Structural basis of mRNA binding by the human FERRY Rab5 effector complex.

The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.

The Rab5 effector FERRY links early endosomes with mRNA localization.

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

Apical bulkheads accumulate as adaptive response to impaired bile flow in liver disease.

Hepatocytes form bile canaliculi that dynamically respond to the signalling activity of bile acids and bile flow. Little is known about their responses to intraluminal pressure. During embryonic development, hepatocytes assemble apical bulkheads that increase the canalicular resistance to intraluminal pressure. Here, we investigate whether they also protect bile canaliculi against elevated pressure upon impaired bile flow in adult liver. Apical bulkheads accumulate upon bile flow obstruction in mouse models and patients with primary sclerosing cholangitis (PSC). Their loss under these conditions leads to abnormally dilated canaliculi, resembling liver cell rosettes described in other hepatic diseases. 3D reconstruction reveals that these structures are sections of cysts and tubes formed by hepatocytes. Mathematical modelling establishes that they positively correlate with canalicular pressure and occur in early PSC stages. Using primary hepatocytes and 3D organoids, we demonstrate that excessive canalicular pressure causes the loss of apical bulkheads and formation of rosettes. Our results suggest that apical bulkheads are a protective mechanism of hepatocytes against impaired bile flow, highlighting the role of canalicular pressure in liver diseases.

Identification of the switch in early-to-late endosome transition.

Sequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Design centering enables robustness screening of pattern formation models.

MOTIVATION: Access to unprecedented amounts of quantitative biological data allows us to build and test biochemically accurate reaction-diffusion models of intracellular processes. However, any increase in model complexity increases the number of unknown parameters and, thus, the computational cost of model analysis. To efficiently characterize the behavior and robustness of models with many unknown parameters remains, therefore, a key challenge in systems biology. RESULTS: We propose a novel computational framework for efficient high-dimensional parameter space characterization of reaction-diffusion models in systems biology. The method leverages the Lp-Adaptation algorithm, an adaptive-proposal statistical method for approximate design centering and robustness estimation. Our approach is based on an oracle function, which predicts for any given point in parameter space whether the model fulfills given specifications. We propose specific oracles to efficiently predict four characteristics of Turing-type reaction-diffusion models: bistability, instability, capability of spontaneous pattern formation and capability of pattern maintenance. We benchmark the method and demonstrate that it enables global exploration of a model's ability to undergo pattern-forming instabilities and to quantify robustness for model selection in polynomial time with dimensionality. We present an application of the framework to pattern formation on the endosomal membrane by the small GTPase Rab5 and its effectors, and we propose molecular mechanisms underlying this system. AVAILABILITY AND IMPLEMENTATION: Our code is implemented in MATLAB and is available as open source under https://git.mpi-cbg.de/mosaic/software/black-box-optimization/rd-parameter-space-screening. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A decade of molecular cell biology: achievements and challenges.

Nature Reviews Molecular Cell Biology celebrated its 10-year anniversary during this past year with a series of specially commissioned articles. To complement this, here we have asked researchers from across the field for their insights into how molecular cell biology research has evolved during this past decade, the key concepts that have emerged and the most promising interfaces that have developed. Their comments highlight the broad impact that particular advances have had, some of the basic understanding that we still require, and the collaborative approaches that will be essential for driving the field forward.

The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development.

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

Correction: Quantification of nematic cell polarity in three-dimensional tissues.

[This corrects the article DOI: 10.1371/journal.pcbi.1008412.].

An endosomal tether undergoes an entropic collapse to bring vesicles together.

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.

Correlative single-molecule localization microscopy and electron tomography reveals endosome nanoscale domains.

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Content-aware image restoration: pushing the limits of fluorescence microscopy.

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration.

The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase in F-actin and phospho-myosin, to compensate an overload of bile acids. These changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to apical F-actin-rich regions and translocates to the nucleus in dependence of the integrity of the actin cytoskeleton. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signaling, we explained this behavior by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status.

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.

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-Navarrete et al., eLife 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 and in-vitro organoids.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.

BACKGROUND & AIMS: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. METHODS: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. RESULTS: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. CONCLUSION: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. LAY SUMMARY: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro.