Meng Carla Wang

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans , kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.

Proteomics is a powerful tool for profiling the protein landscape underlying cellular and physiological processes. Advances in mass spectrometry-based methods, such as tandem-mass-tags (TMT) isobaric labeling-based proteomics at both MS2 and MS3, label-free quantification (LFQ) with data-dependent-acquisition (DDA) or data-independent acquisition (DIA)-based proteomics, and stable isotope labeling with amino acid (SILAC) spike-in-assisted proteomics, enable quantitative proteome comparisons across biological conditions. These five quantitative proteomic methods offer distinct advantages in coverage, throughput, and quantitative reproducibility. Here, we treated them as orthogonal approaches, retaining protein candidates with significant, consistent-direction changes supported across multiple independently instrumented methods to identify high-confidence protein-level changes associated with longevity. Insulin/insulin-like growth factor signaling (IIS) is a highly conserved longevity regulatory pathway from Caenorhabditis elegans to humans. By quantitatively profiling IIS mutants in C. elegans and performing genetic screening, we obtained a systems-view of proteomic changes linked to longevity and identified new longevity regulators. This systematic cross-method comparison also serves as a valuable resource for guiding the selection of quantitative proteomics approaches.

High-spatial-resolution in situ mapping of biomolecules within tissue reveals critical insights into the complex molecular landscape and spatial organization of biological systems. Mass spectrometry imaging (MSI) is a powerful tool for spatially resolved molecular analysis of biological samples, with ongoing demand for improved spatial resolution. Tissue expansion combined with MSI (TEMI) is a recently developed approach that enables multiomics molecular mapping across various biological tissues with significantly improved spatial resolution. Unlike conventional methods that depend on instrument-based enhancements in spatial resolution, TEMI physically enlarges tissue samples via harsh-condition-free hydrogel expansion, achieving more than 3.5-fold increase in effective imaging resolution using standard MSI instrumentation. TEMI delivers single-cell spatial resolution in tissue samples and enables detection of biomolecular heterogeneity that remains uncharacterizable in unexpanded tissue using conventional MSI. Notably, TEMI supports high-spatial-resolution mapping of multiple biomolecular classes—including lipids, metabolites, N-glycans, peptides and proteins—within a single tissue sample. Here, we provide a detailed, step-by-step guide for TEMI, including hydrogel-based tissue expansion under mild conditions, cryosectioning of the expanded tissue-hydrogel sample, a comprehensive experimental workflow for multiomics TEMI on a single tissue section, data acquisition and visualization pipelines, as well as troubleshooting tips. Overall, we demonstrate that TEMI overcomes the long-standing spatial limitations of MSI without requiring hardware modifications, ensuring compatibility with existing MSI instruments and promoting broad accessibility and adoption within the research community.

Protein assemblies, including aggregates and condensates, are closely linked to health and diseases. We demonstrate boxcar-enhanced Fluorescence-detected mid-Infrared photothermaL Microscopy (FILM), using two model species, Caenorhabditis elegans and Saccharomyces cerevisiae , to quantitatively resolve these protein states in vivo by imaging β-sheet and α-helix secondary structures and analyzing their ratios. This method directly distinguishes polyglutamine (PolyQ) protein aggregates, α-synuclein protein condensates, and P-granule condensates implicated in neurodegenerative diseases and embryonic development in live organisms. It further enables the unraveling of protein assembly dynamics and their physio-pathological roles, such as age-related progression of PolyQ from condensates to aggregates.

Metabolism unfolds within specific organelles in eukaryotic cells. Lysosomes are highly metabolically active organelles, and their metabolic states dynamically influence signal transduction, cellular homeostasis and organismal physiopathology. Despite the importance of lysosomal metabolism, a method for its in vivo measurement is currently lacking. Here we report a fluorescence-detected mid-infrared photothermal microscope (FILM) implemented with optical boxcar demodulation, artificial intelligence-assisted data denoising and spectral deconvolution, to map metabolic activity and composition of individual lysosomes in living cells and organisms. Using this method, we uncovered lipolysis and proteolysis heterogeneity across lysosomes within the same cell, as well as early-onset lysosomal dysfunction during organismal aging. In addition, we discovered organelle-level metabolic changes associated with diverse lysosomal storage diseases. This method holds the broad potential to profile metabolic fingerprints of individual organelles within their native context and quantitatively assess their dynamic changes under different physiological and pathological conditions, providing a high-resolution chemical cellular atlas.

Metabolism unfolds within specific organelles in eukaryotic cells. Lysosomes are highly metabolically active organelles, and their metabolic states dynamically influence signal transduction, cellular homeostasis, and organismal physiopathology. Despite the importance of lysosomal metabolism, a method for its in vivo measurement is currently lacking. Here, we report a Fluorescence-detected mid-Infrared photothermaL Microscope (FILM) implemented with optical boxcar demodulation, AI-assisted data denoising, and spectral deconvolution, to map metabolic activity and composition of individual lysosomes in living cells and organisms. Using this method, we uncovered lipolysis and proteolysis heterogeneity across lysosomes within the same cell, as well as early-onset lysosomal dysfunction during organismal aging. Additionally, we discovered organelle-level metabolic changes associated with diverse lysosomal storage diseases. This method holds the broad potential to profile metabolic fingerprints of individual organelles within their native context and quantitatively assess their dynamic changes under different physiological and pathological conditions, providing a high-resolution chemical cellular atlas.

