The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer’s disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated ...amyloid beta (Aβ) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aβ pathology and neurodegeneration but reduced Iba1+ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33−/− and downregulated in 5xFAD;TREM2−/− mice. Differential gene expression in 5xFAD;CD33−/− microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1β/IL-1RN axis and a gene set in the “receptor activity chemokine” cluster. Our results should facilitate AD therapeutics targeting these receptors.
•Mitigation of Aβ pathology in 5xFAD;CD33−/− mice is abrogated by knocking out TREM2•Reduction of Iba1+ cells in 5xFAD;TREM2−/− mice is not rescued by knocking out CD33•CD33 and TREM2 knockout increase and reduce microglial activation, respectively•Gene expression changes in 5xFAD;CD33−/− microglia depend on the presence of TREM2
Microglial receptors CD33 and TREM2 exhibit opposite effects on Aβ pathology and microglial activity in AD mice. In crosstalk between these receptors, TREM2 is required for differential gene expression in 5xFAD mice deficient for CD33 and acts downstream of CD33.
The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions ...accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.
We present a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection at ultra-high analytical throughput. The imaging platform ...is capable of multiparametric fluorescence quantification and sub-cellular localization of these structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of the approach through the analysis and localization of P-bodies and stress granules in yeast and human cells using fluorescence and bright-field detection at analytical throughputs in excess of 60,000 and 400,000 cells/s, respectively. Results highlight the utility of our imaging flow cytometer in directly investigating phase-separated compartments within cellular environments and screening rare events at the sub-cellular level for a range of diagnostic applications.
Display omitted
•A high-throughput (60,000 cells/s) method for fluorescence imaging flow cytometry•Collection of multi-color fluorescence blur-free images with high sensitivity•Sub-cellular analysis of structures down to 500 nm with microscopy image quality•High-throughput localization analysis of phase-separated compartments in cells
Holzner et al. describe a microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection, enabling sub-cellular multi-color analysis at throughputs above 60,000 cells/s. This imaging platform notably enables ultra-high-throughput colocalization analysis of fluorescent proteins within phase-separated compartments at a resolution down to 500 nm in eukaryotic cells.
Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing ...homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.
Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular ...regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.
Protein aggregates, and in particular amyloids, are generally considered to be inherently irreversible aberrant clumps, and are often associated with pathologies, such as Alzheimer's disease, ...Parkinson's disease, or systemic amyloidosis. However, recent evidence demonstrates that some aggregates are not only fully reversible, but also perform essential physiological functions. Despite these new findings, very little is known about how these functional protein aggregates are regulated in a physiological context. Here, we take the yeast pyruvate kinase Cdc19 as an example of a protein forming functional, reversible, solid, amyloid-like aggregates in response to stress conditions. Cdc19 aggregation is regulated via an aggregation-prone low complexity region (LCR). In favorable growth conditions, this LCR is prevented from aggregating by phosphorylation or oligomerization, while upon glucose starvation it becomes exposed and allows aggregation. We suggest that LCR phosphorylation, oligomerization or partner-binding may be general and widespread mechanisms regulating LCR-mediated reversible protein aggregation. Moreover, we show that, as predicted by computational tools, Cdc19 forms amyloid-like aggregates in vitro. Interestingly, we also observe striking similarities between Cdc19 and its mammalian counterpart, PKM2. Indeed, also PKM2 harbors a LCR and contains several peptides with high amyloidogenic propensity, which coincide with known phosphorylation sites. Thus, we speculate that the formation of reversible, amyloid-like aggregates may be a general physiological mechanism for cells to adapt to stress conditions, and that the underlying regulatory mechanisms may be conserved from yeast to humans.
Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and ...heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical–chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro. Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease–associated Aβ42 peptide, particularly secondary β-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.
Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular ...functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.
Aggregation-prone proteins (APPs) have been implicated in numerous human diseases but the underlying mechanisms are incompletely understood. Here we comparatively analysed cellular responses to ...different APPs. Our study is based on a systematic proteomic and phosphoproteomic analysis of a set of yeast proteotoxicity models expressing different human disease-related APPs, which accumulate intracellular APP inclusions and exhibit impaired growth. Clustering and functional enrichment analyses of quantitative proteome-level data reveal that the cellular response to APP expression, including the chaperone response, is specific to the APP, and largely differs from the response to a more generalized proteotoxic insult such as heat shock. We further observe an intriguing association between the subcellular location of inclusions and the location of the cellular response, and provide a rich dataset for future mechanistic studies. Our data suggest that care should be taken when designing research models to study intracellular aggregation, since the cellular response depends markedly on the specific APP and the location of inclusions. Further, therapeutic approaches aimed at boosting protein quality control in protein aggregation diseases should be tailored to the subcellular location affected by inclusion formation.
We have examined the global cellular response, in terms of protein abundance and phosphorylation changes, to the expression of five human neurodegeneration-associated, aggregation-prone proteins (APPs) in a set of isogenic yeast models. Our results show that the cellular response to each APP is unique to that protein, is different from the response to thermal stress, and is associated with processes at the subcellular location of APP inclusion formation. These results further our understanding of how cells, in a model organism, respond to expression of APPs implicated in neurodegenerative diseases like Parkinson's, Alzheimer's, and ALS. They have implications for mechanisms of toxicity as well as of protective responses in the cell. The specificity of the response to each APP means that research models of these diseases should be tailored to the APP in question. The subcellular localization of the response suggest that therapeutic interventions should also be targeted within the cell.
Display omitted
•Proteomics and phosphoproteomics on yeast strains expressing aggregation-prone proteins•Catalogues cellular response to 5 such proteins associated with neurodegenerative disease.•Cellular response to expression of aggregation-prone proteins is specific to the protein.•The response to aggregation-prone proteins also differs from global thermal unfolding.•The response is associated with processes at the subcellular location of inclusions.
PDF
