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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.

ATPases are a group of enzymes that can cyclically convert the free energy of ATP hydrolysis into mechanical work. GTPases are another class of enzymes that are predominantly associated with signal transduction processes, but their role in mechanotransduction is less established. It was previously shown that the binding of the GTPase Rab5 to the tethering protein EEA1 induces a large conformational change in EEA1 from a rigid, extended to a flexible, collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer. It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperone proteins. Here we show that EEA1 in a bulk solution can undergo multiple flexibility transition cycles driven by the energetics of Rab5 binding and unbinding as well as GTP hydrolysis. Each cycle can perform up to 20kBT of mechanical work. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from the Rab5 GTPase cycle. We conclude that tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

Three dimensional (3D) geometrical models are not only a powerful tool for quantitatively characterizing complex tissues but also useful for probing structure-function relationships in a tissue. However, these models are generally incomplete due to experimental limitations in acquiring multiple (>4) fluorescent channels simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we performed deep-tissue immunostaining, multiphoton microscopy, deeplearning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatio-temporal singlecell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the structural role of KCs in the murine liver homeostasis. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs alters the number and morphology of the HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, under both physiological and pathological conditions.

The recognition of vesicles by their correct target compartment depends on the pairing of small GTPases with effector proteins (1–3). Whereas ATPases can cyclically convert the free energy of ATP hydrolysis into mechanical work, the function of small GTPases is predominantly associated with signal transduction processes, and their role in mechano-transduction is less established (4–6). However, binding of the GTPase Rab5 to the long coiled-coil tethering protein EEA1 on an early endosome induces a rigidity transition resulting in a large conformational change in EEA1 from a rigid and extended to a flexible and collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer (7). It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperones. Here, we use fluorescence correlation spectroscopy to reveal that EEA1 in bulk solution can undergo multiple flexibility transition cycles that are associated with the binding and release of Rab5(GTP) and Rab5(GDP). Using semi-flexible polymer theory we provide evidence that the cyclic transitioning of Rab5-EEA1 between extended and collapsed conformations is driven by the energetics of Rab5 binding/unbinding and GTP hydrolysis. Cyclic flexibility transitions represent a complete mechanical work cycle that is able to perform up to 20 kBT of mechanical work per cycle, against an opposing force. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from GTP hydrolysis by Rab5. We conclude that coiled-coil tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

Paramasivam et al. compared the endosomal trafficking of six LNP-mRNA formulations. LNP-mRNA accumulates in large endosomes impaired in acidification and unproductive for delivery. Super-resolution microscopy allowed visualization of single LNP-mRNA within endosomes and escape of mRNA from endosomal recycling tubules., Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.

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.

© 2022 The Authors. This is an open access article under the CC BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/)., Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development., This work was supported by grants from the la Caixa Banking Foundation and Fundação para a Ciência e a Tecnologia (HR17-00264 and PTDC/SAU-PAR/30751/2017 respectively, both to M.M.M.). A.L and S.S.B were sponsored by Fundação para a Ciência e a Tecnologia fellowships (PD/BD/114036/2015, SFRH/BD/114464/2016, respectively). V.S acknowledges funding from the Science and Engineering Research Board (SERB) (EMR/2016/006810), Department of Science and Technology (DST), Government of India. V.P. and C.S. were supported by GRK2581 (P6) SPHINGOINF of the Growing Spine Foundation (DFG).

Hydrogen deuterium exchange mass spectrometry (HDX-MS) gives insight into the structure of proteins. By monitoring the rate of exchange of the amide hydrogens on the protein backbone with deuterium atoms in the solvent, one can determine if a given region is highly structured or dynamic, map binding sites of interacting molecules or determine if a binding event is associated with allosteric structural alterations in a protein. Herein, we illustrate the use of this technique to monitor the nucleotide exchange process in Rab5, using the guanine nucleotide exchange factor (GEF)-effector complex, Rabex5:Rabaptin5. By simultaneously monitoring the HDX in Rab5, Rabex5 and Rabaptin5, we can directly visualize nucleotide exchange in Rab5, gain mechanistic insights into the exchange reaction and, by witnessing the transfer of Rab5 from Rabex5 to Rabaptin5, provide direct evidence for the positive feedback loop generated by a GEF-effector complex. HDX-MS can be used to monitor a variety of Rab protein-effector and -regulator interactions and be widely applied to other enzymatic processes as well.

