Cell Reports Methods最新文献

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (k_flux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in k_flux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.

Continual advancements in genomics have led to an ever-widening disparity between the rate of discovery of genetic variants and our current understanding of their functions and potential roles in disease. Systematic methods for phenotyping DNA variants are required to effectively translate genomics data into improved outcomes for patients with genetic diseases. To make the biggest impact, these approaches must be scalable and accurate, faithfully reflect disease biology, and define complex disease mechanisms. We compare current methods to analyze the function of variants in their endogenous DNA context using genome editing strategies, such as saturation genome editing, base editing and prime editing. We discuss how these technologies can be linked to high-content readouts to gain deep mechanistic insights into variant effects. Finally, we highlight key challenges that need to be addressed to bridge the genotype to phenotype gap, and ultimately improve the diagnosis and treatment of genetic diseases.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 μm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.

Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.

The pathogenesis of Alzheimer disease (AD) involves complex gene regulatory changes across different cell types. To help decipher this complexity, we introduce single-cell Bayesian biclustering (scBC), a framework for identifying cell-specific gene network biomarkers in scRNA and snRNA-seq data. Through biclustering, scBC enables the analysis of perturbations in functional gene modules at the single-cell level. Applying the scBC framework to AD snRNA-seq data reveals the perturbations within gene modules across distinct cell groups and sheds light on gene-cell correlations during AD progression. Notably, our method helps to overcome common challenges in single-cell data analysis, including batch effects and dropout events. Incorporating prior knowledge further enables the framework to yield more biologically interpretable results. Comparative analyses on simulated and real-world datasets demonstrate the precision and robustness of our approach compared to other state-of-the-art biclustering methods. scBC holds potential for unraveling the mechanisms underlying polygenic diseases characterized by intricate gene coexpression patterns.

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.

Tissue infiltration by circulating leukocytes occurs via adhesive interactions with the local vasculature, but how the adhesive quality of circulating cells guides the homing of specific phenotypes to different vascular microenvironments remains undefined. We developed an optofluidic system enabling fluorescent labeling of photoactivatable cells based on their adhesive rolling velocity in an inflamed vasculature-mimicking microfluidic device under physiological fluid flow. In so doing, single-cell level multidimensional profiling of cellular characteristics could be characterized and related to the associated adhesive phenotype. When applied to CD8⁺ T cells, ligand/receptor expression profiles and subtypes associated with adhesion were revealed, providing insight into inflamed tissue infiltration capabilities of specific CD8⁺ T lymphocyte subsets and how local vascular microenvironmental features may regulate the quality of cellular infiltration. This methodology facilitates rapid screening of cell populations for enhanced homing capabilities under defined biochemical and biophysical microenvironments, relevant to leukocyte homing modulation in multiple pathologies.