recent advances in high-resolution imaging and digital reconstruction of adult mice liver tissue. We find that the topology of the three-dimensional sinusoidal network reflects its two design requirements of a space-filling network that connects all hepatocytes, while using shortest transport routes: sinusoidal networks are sub-graphs of the Delaunay graph of their set of branching points, and also contain the corresponding minimum spanning tree, both to good approximation. To overcome the spatial limitations of experimental samples and generate arbitrarily-sized networks, we developed a network generation algorithm that reproduces the statistical features of 0.3-mm-sized samples of sinusoidal networks, using multi-objective optimization for node degree and edge length distribution. Nematic order in these simulated networks implies anisotropic transport properties, characterized by an empirical linear relation between a nematic order parameter and the anisotropy of the permeability tensor. Under the assumption that all sinusoid tubes have a constant and equal flow resistance, we predict that the distribution of currents in the network is very inhomogeneous, with a small number of edges carrying a substantial part of the flow-a feature known for hierarchical networks, but unexpected for plexus-like networks. We quantify network resilience in terms of a permeability-at-risk, i.e., permeability as function of the fraction of removed edges. We find that sinusoidal networks are resilient to random removal of edges, but vulnerable to the removal of high-current edges. Our findings suggest the existence of a mechanism counteracting flow inhomogeneity to balance metabolic load on the liver

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi. Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity characteristic of epithelial cells. Whether cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary mouse hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Timecourse RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or by increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feed back to hepatoblast-to-hepatocyte differentiation