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

Early disease diagnosis is key to the effective treatment of diseases. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D (three-dimensional) structural changes resulting from functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of morphometric cellular and tissue parameters correlated with disease progression, and discover profound topological defects in the 3D bile canalicular (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and micro-cholestasis, consistent with elevated cholestatic biomarkers in patients’ sera. Our spatially resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multiparametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology. A combination of high-resolution imaging and modeling approaches facilitates the study of the mechanisms and clinical progression of non-alcoholic fatty liver disease in humans.

International audience; 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.

Long-distance axonal trafficking plays a critical role in neuronal function, and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

Belicova et al. report previously unrecognized apical membrane extensions, forming a pattern reminiscent of the bulkheads of boats, that determine the anisotropic expansion of hepatocyte lumina. Loss of the bulkheads caused by Rab35 silencing leads to re-engineering of epithelial polarity and liver tissue architecture., Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis., Graphical Abstract

Detection of nucleic acids within sub-cellular 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 important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods 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 immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be 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.

Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction leads to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, where two arm-like appendages of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. The coiled-coil domains of Fy-2 are flexible and project into opposite directions from the core. 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. The FERRY structure provides novel mechanistic insights into long-distance mRNA transport.

We present a novel method for model-based segmentation of extended, blob-like objects on curved surfaces. Our method addresses several challenges arising when imaging curved biological membrane, such as out-of-membrane signal and geometry-induced background variations. We use a particle-based reconstruction of the membrane geometry, moment-conserving intensity interpolation from pixels to surface particles, and model-based in-surface segmentation. Our method denoises and deconvolves images, corrects for background variations, and quantifies the number, size, and intensity of segmented objects. We benchmark the accuracy of the method and present two applications to (1) neuroepithelial focal adhesion sites during optic cup morphogenesis in zebrafish and (2) reconstituted membrane domains bearing the small GTPase Rab5 on spherical beads.

Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1–3. The mechanisms governing anisotropic lumen expansion remain elusive4–6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) gives insight into the structure of proteins. By monitoring the rate of exchange of the amide hydrogens on the protein backbone with deuterium atoms in the solvent, one can determine if a given region is highly structured or dynamic, map binding sites of interacting molecules or determine if a binding event is associated with allosteric structural alterations in a protein. Herein, we illustrate the use of this technique to monitor the nucleotide exchange process in Rab5, using the guanine nucleotide exchange factor (GEF)-effector complex, Rabex5:Rabaptin5. By simultaneously monitoring the HDX in Rab5, Rabex5 and Rabaptin5, we can directly visualize nucleotide exchange in Rab5, gain mechanistic insights into the exchange reaction and, by witnessing the transfer of Rab5 from Rabex5 to Rabaptin5, provide direct evidence for the positive feedback loop generated by a GEF-effector complex. HDX-MS can be used to monitor a variety of Rab protein-effector and -regulator interactions and be widely applied to other enzymatic processes as well.

Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines, in primary human adipocytes, fibroblasts and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery and different LNP vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By super-resolution microscopy we could resolve single LNP-mRNA within sub-endosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.

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., Author summary Cell polarity enables cells to carry out specific functions. Cell polarity is characterized by the formation of different plasma membrane domains, each with specific composition of proteins, phospholipids and cytoskeletal components. In simple epithelial sheets, or tube-like tissues such as kidney, epithelial cells are known to display a single apical domain, facing a lumenal cavity, and a single basal domain on the opposite side of the cell, facing a basal layer of extracellular matrix. This apico-basal polarity defines a vector of cell polarity, which provides a direction of fluid transport, e.g., from the basal side of the sheet to the lumen-facing side. In more complex, three-dimensional epithelial tissues, such as liver tissue with its complex network of blood-transporting sinusoids, the membrane domains of hepatocyte cells display more intricate patterns, including rings and antipodal pairs of apical membrane. Here, we develop a mathematical framework to precisely characterize and quantify complex polarity patterns. Thereby, we reveal ordered patterns of cell polarity that span across a liver lobule. Our new method builds on physical concepts originally developed for ordered phases of liquid crystals. It provides a versatile tool to characterize the spatial organization of a complex three-dimensional tissue.

Imaging biological systems at subcellular resolution and across scales is essential to under-standing how cells form tissues, organs, and organisms. However, existing large-scale optical techniques often require harsh tissue-clearing methods that cause significant morphological changes, compromise the integrity of cell membranes, and reduce the signal of fluorescent proteins. Here, we demonstrate multifocal two-photon microscopy that enables imaging mesoscopic scattering samples in their native tissue environment at high resolution and high speed.

