It is well established that glutathione (GSH) is mainly utilized by glutathione peroxidase 4 (GPX4) to diminish peroxidized phospholipids and defend against ferroptosis. However, we found that GSH robustly protects a variety of GPX4 knockout (KO) cells from ferroptosis, thus indicating that there is an alternative pathway for GSH to suppress ferroptosis. By carrying out genome-wide CRISPR-Cas9 screening, we identified that ferroptosis suppressor protein 1 (FSP1) is the key factor mediating the GPX4-independent protective effect of GSH. Mechanistically, FSP1 uses GSH to generate reduced ubiquinone, which scavenges oxidized lipids and suppresses ferroptosis. Inhibition of FSP1 abolishes this GSH-dependent ferroptosis protection both in vitro and in vivo. Distinct from NAD(P)H-mediated ubiquinone reduction, GSH-stimulated generation of reduced ubiquinone via FSP1 is independent of flavin adenine dinucleotide (FAD). Together, our work clarifies a new mechanism of GSH-mediated ferroptosis suppression, thus highlighting the potential that targeting GSH metabolism should be beneficial for ferroptosis-related diseases whether or not it is related to GPX4.

How gene expression programs are coordinated across space and time to build a complete plant remains poorly understood. Here, we combine high-resolution spatial transcriptomics, single-nucleus RNA sequencing, and a gap-free reference genome to build a 3D spatiotemporal atlas of rice, spanning 10 organs and 61 stages from seed to seed. Our datasets profile 851,725 nuclei and 347,640 spatial bins, defining 119 cell types and 133 subtypes across the rice lifecycle. Through multimodal integration, we reconstruct an inferred developmental trajectory graph that links cell states from the proembryo to mature organs and identifies trajectory-associated regulators. We further implicate the pleiotropic regulator OsARF1 in distinct spatial gene programs across developmental contexts. We also uncover widespread axis-associated transcriptional asymmetry, including dorsoventral endosperm programs that partition starch and protein accumulation. Collectively, this high-resolution atlas provides a spatiotemporal framework for understanding rice development and guiding future crop improvement.

Forward genetic screening links genotype to phenotype by introducing random genetic perturbations and identifying phenotype-altering mutations. Although genome-scale screens are routine for simple phenotypes in cultured cells, extending them to complex image-based phenotypes remains challenging. Here, we present spatially resolved CRISPR screening (SPARCS), a microscopy-based platform for forward genetic screening on single-cell images. SPARCS physically isolates mutants in situ by automated laser microdissection, enabling image-based screening at unprecedented scale with multimodal hit phenotyping. We demonstrate SPARCS in genome-wide CRISPR knockout screens of autophagosome formation and activation of the immune sensor STING across 70 million cells. Via machine learning-based image analysis, SPARCS recovered most macroautophagy genes and identified GPHR as a pH-dependent regulator of STING. Mass spectrometry-based proteomics of isolated hit cells revealed endoplasmic reticulum (ER)/Golgi disruption and nominated additional STING regulators via in silico perturbation modeling. These results establish SPARCS as a scalable platform for genome-wide genetic screening of complex cellular phenotypes with a proteome-level readout.

Metabolic competition between tumors and T cells drives immune evasion, but the transporters and mechanisms remain largely elusive. Here, we establish the CareSLCs platform and identify that SLC52A3-mediated vitamin B2 (VB2) uptake is indispensable for T cell antitumor immunity. Mechanistically, VB2 deficiency impairs mitochondrial respiration and glutathione regeneration, leading to mitophagy, labile iron accumulation, and lipid peroxidation that trigger T cell ferroptosis. Unexpectedly, tumors preferentially employ SLC52A2, not SLC52A3, to scavenge VB2 and outcompete T cells. Tumor SLC52A2 ablation enhances T cell function in immunocompetent hosts without appreciably affecting intrinsic tumor growth. Clinically, high tumoral SLC52A2 correlates with T cell dysfunction and poor survival, whereas dietary VB2 intake is associated with reduced cancer risks. Thus, VB2 supplementation or SLC52A3 overexpression augments CAR-T cell antitumor efficacy. Our results suggest that tumoral VB2 competition establishes a metabolic checkpoint driving T cell ferroptosis and that enhancing VB2 uptake reinvigorates T cells to improve cancer immunotherapy.

Sex differences in cancer therapy response remain poorly understood. Using a "phase II-like preclinical trial" across 55 CLDN18.2+ pancreatic cancer patient-derived xenografts, we identify marked female-biased resistance to CLDN18.2-targeted antibody-drug conjugate (ADC) therapy despite equivalent antigen expression. Mechanistically, estrogen signals through GPER1 to upregulate TRAF6, redirecting tumor-intrinsic STING signaling from IRF3-mediated interferon production toward nuclear factor κB (NF-κB) activation. This drives CXCL1 secretion, neutrophil recruitment, and neutrophil extracellular trap (NET) formation. NETs form a charged extracellular network that restricts antibody access to tumor-cell-surface antigens-a mechanism we term functional antigen shielding-reducing ADC cytotoxicity independently of antigen expression. The NET burden also correlated with poorer responses to trastuzumab, cetuximab, and enfortumab vedotin across three clinical cohorts. Pharmacologic NET disruption with sivelestat restored ADC efficacy in female mice. These findings reveal an estrogen-driven resistance mechanism with broad implications for antibody-based cancer therapy.

