Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Therapy Development for Microvillus Inclusion Disease using Patient-derived Enteroids.

Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na + /H + exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Inhibition of Notch signaling with the γ-secretase inhibitor, DAPT, recovered apical brush border structure and functional Na + /H + exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID. Conflict-of-interest statement: The authors have declared that no conflict of interest exists.

High-Content Imaging Platform to Discover Chemical Modulators of Plasma Membrane Rafts.

Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.

Cholera Toxin as a Probe for Membrane Biology.

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein µNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus µNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance.

The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin.

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons.

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors.

Confocal Microscopy of Reovirus Transport in Living Dorsal Root Ganglion Neurons.

Neurotropic reoviruses repurpose host machinery to traffic over long distances in neuronal processes and access distal replication sites. Understanding mechanisms of neuronal transmission is facilitated by using simplified in vitro primary neuronal culture models. Advances in the design of compartmentalized microfluidic devices lend robustness to neuronal culture models by enabling compartmentalization and manipulation of distinct neuronal processes. Here, we describe a streamlined methodology to culture sensory neurons dissociated from dorsal root ganglia of embryonic rats in microfluidic devices. We further describe protocols to exogenously label reovirus and image, track, and analyze transport of single reovirus particles in living neurons. These techniques can be adapted to study directed axonal transport of other neurotropic viruses and neuronal factors involved in signaling and pathology.

Chikungunya virus replication in skeletal muscle cells is required for disease development.

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell-specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of Il1b, Il6, Ip10, and Tnfa transcripts. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

Helicobacter pylori VacA is a secreted pore-forming toxin that induces cell vacuolation in vitro and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NH4Cl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the hypothesis that NH4Cl augments VacA toxicity by altering the intracellular trafficking of VacA or inhibiting intracellular VacA degradation. We observed VacA colocalization with LAMP1- and LC3-positive vesicles in both the presence and absence of NH4Cl, indicating that NH4Cl does not alter VacA trafficking to lysosomes or autophagosomes. Conversely, we found that supplemental NH4Cl significantly increases the intracellular stability of VacA. By conducting experiments using chemical inhibitors, stable ATG5 knockdown cell lines, and ATG16L1 knockout cells (generated using CRISPR/Cas9), we show that VacA degradation is independent of autophagy and proteasome activity but dependent on lysosomal acidification. We conclude that weak bases like ammonia, potentially generated during H. pylori infection by urease and other enzymes, enhance VacA toxicity by inhibiting toxin degradation.

Dynamic pattern generation in cell membranes: Current insights into membrane organization.

It has been two decades since the lipid raft hypothesis was first presented. Even today, whether these nanoscale cholesterol-rich domains are present in cell membranes is not completely resolved. However, especially in the last few years, a rich body of literature has demonstrated both the presence and the importance of non-random distribution of biomolecules on the membrane, which is the focus of this review. These new developments have pushed the experimental limits of detection and have brought us closer to observing lipid domains in the plasma membrane of live cells. Characterization of biomolecules associated with lipid rafts has revealed a deep connection between biological regulation and function and membrane compositional heterogeneities. Finally, tantalizing new developments in the field have demonstrated that lipid domains might not just be associated with the plasma membrane of eukaryotes but could potentially be a ubiquitous membrane-organizing principle in several other biological systems. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA.

Helicobacter pylori, a Gram-negative bacterium, is a well-known risk factor for gastric cancer. H. pylori vacuolating cytotoxin A (VacA) is a secreted pore-forming toxin that induces a wide range of cellular responses. Like many other bacterial toxins, VacA has been hypothesized to utilize lipid rafts to gain entry into host cells. Here, we used giant plasma membrane vesicles (GPMVs) as a model system to understand the preferential partitioning of VacA into lipid rafts. We show that a wild-type (WT) toxin predominantly associates with the raft phase. Acid activation of VacA enhances binding of the toxin to GPMVs but is not required for raft partitioning. VacA mutant proteins with alterations at the amino terminus (resulting in impaired membrane channel formation) and a nonoligomerizing VacA mutant protein retain the ability to preferentially associate with lipid rafts. Consistent with these results, the isolated VacA p55 domain was capable of binding to lipid rafts. We conclude that the affinity of VacA for rafts is independent of its capacity to oligomerize or form membrane channels.

Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains.

Current models of lipid rafts propose that lipid domains exist as nanoscale compositional fluctuations and these fluctuations can potentially be stabilized into larger domains, consequently better compartmentalizing cellular functions. However, the mechanisms governing stabilized raft assembly and function remain unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) that binds up to five GM1 glycosphingolipids to enter host cells. We show that when applied to cell-derived giant plasma membrane vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning to the ordered phase compared to wild-type CTxB with five binding sites. Moreover, monovalent CTxB does not stabilize membrane domains, unlike wild-type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a lipid crosslinking mechanism and establish a role for crosslinking in the partitioning of CTxB to ordered domains.

Caveolin-1 is an aggresome-inducing protein.

Caveolin-1 (Cav1) drives the formation of flask-shaped membrane invaginations known as caveolae that participate in signaling, clathrin-independent endocytosis and mechanotransduction. Overexpression or mutations of Cav1 can lead to its mistrafficking, including its accumulation in a perinuclear compartment previously identified as the Golgi complex. Here, we show that in the case of overexpressed Cav1-GFP, this perinuclear compartment consists of cytoplasmic inclusion bodies generated in response to the accumulation of aggregates of misfolded proteins, known as aggresomes. Aggresomes containing Cav1-GFP are encased within vimentin cages, form in a microtubule-dependent manner, and are enriched in a number of key regulators of protein turnover, including ubiquitin, VCP/p97 and proteasomes. Interestingly, aggresome induction was cell-type dependent and was observed for many but not all Cav1 constructs tested. Furthermore, endogenous Cav1 accumulated in aggresomes formed in response to proteosomal inhibition. Our finding that Cav1 is both an aggresome-inducing and aggresome-localized protein provides new insights into how cells handle and respond to misfolded Cav1. They also raise the possibility that aggresome formation may contribute to some of reported phenotypes associated with overexpressed and/or mutant forms of Cav1.

Analysis of diffusion in curved surfaces and its application to tubular membranes.

Diffusion of particles in curved surfaces is inherently complex compared with diffusion in a flat membrane, owing to the nonplanarity of the surface. The consequence of such nonplanar geometry on diffusion is poorly understood but is highly relevant in the case of cell membranes, which often adopt complex geometries. To address this question, we developed a new finite element approach to model diffusion on curved membrane surfaces based on solutions to Fick's law of diffusion and used this to study the effects of geometry on the entry of surface-bound particles into tubules by diffusion. We show that variations in tubule radius and length can distinctly alter diffusion gradients in tubules over biologically relevant timescales. In addition, we show that tubular structures tend to retain concentration gradients for a longer time compared with a comparable flat surface. These findings indicate that sorting of particles along the surfaces of tubules can arise simply as a geometric consequence of the curvature without any specific contribution from the membrane environment. Our studies provide a framework for modeling diffusion in curved surfaces and suggest that biological regulation can emerge purely from membrane geometry.

Characterization of a caveolin-1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin-1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C-terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae-associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C-terminus of wild-type CAV1 in caveolae, reduced colocalization of cavin-1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 -/- mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.