Hippocampal neurons’ cytosolic and membrane-bound ribosomal transcript profiles are differentially regulated by learning and subsequent sleep

The hippocampus is essential for consolidating transient experiences into long-lasting memories. Memory consolidation is facilitated by postlearning sleep, although the underlying cellular mechanisms are largely unknown. We took an unbiased approach to this question by using a mouse model of hippocampally mediated, sleep-dependent memory consolidation (contextual fear memory). Because synaptic plasticity is associated with changes to both neuronal cell membranes (e.g., receptors) and cytosol (e.g., cytoskeletal elements), we characterized how these cell compartments are affected by learning and subsequent sleep or sleep deprivation (SD). Translating ribosome affinity purification was used to profile ribosome-associated RNAs in different subcellular compartments (cytosol and membrane) and in different cell populations (whole hippocampus, Camk2a+ neurons, or highly active neurons with phosphorylated ribosomal subunit S6 [pS6+]). We examined how transcript profiles change as a function of sleep versus SD and prior learning (contextual fear conditioning; CFC). While sleep loss altered many cytosolic ribosomal transcripts, CFC altered almost none, and CFC-driven changes were occluded by subsequent SD. In striking contrast, SD altered few transcripts on membrane-bound (MB) ribosomes, while learning altered many more (including long non-coding RNAs [lncRNAs]). The cellular pathways most affected by CFC were involved in structural remodeling. Comparisons of post-CFC MB transcript profiles between sleeping and SD mice implicated changes in cellular metabolism in Camk2a+ neurons and protein synthesis in highly active pS6+ (putative “engram”) neurons as biological processes disrupted by SD. These findings provide insights into how learning affects hippocampal neurons and suggest that the effects of SD on memory consolidation are cell type and subcellular compartment specific.

SubcellulaRVis: an app to visualize subcellular compartment enrichment

High-throughput 'omics methods result in lists of differentially regulated or expressed genes or proteins, whose function is generally studied through statistical methods such as enrichment analyses. One aspect of protein regulation is subcellular localization, which is crucial for their correct processing and function and can change in response to various cellular stimuli. Enrichment of proteins for subcellular compartments is often based on Gene Ontology Cellular Compartment annotations. Results of enrichment are typically visualized using bar-charts, however enrichment analyses can result in a long list of significant annotations which are highly specific, preventing researchers from gaining a broad understanding of the subcellular compartments their proteins of interest may be located in. Schematic visualization of known subcellular locations has become increasingly available for single proteins via the UniProt and COMPARTMENTS platforms. However, it is not currently available for a list of proteins (e.g. from the same experiment) or for visualizing the results of enrichment analyses. To generate an easy-to-interpret visualization of protein subcellular localization after enrichment we developed the SubcellulaRVis web app, which visualizes the enrichment of subcellular locations of gene lists in an easy and impactful manner. SubcellulaRVis projects the results of enrichment analysis on a graphical representation of a eukaryotic cell. Implemented as a web app and an R package, this tool is user-friendly, provides exportable results in different formats, and can be used for gene lists derived from multiple organisms. Here, we show the power of SubcellulaRVis to assign proteins to the correct subcellular compartment using gene list enriched in previously published spatial proteomics datasets. We envision SubcellulaRVis will be useful for cell biologists with limited bioinformatics expertise wanting to perform precise and quick enrichment analysis and immediate visualization of gene lists.

Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML. The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations. Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models. We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1. We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A). To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B). We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C). In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D). In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E). Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers. Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. Figure 1 Figure 1. Disclosures Stahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Chlamydomonas State Transition Is Quiet Around Pyrenoid and Independent from Thylakoid Deformation

Photosynthetic organisms have developed a rapid regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the ultrastructural dynamics of the thylakoid membrane to the ST mechanism, how ST is related to the ultrastructural dynamic of the thylakoid in Chlamydomonas remains elusive. To clarify the above-mentioned relation, here we used two specialized microscope techniques, observation via the excitation-spectral microscope (ESM) developed recently by us and the super-resolution imaging based on structured illumination microscopy (SIM). The ESM observation revealed a highly reversible rearrangement of LHCII-related fluorescence. More importantly, it clarified lower ST activity in the region surrounding the pyrenoid, which is the specific subcellular compartment associated with the carbon-fixation reaction. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations induced by the ST-inducing illumination. Fine irreversible thylakoid transformation was also observed for the Stt7-kinase-lacking mutant. This result, together with the nearly equal structural changes in the less active ST regions around the pyrenoid, suggested the independence of the observed fine structural changes from the LHCII phosphorylation.