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., Author summary The liver is the largest metabolic organ of the human body and pivotal for blood detoxification and the uptake of many medically relevant drugs. Inside liver tissue, a dense meshwork of blood micro-vessels, the sinusoidal network, contacts every hepatocyte cell. The architecture of these networks, and especially how their function responds to local damage is not well understood. Previous theoretical work addressed network resilience in simple two-dimensional networks such as vein networks of leaves but not in three-dimensional networks, partly due to the considerable difficulties of imaging three-dimensional tissues. Here, we build on unprecedented advances in imaging mouse liver tissue. By simulating arbitrarily-sized networks that faithfully reproduce the statistical features of spatially restricted experimental samples, we computationally characterize the relation between a weak alignment of the network along the flow direction and enhanced transport along this direction. A simple transport model predicts an inhomogeneous distribution of flow in the sinusoidal network. Concomitantly, these networks are resilient against the random removal of edges, but vulnerable against removal of ‘highway’ edges that carried a high flow in the unperturbed state. We speculate that yet unknown adaptive mechanisms balance the distribution of flows and thereby increase the resilience of this physiologically important network.

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., This study shows 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 and activates YAP, by a mechanism dependent on the actin cytoskeleton.

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.

Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effector binding, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers as a model system for membrane patterning. We show that there is a “handover” of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Surprisingly, a lipid membrane composition mimicking that of the early endosome was required for Rab5 patterning. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

Fluorescently targeted proteins are widely used for studies of intracellular organelles dynamic. Peripheral proteins are transiently associated with organelles and a significant fraction of them are located at the cytosol. Image analysis of peripheral proteins poses a problem on properly discriminating membrane-associated signal from the cytosolic one. In most cases, signals from organelles are compact in comparison with diffuse signal from cytosol. Commonly used methods for background estimation depend on the assumption that background and foreground signals are separable by spatial frequency filters. However, large non-stained organelles (e.g., nuclei) result in abrupt changes in the cytosol intensity and lead to errors in the background estimation. Such mistakes result in artifacts in the reconstructed foreground signal. We developed a new algorithm that estimates background intensity in fluorescence microscopy images and does not produce artifacts on the borders of nuclei.

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

High-content screens (HCS) using chemical and genomic interference based on light microscopy and quantitative image analysis yielded a large amount of multi-parametric (MP) phenotypic data. Such data-sets hold great promise for the understanding of cellular mechanisms by systems biology. However, extracting functional information from data-sets, such as links between cellular processes and the functions of unknown genes, remains challenging. The limitation of HCS analysis lies in the complexity of cellular organization. Here, we assumed that cellular processes have a modular structure, and deconvolved the MP data into separate signals from different cellular modules by Blind Source Separation. We applied a combination of quantitative MP image analysis (QMPIA) and Independent Component Analysis (ICA) to an image-based HCS of endocytosis, the process whereby cells uptake molecules from the outside and distribute them to different sub-cellular organelles. We named our approach Independent Phenotypes Analysis (IPA). Phenotypic traits revealed by IPA are interpretable in terms of perturbation of specific endosomal populations (e.g. specific cargo, specific molecular markers) and of specific functional modules (early stages of endocytosis, recycling, cell cycle, etc.). The profile of perturbation of each gene in such basic phenotypic coordinates intrinsically suggest its possible mode of action.

A quantitative understanding of complex biological systems such as tissues requires reconstructing the structure of the different components of the system. Fluorescence microscopy provides the means to visualize simultaneously several tissue components. However, it can be time consuming and is limited by the number of fluorescent markers that can be used. In this study, we describe a toolbox of algorithms based on convolutional neural networks for the prediction of 3D tissue structures by learning features embedded within single-marker images. As proof of principle, we aimed to predict the network of bile canaliculi (BC) in liver tissue using images of the cortical actin mesh as input. The actin meshwork has a characteristic organization in specific cellular domains, such as BC. However, the use of manually selected features from images of actin is not sufficient to properly reconstruct BC structure. Our deep learning framework showed a remarkable accuracy for the prediction of BC network and was successfully adapted (i.e. transfer learning) to predict the sinusoidal network. This approach allows for a complete reconstruction of tissue microarchitecture using a single fluorescent marker.