Metabolic processes shape ageing and longevity at multiple levels. Emerging evidence shows that many of these processes are orchestrated within and between cellular organelles. Organelles function not only as metabolic reactors but also as signalling hubs, and their coordination plays crucial roles in maintaining cellular homeostasis and promoting organismal fitness. Rather than acting in isolation, organelles engage in dynamic crosstalk through membrane contact sites, metabolite exchange and signalling interplay. In recent years, organelles have been increasingly recognized as critical regulators of ageing and longevity. Here we summarize age-related organellar changes, highlight organelle-mediated intra- and intercellular signalling communication in lifespan and healthspan regulation, and discuss the active roles of organelles in microbiome–host interactions and transgenerational inheritance in regulating longevity. We further outline how longevity-promoting interventions influence organelles, and provide perspectives on how future technological advances may further accelerate progress in this emerging research topic.

Mammalian development takes place inside the maternal uterus, creating technological constraints that make difficult the study of embryogenesis in live developing embryos. A central challenge for understanding the role of metabolism in mammalian development is discriminating placental and uterine-regulated signals from embryo-intrinsic processes independent of maternal influence, a process that until now has remained inseparable during gastrulation and organogenesis ^1–3 . Ex utero culture systems allowing continuous growth of embryos during pre-gastrulation to organogenesis ^4,5 offer a promising solution to this challenge. Here, we present optimized ex utero culture platforms that support faithful development of mouse embryos from gastrulation (embryonic day 6.5/7.5) through the fetal period (embryonic day ∼12.5) and harnessed these platforms for dissecting metabolic transitions in vivo during embryogenesis independently of uterus and placenta. We characterized the metabolome of in utero and ex utero whole embryos, fetal organs and culture medium between embryonic days E6.5 and E12.5 by liquid chromatography mass-spectrometry (LC-MS) metabolomics, isotope tracing, and single cell transcriptomics. These datasets present a comprehensive overview of the dynamic embryonic metabolism during gastrulation and organogenesis in utero and ex utero. This analysis revealed that the midgestational metabolic switch occurring at E10.5-E11.5 is faithfully recapitulated ex utero, indicating that this transition is intrinsically programmed in embryonic tissues and does not require direct maternal or placental cues. Notably, oxygen availability modulated the extent of this transition, but elevated oxygen was insufficient to induce it prematurely, demonstrating that the switch is developmentally timed and only partially environmental-responsive. We further harnessed the ex utero platform for identifying and perturbing a mitochondrial redox shift at E7.5-E8.5 that is critical for developmental progress after gastrulation. These findings uncover the remarkable metabolic plasticity of the mammalian embryo, demonstrating its capacity to sustain growth independently of maternal inputs from the establishment of the body plan through the onset of the fetal period. Moreover, they highlight the use of long-term ex utero culture as a unique framework for dissecting the mechanisms that shape embryogenesis under physiological and experimentally perturbed conditions, while functionally uncoupling embryonic programs from maternal and placental influences.

The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in Caenorhabditis elegans . Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs) - arx-2 (Arp2/3), unc-60 (cofilin), and lev-11 (tropomyosin) - led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Bulk and single-nucleus RNA-sequencing revealed that ABP knockdowns elicited a strongly “aged” transcriptome. Actin dysfunction broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely-tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common ACTB polymorphisms correlate with differences in age-related decline in gait speed, suggesting evolutionary conservation of actin’s role in healthy aging. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how actin integrity intersects with diverse aging pathways across tissues and scales. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics contribute to aging.

Microbiota-derived metabolites have emerged as key regulators of longevity. The metabolic activity of the gut microbiota, influenced by dietary components and ingested chemical compounds, profoundly impacts host fitness. While the benefits of dietary prebiotics are well-known, chemically targeting the gut microbiota to enhance host fitness remains largely unexplored. Here, we report a novel chemical approach to induce a pro-longevity bacterial metabolite in the host gut. We discovered that wild-type Escherichia coli strains overproduce colanic acids (CAs) when exposed to a low dose of cephaloridine, leading to an increased life span in the host organism Caenorhabditis elegans . In the mouse gut, oral administration of low-dose cephaloridine induced transcription of the capsular polysaccharide synthesis ( cps ) operon responsible for CA biosynthesis in commensal E. coli at 37 °C, and attenuated age-related metabolic changes. We also found that low-dose cephaloridine overcomes the temperature-dependent inhibition of CA biosynthesis and promotes its induction through a mechanism mediated by the membrane-bound histidine kinase ZraS, independently of cephaloridine’s known antibiotic properties. Our work lays a foundation for microbiota-based therapeutics through chemical modulation of bacterial metabolism and highlights the promising potential of leveraging bacteria-targeting drugs in promoting host longevity.