Enhanced enzymatic production of cholesteryl 6ʹ-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori

Background During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6ʹ-O-acyl-α-d-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. Methods We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. Results The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. Conclusions Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Methyl jasmonate elicits distinctive hydrolyzable tannin, flavonoid, and phyto-oxylipin responses in pomegranate (Punica granatum L.) leaves

Main conclusion Transcriptome and biochemical analyses suggested that, while suppression of multiple flavonoids and anthocyanins occurs at least partially at the transcriptional level, increased biosynthesis of non-jasmonate phyto-oxylipins is likely controlled non-transcriptionally. Abstract Methyl jasmonate (MeJA) produced in plants can mediate their response to environmental stresses. Exogenous application of MeJA has also shown to activate signaling pathways and induce phytoalexin accumulation in many plant species. To understand how pomegranate plants respond biochemically to environmental stresses, metabolite analysis was conducted in pomegranate leaves subjected to MeJA application and revealed unique changes in hydrolyzable tannins, flavonoids, and phyto-oxylipins. Additionally, transcriptome and real-time qPCR analyses of mock- and MeJA-treated pomegranate leaves identified differentially expressed metabolic genes and transcription factors that are potentially involved in the control of hydrolyzable tannin, flavonoid, and phyto-oxylipin pathways. Molecular, biochemical, and bioinformatic characterization of the only lipoxygenase with sustained, MeJA-induced expression showed that it is capable of oxidizing polyunsaturated fatty acids, though not located in the subcellular compartment where non-jasmonate (non-JA) phyto-oxylipins were produced. These results collectively suggested that while the broad suppression of flavonoids and anthocyanins is at least partially controlled at the transcriptional level, the induced biosynthesis of non-JA phyto-oxylipins is likely not regulated transcriptionally. Overall, a better understanding of how pomegranate leaves respond to environmental stresses will not only promote plant health and productivity, but also have an impact on human health as fruits produced by pomegranate plants are a rich source of nutritional compounds.

Optimization of ROS measurement and localization in plant tissues: challenges and solutions v1

During the last decade, there has been a huge interest in understanding the role of reactive oxygen species (ROS) in plant signalling transduction pathways. This understanding requires precise quantification of ROS levels in each cell and each cellular compartment. However, the current methods of ROS detection and measuring are limited. This paper revisits the existing ROS detection methods and discuss general guidelines for applying them to specific cases. Introduction All plants require molecular oxygen for survival (Mittler, 2017). ROS formation naturally occurred during electron transport through all membranes which, in turn, regulate DNA repair systems, cell cycle, phytohormone-dependent signalling and pathogen integration (Huang et al., 2019). In the non-photosynthetic plant tissue, the mitochondrial electron transport system of oxidative phosphorylation is the major site for ROS generation (Dourmap et al., 2020). While in photosynthetic tissue, electron transport between stroma and thylakoid is the primary ROS source (Asada, 2006). On plasma membranes and on endoplasmic reticulum membranes, ROS is mainly produced via NADPH oxidases (Foreman et al., 2003).Cell wall peroxidases are another source of apoplastic ROS (Torres, 2010). In addition, peroxisomes can be considered as the major site of intracellular hydrogen peroxide (H2O2) production (Sandalio et al., 2021). Major ROS produced by cellular processes are superoxide (O2-), H2O2, and hydroxyl radical (∙OH). Superoxide is rapidly converted to H2O2 by superoxide dismutase enzymes (SODs; Cu/Zn-SOD in chloroplasts and cytoplasm, Fe-SOD and Mn-SOD in mitochondria). Hydroxyl radicals are thus generated in the cell wall, plasma membrane, and intracellularly by a range of peroxidases, superoxide dismutases, NADPH oxidases, and transition metal catalysts (Richards et al., 2015). Because of the cellular and biochemical damage caused by oxidative stress (Huang et al., 2019), ROS levels should be precisely controlled in each subcellular compartment and each cell type. ROS are highly reactive molecules rapidly subjected to scavenging or degradation, in processes that are highly sensitive to any environmental change, therefore making ROS extremely unstable and difficult to directly detect. Transferring of the plants to buffers with non-physiological pH can be considered as an abiotic stress factor and it eventually might change endogenous ROS levels (Choudhury et al., 2017). However, many established protocols for ROS measurement (Dunand et al., 2007, Jambunathan, 2010, Rodríguez & Taleisnik, 2012) included the soaking of plant tissues on non-physiological buffers, which might alter steady-state ROS levels. Several methods have been used for ROS localization and they rely on histochemistry, fluorescent dyes, and spectrophotometric measurements (Mittler et al., 2011). Histochemistry Histochemical methods are based on the oxidation of dyes in the presence of ROS, resulting in the production of insoluble precipitates. For example, nitro blue tetrazolium (NBT) chloride reacted with O2- to generate water-insoluble di-formazan, while 3-3-diaminobenzidine (DAB) is oxidized by H2O2 in the presence of peroxidases with formation of a dark-brown precipitate (Jambunathan, 2010). Fluorescent dyes Some chemical dyes became fluorescent after oxidation by ROS, like H2DCFDA, DHE or Amplex red (Ortega-Villasante et al., 2016). These dyes can be used for direct ROS localization. Spectrophotometric methods They allow to quantitatively determine ROS level after tissue homogenization, such as the determination of H2O2 levels with 3,5-dichloro-2-hydroxybenzensulfonic acid (DCHBS)in conjunction with 4-aminoantipyrine (AAP) (Van Gestelen et al., 1998). There methods were summarised in the graphical abstracts. Here we provide several detailed protocols for ROS localization and quantification under physiological conditions, aimed to improve current methods and to minimize artefacts.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.