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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.

Funder: This study received Portuguese national funds from FCT - Foundation for Science and Technology through project UIDB/04326/2020, UIDP/04326/2020 and LA/P70101/2020, and from the operational programmes CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER-022121, Funder: UK Medical Research Council, 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.

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., Alpha-1 Foundation Grifols Alpha-1-Antitrypsin Laurell’s Training Award National Biofilms Innovation Centre MINECO MedImmune Infinitus NIH The American Cancer Society The Pershing Square Sohn Cancer Research Award The Chan Zuckerberg Initiative UK Dementia Research Institute grant Canadian Institutes of Health Research US Alzheimer Society Integrated Biological Imaging Network

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 within minutes 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 re-positioning of lysosomes, which leads to both a redistribution of the ER tubules and its global morphology. 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.

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. Using super-resolution microscopy, tracking of single particle trajectories of endoplasmic reticulum (ER) luminal proteins traversing tubular ER, and measuring ER dynamics, Holcman et al. show that ER content is propelled by active luminal flow.

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 (FLIM). ROVI visualised spatial and temporal dynamics of microviscosity with sub-organellar 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 amongst individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarisation. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.

Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.

$\textbf{Background:}$ The fate of hydrogen peroxide (H$_2$O$_2$) 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 H$_2$O$_2$ produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H$_2$O$_2$ probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione’s role in ER H$_2$O$_2$ metabolism. $\textbf{Results:}$ Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H$_2$O$_2$ in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H$_2$O$_2$ production, ER-localized TriPer detected an increase in the luminal H$_2$O$_2$ signal upon induction of pro-insulin (a disulfide-bonded protein of pancreatic β-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 H$_2$O$_2$ signal and eroded β-cell viability. $\textbf{Conclusions:}$ A tri-cysteine system with a single peroxidatic thiol enables H$_2$O$_2$ detection in oxidizing milieux such as that of the ER. Tracking ER H$_2$O$_2$ in live pancreatic β-cells points to a role for glutathione in H$_2$O$_2$ turnover., This work is supported by grants from the Wellcome Trust (Wellcome 200848/Z/16/Z, WT: UNS18966), Fundação para a Ciência e Tecnologia, Portugal (PTDC/QUI/BIQ/119677/2010 and UID/BIM/04773/2013-CBMR), European Commission (EU FP7 Beta-Bat No: 277713), EPSRC (1503478), MRC (MR/K015850/1), and a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140). DR is a Wellcome Trust Principal Research Fellow.

Fluorescent lifetime imaging of an ER-tuned redox-responsive probe revealed an unanticipated stability of ER thiol redox to fluctuations in unfolded protein load, in contrast with sensitivity to lumenal calcium., Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. In vivo lifetime imaging by time-correlated single-photon counting (TCSPC) recorded subtle changes in ER redox poise induced by exposure of mammalian cells to a reducing environment but revealed an unanticipated stability of redox to fluctuations in unfolded protein load. By contrast, TCSPC of roGFPiE uncovered a hitherto unsuspected reductive shift in the mammalian ER upon loss of luminal calcium, whether induced by pharmacological inhibition of calcium reuptake into the ER or by physiological activation of release channels. These findings recommend fluorescent lifetime imaging as a sensitive method to track ER redox homeostasis in mammalian cells.

Tracking the kinetics of equilibration of H2O2 between compartments reveals unexpected isolation of the endoplasmic reticulum and hints at a hitherto unsuspected local source of peroxide., The endoplasmic reticulum (ER)–localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H2O2) in the ER lumen. We report on a simple kinetic technique to track H2O2 equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondrial H2O2 pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H2O2 attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H2O2 in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H2O2 by PRDX4 and a parallel slow H2O2-independent pathway for disulfide formation.

SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using ‘FLIP’ (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2., Exportin interacts with FTO and this interaction might be involved in the nucelocytoplasmic shuttling of FTO in the cell.

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.