Oxidative phosphorylation (OXPHOS) is a key metabolic process that couples redox energy to ATP production. While some core OXPHOS complex subunits are found across all domains of life, many have diverged or expanded across evolution-as seen in the protozoan pathogen Acanthamoeba castellanii. By integrating cryo-electron microscopy of unenriched mitochondrial lysate with mass spectrometry proteomics, we resolved the structures of endogenous mitochondrial ATP synthase (complex V), Hsp60, and respiratory complex III from Acanthamoeba. We capture Acanthamoeba ATP synthase in an IF1-inhibited state and reveal how Acanthamoeba-specific subunits and extensions stabilize the molecular machine, which includes a β subunit extension that interfaces with the peripheral stalk. Additionally, we characterize an active malate dehydrogenase (MDH) dimer structurally integrated within the ATP synthase peripheral stalk, thus revealing a direct protein tether between OXPHOS and the tricarboxylic acid cycle. Together, these findings provide structural insight into lineage-specific adaptations in Acanthamoeba that may tune protozoan metabolism.

Mitochondria arose from the endosymbiosis of a bacterium with an archaea-related host cell about 2 billion years ago. To understand their origins and evolution, we compared experimentally defined mitoproteomes from the MitoCarta Tree of Life project. Across eight organisms, we identified 8,619 distinct mitochondrial proteins within 3,199 families, of which 43% lack Pfam domains. We report 33 protein families conserved in eukaryotic pathogens yet absent in humans, representing promising candidate targets for protozoan infectious diseases. Leveraging our experimentally defined mitoproteomes, we retrained a classifier based on a protein language model to predict mitoproteomes of ∼200 eukaryotes. From this expanded set, we detail the evolutionary trajectories of mitochondria, ranging from clade-specific gene family expansions to extreme mitoproteome reductions seemingly en route to complete organelle loss. Finally, we reconstruct the last eukaryotic common ancestor (LECA) mitoproteome, revealing that LECA possessed a complex mitochondrion capable of both aerobic and anaerobic metabolism.

Plant mitochondria share many features with animal mitochondria but also possess unique traits, including presence of both circular and linear mitochondrial DNA, RNA editing, a branched electron transport chain, and metabolic pathways supporting photosynthesis. An accurate mitochondrial proteome is crucial for studying these conserved and lineage-specific functions. Here, we introduce a novel approach to defining plant mitoproteomes that combines serial mitochondrial enrichment, mass spectrometry, and protein correlation profiling. This approach allows us to experimentally define a mitochondrial protein inventory of 1,462 proteins with high sensitivity and specificity. Integration of our experimental inventory with literature and microscopy validation yields the Arabidopsisthaliana MitoCarta (AtMitoCarta) atlas of 1,609 mitochondrial proteins, including 236 newly defined proteins and 147 proteins of unknown functions. A comparative analysis across eukaryotic organisms reveals that 15% of mitochondrial protein families are plant specific, including expanded pentatricopeptide repeat proteins. The AtMitoCarta inventory and the experimental workflow applicable to wild-type plants will enable comparative studies of plant mitochondria.

Acanthamoeba castellanii causes infectious blindness and resides in a key evolutionary outgroup to humans and fungi. Its divergent mitochondria are of outstanding interest due to their predicted aerobic and anaerobic functions with potential for drug targeting. However, a detailed delineation of its bioenergetic machinery, its activities, and their regulation remains lacking. Here, we integrate mitochondrial immunoprecipitation, density gradient purification, mass spectrometry, protein correlation profiling, and microscopy to generate a high-confidence inventory of the Acanthamoeba mitoproteome. The resulting AcMitoCarta contains 1,122 proteins, including 381 lacking readily identifiable homologs in human and yeast mitochondria. Complexome analysis highlights 20 macromolecular assemblies. Complementary proteomic and transcriptomic profiling reveals extensive rewiring of the organelle's bioenergetic machinery by oxygen, including induction of an anaerobic pyruvate:ferredoxin oxidoreductase-to-hydrogenase pathway under anoxia. We experimentally demonstrate that Acanthamoeba can produce H2 gas under anoxic conditions via a mitochondria-localized, oxygen-labile hydrogenase. AcMitoCarta establishes a framework for dissecting the interplay between aerobic and anaerobic energy metabolism.

Our textbook view of mitochondria, which has been shaped by studies in animals and yeast, fails to do justice to the organelle's broader eukaryotic diversity. While most mitochondria retain a tiny genome, the overwhelming majority of their proteins are nuclear-encoded and vary extensively across lineages. To map this diversity, the MitoCarta Tree of Life Consortium developed experimental and computational workflows to generate high-accuracy mitochondrial proteomes across diverse eukaryotes-helping to lay a foundation for comparative mitochondrial biology with broad implications for physiology, evolution, and disease.

In this issue of Cell, Chen et al. chart mitochondrial proteome diversity across eukaryotes, revealing an unexpectedly complex ancestral proteome alongside extensive lineage-specific innovation. Their broad phylogenetic reconstruction provides new insights into the mitochondrial proteome of the last eukaryotic common ancestor while unearthing a wealth of unexplored mitochondrial biology outside of traditional model systems.

What's the highest temperature at which complex life can grow? It's an old question. Here, Rappaport and colleagues describe a thermophilic amoeba that breaks the previous record, which was set at ∼60°C, and suggest the beginnings of an answer to what innovations enable this organism to take the heat.

In this issue of Cell, Liu et al. introduce ULTRA, which combines rapid tissue clearing, single-channel stimulated Raman scattering microscopy, and AI to generate cell-resolved three-dimensional virtual histology of intact glioma specimens in about 30 minutes-fast enough to expose depth-dependent infiltration before surgery ends.