Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals.

The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology.

The ability to quantify structural changes of the endoplasmic reticulum (ER) is crucial for understanding the structure and function of this organelle. However, the rapid movement and complex topology of ER networks make this challenging. Here, we construct a state-of-the-art semantic segmentation method that we call ERnet for the automatic classification of sheet and tubular ER domains inside individual cells. Data are skeletonized and represented by connectivity graphs, enabling precise and efficient quantification of network connectivity. ERnet generates metrics on topology and integrity of ER structures and quantifies structural change in response to genetic or metabolic manipulation. We validate ERnet using data obtained by various ER-imaging methods from different cell types as well as ground truth images of synthetic ER structures. ERnet can be deployed in an automatic high-throughput and unbiased fashion and identifies subtle changes in ER phenotypes that may inform on disease progression and response to therapy.

Intracellular Aß42 Aggregation Leads to Cellular Thermogenesis.

The aggregation of Aß42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aß42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aß42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aß42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aß peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aß peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2.

BACKGROUND: The fate of hydrogen peroxide (H2O2) in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H2O2 produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H2O2 probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H2O2 metabolism. RESULTS: Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide-bonded protein of pancreatic ß-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in the lumen of the ER further enhanced the luminal H2O2 signal and eroded ß-cell viability. CONCLUSIONS: A tri-cysteine system with a single peroxidatic thiol enables H2O2 detection in oxidizing milieux such as that of the ER. Tracking ER H2O2 in live pancreatic ß-cells points to a role for glutathione in H2O2 turnover.

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum.

BACKGROUND: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. RESULTS: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca(2+) concentrations approached those normally found in the ER lumen ([Ca(2+)]K(0.5max) = 190 µM). CONCLUSIONS: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.

A shared endoplasmic reticulum-associated degradation pathway involving the EDEM1 protein for glycosylated and nonglycosylated proteins.

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF(Fbs2) E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.

Non-esterified fatty acid palmitate facilitates oxidative endoplasmic reticulum stress and apoptosis of ß-cells by upregulating ERO-1α expression.

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to ß-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1ß, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic ß-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent ß-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated ß-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic ß-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and ß-cell failure.

Cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy in mouse models.

Aggregates of the protein tau are proposed to drive pathogenesis in neurodegenerative diseases. Tau can be targeted by using passively transferred antibodies (Abs), but the mechanisms of Ab protection are incompletely understood. In this work, we used a variety of cell and animal model systems and showed that the cytosolic Ab receptor and E3 ligase TRIM21 (T21) could play a role in Ab protection against tau pathology. Tau-Ab complexes were internalized to the cytosol of neurons, which enabled T21 engagement and protection against seeded aggregation. Ab-mediated protection against tau pathology was lost in mice that lacked T21. Thus, the cytosolic compartment provides a site of immunotherapeutic protection, which may help in the design of Ab-based therapies in neurodegenerative disease.

High-throughput super-resolution single-particle trajectory analysis reconstructs organelle dynamics and membrane reorganization.

Super-resolution imaging can generate thousands of single-particle trajectories. These data can potentially reconstruct subcellular organization and dynamics, as well as measure disease-linked changes. However, computational methods that can derive quantitative information from such massive datasets are currently lacking. We present data analysis and algorithms that are broadly applicable to reveal local binding and trafficking interactions and organization of dynamic subcellular sites. We applied this analysis to the endoplasmic reticulum and neuronal membrane. The method is based on spatiotemporal segmentation that explores data at multiple levels and detects the architecture and boundaries of high-density regions in areas measuring hundreds of nanometers. By connecting dense regions, we reconstructed the network topology of the endoplasmic reticulum (ER), as well as molecular flow redistribution and the local space explored by trajectories. The presented methods are available as an ImageJ plugin that can be applied to large datasets of overlapping trajectories offering a standard of single-particle trajectory (SPT) metrics.

Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP.

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.

Z-α1-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state.

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.

THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC.

Intracellular Sources of ROS/H2O2 in Health and Neurodegeneration: Spotlight on Endoplasmic Reticulum.

Reactive oxygen species (ROS) are produced continuously throughout the cell as products of various redox reactions. Yet these products function as important signal messengers, acting through oxidation of specific target factors. Whilst excess ROS production has the potential to induce oxidative stress, physiological roles of ROS are supported by a spatiotemporal equilibrium between ROS producers and scavengers such as antioxidative enzymes. In the endoplasmic reticulum (ER), hydrogen peroxide (H2O2), a non-radical ROS, is produced through the process of oxidative folding. Utilisation and dysregulation of H2O2, in particular that generated in the ER, affects not only cellular homeostasis but also the longevity of organisms. ROS dysregulation has been implicated in various pathologies including dementia and other neurodegenerative diseases, sanctioning a field of research that strives to better understand cell-intrinsic ROS production. Here we review the organelle-specific ROS-generating and consuming pathways, providing evidence that the ER is a major contributing source of potentially pathologic ROS.

The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes.

The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains: sheets and interconnected tubules. These domains undergo dynamic reshaping in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status: The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven repositioning of lysosomes, which leads to both a redistribution of the ER tubules and a change of its global morphology. Therefore, lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.

Single particle trajectories reveal active endoplasmic reticulum luminal flow.

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell1,2. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure-function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders3,4. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.

An Optical Technique for Mapping Microviscosity Dynamics in Cellular Organelles.

Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment-sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment-specific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.

ER-resident antioxidative GPx7 and GPx8 enzyme isoforms protect insulin-secreting INS-1E ß-cells against lipotoxicity by improving the ER antioxidative capacity.

Increased circulating levels of saturated fatty acids (FFAs) and glucose are considered to be major mediators of ß-cell dysfunction and death in T2DM. Although it has been proposed that endoplasmic reticulum (ER) and oxidative stress play a crucial role in gluco/lipotoxicity, their interplay and relative contribution to ß-cell dysfunction and apoptosis has not been fully elucidated. In addition it is still unclear how palmitate - the physiologically most abundant long-chain saturated FFA - elicits ER stress and which immediate signals commit ß-cells to apoptosis. To study the underlying mechanisms of palmitate-mediated ER stress and ß-cell toxicity, we exploited the observation that the recently described ER-resident GPx7 and GPx8 are not expressed in rat ß-cells. Expression of GPx7 or GPx8 attenuated FFAs-mediated H2O2 generation, ER stress, and apoptosis induction. These results could be confirmed by a H2O2-specific inactivating ER catalase, indicating that accumulation of H2O2 in the ER lumen is critical in FFA-induced ER stress. Furthermore, neither the expression of GPx7 nor of GPx8 increased insulin content or facilitated disulfide bond formation in insulin-secreting INS-1E cells. Hence, reduction of H2O2 by ER-GPx isoforms is not rate-limiting in oxidative protein folding in rat ß-cells. These data suggest that FFA-mediated ER stress is partially dependent on oxidative stress and selective expression of GPx7 or GPx8 improves the ER antioxidative capacity of rat ß-cells without compromising insulin production and the oxidative protein folding machinery.

Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.

Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor-acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor-acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET.

Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD.

Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.