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 sub-domains 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-colour 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 labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains 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.SynopsisSuborganelle compartmentalization cannot be resolved by diffraction limited light microscopy and not interpreted without knowledge of the underlying ultrastructure. This work shows a novel superCLEM workflow that combines multi-colour single-molecule localization-microscopy with electron tomography to map several functional domains on early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nano-domains largely devoid from cargo molecules Transferrin and EGF and opens new possibilities to perform structure-function analysis of organelles at the nanoscale.

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, which is controlled by Hippo signalling. 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 of F-actin and phospho-Myosin, to compensate an overload of bile acids. Interestingly, these changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to the apical F-actin-rich region and translocates to the nucleus in dependence of the acto-myosin system. 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 signalling, we explained this behaviour 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.

Early disease diagnosis is key to the effective treatment of diseases. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D (three-dimensional) structural changes resulting from functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of morphometric cellular and tissue parameters correlated with disease progression, and discover profound topological defects in the 3D bile canalicular (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and micro-cholestasis, consistent with elevated cholestatic biomarkers in patients' sera. Our spatially resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multiparametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be autoinhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical crosslinking mass spectrometry and integrative modeling techniques. Our results are inconsistent with the previous model of autoinhibition. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the Ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that Ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

Background & Aims The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. Methods Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). Results The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1 −/− Mst2 −/− liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53 LSL/LSL mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. Conclusion Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells as generally assumed, since silencing Integrin-ß1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS., eLife digest The inside of a human cell is divided into compartments called organelles, which are surrounded by membranes. Each organelle plays a specific role in keeping the cell healthy and also has unique mix of molecular markers on its surface. These markers allow other molecules to identify the different organelles, meaning that specific organelles can communicate with each other and coordinate their activities. One way that organelles can do this is via so-called membrane contact sites, which are small areas where the compartments’ outer membranes come close together. Mitochondria are organelles that release energy inside human cells. These compartments also work to keep the levels of toxic chemicals called reactive oxygen species in the cell within a safe range. This is important because cells can die if these levels become too high – a state known as oxidative stress. Mitochondria also communicate with other organelles called endosomes, which receive materials from the cell surface, sort and direct them to different destinations throughout the cell. In many diseases affecting the nervous system, the mitochondria and endosomes in nerve cells do not work properly. These cells also have higher than normal levels of oxidative stress. Hsu et al. therefore wanted to find out if mitochondria and endosomes worked together to help cells to cope with this kind of stress. Hsu et al. triggered oxidative stress in human cancer cells by exposing them first to a dye that stained the mitochondria and then to intense light. In stressed cells, a subset of endosomes called early endosomes formed many more membrane contact sites with mitochondria than in non-stressed cells. At the same time, the protein Rab5, usually found on early endosomes, relocated to the surface of mitochondria. Human cells previously engineered to produce larger than normal amounts of Rab5 were also more likely to survive oxidative stress. These experiments suggested that early endosomes cooperate with mitochondria, via Rab5, to protect cells from oxidative stress. So, how is Rab5 relocated to mitochondria? Hsu et al. searched for activators of Rab5 and found that Alsin also migrated to mitochondria in stressed cells. The gene for Alsin is also mutated in amyotrophic lateral sclerosis (ALS), a degenerative nerve disorder that remains poorly understood. Next, Hsu et al. deleted the gene for Alsin from human stem cells growing in the laboratory and coaxed these cells into becoming nerve cells. Experiments with these cells showed that the absence of Alsin prevented Rab5 from moving to the mitochondria. Nerve cells lacking Alsin were also more susceptible to oxidative stress than normal cells. Together, these findings show that early endosomes work with mitochondria to sense and ward off oxidative stress. They also reveal an unexpected connection between this process and a gene mutated in ALS. Further experiments are now needed to explore if problems with endosomes or mitochondria, and specifically with molecules like Alsin and Rab5, are responsible for other neurodegenerative disorders, like Parkinson’s disease and Huntington’s disease.

APPL1 vesicles represent a distinct population of Rab5-positive early endosomes that undergoes fusion and fission and sorts and exchanges cargo with EEA1 endosomes, suggesting a new organization of the endocytic pathway., Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.

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. Content-aware image restoration (CARE) uses deep learning to improve microscopy images. CARE bypasses the trade-offs between imaging speed, resolution, and maximal light exposure that limit fluorescence imaging to enable discovery.