Gunar Fabig

When eukaryotic cells divide, a mitotic spindle, composed of microtubule filaments interacting with molecular motors, self-assembles and is responsible for aligning and segregating chromosomes to the opposite daughter cells. Recent structural studies revealed that spindles are composed of short microtubules that do not span the full pole-to-chromosome distance. It remains unclear how these short, disconnected microtubules collectively assemble a bipolar spindle. Using cryo-electron tomography to map microtubule polarity in human spindles, we find that microtubules organize with consistent 8 nm wall-to-wall spacing. Spacing scales inversely with local microtubule density, consistent with organization by density-driven steric interactions, analogous to liquid crystal ordering. We find that the Eg5 motor establishes local antiparallel overlap independently of spindle bipolarity, while a balance of Eg5 and dynein regulates microtubule density to maintain spindle architecture. Together, these findings reveal the mesoscale principles by which cells build complex, self-healing cellular architectures from nanoscale building blocks.

How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology. Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2- to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation with large-scale electron tomography, we find that mid-spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size. Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces spindle pole-to-pole dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo, spindle-length scaling across nematode species spanning ~100 million years of divergence, and spindle rotation in human cells. Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species.

Microtubule asters are central to positioning organelles and organizing intracellular architectures. We show that the multi-stage choreography of microtubule asters which positions pronuclei during the first cell division in Caenorhabditis elegans , is governed by a cellular analog of geometric optics. Large-scale electron tomography reveals that astral microtubules reach cortical and pronuclear surfaces largely along line-of-sight trajectories, leaving complementary regions shadowed and inaccessible. Laser ablation identifies surface-anchored motors pulling on microtubules as the dominant drivers of motion. We develop a biophysical model incorporating dynamic terminator curves that separate microtubule-accessible and -inaccessible surfaces, and quantitatively recapitulate aster separation, pronuclear migration, centering, and rotation in control and genetically perturbed embryos. More broadly, robust positioning emerges from a feedback loop wherein intracellular geometry gates microtubule access, thus sculpting the distribution and magnitude of pulling forces, which in turn reshape that geometry.

Lipids and proteins compartmentalize biological membranes into nanoscale domains, which are crucial for signalling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labelling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.

Mitochondrial dysfunction contributes to impaired myocardial energetics and performance in heart failure with preserved ejection fraction (HFpEF). Elamipretide (Ela) enhances mitochondrial bioenergetics in preclinical models, yet its relevance in HFpEF remains unclear. This study examined the effects of Ela on cardiac mitochondrial function, structure, and cardiovascular performance in a rodent HFpEF model. Female obese ZSF1 rats received vehicle or Ela for 12 weeks, with age-matched lean rats as controls. Cardiac function and hemodynamics were assessed by echocardiography and pressure–volume analysis. Mitochondrial respiration was measured in permeabilized fibers and ultrastructure evaluated by transmission electron microscopy. Molecular and histological analyses included cardiolipin lipidomics and mRNA/protein profiling of hypertrophic, fibrotic, and inflammatory markers. Ela modestly improved complex I and II respiration, whereas mitochondrial ultrastructure, cardiolipin composition, and tafazzin expression were unchanged. Diastolic dysfunction persisted, reflected by unchanged E/é, ventricular stiffness factor β, and titin phosphorylation. Compared to untreated HFpEF, systolic performance showed a mild decline, with small reductions in LV ejection fraction and end-systolic elastance. Accordingly, cardiac remodeling, including hypertrophy, fibrosis, and inflammatory activation, remained unaltered. Vascular stiffness slightly increased, while carotid reactivity and morphology were preserved. In conclusion, despite enhanced mitochondrial respiration following Ela treatment, no functional or structural benefits were observed in experimental HFpEF, suggesting limited therapeutic efficacy once HFpEF is established.

The cell biology literature of the 19th and early-to-mid 20th centuries is full of the seminal works that were critical for the development of our current understanding of biology but were done using model systems that are no longer commonly used. We argue that these historical papers are ideal for the development of course-based research experiences, both because the background work is conceptually and technically straightforward, and because the study of the abandoned systems stopped prior to the availability of modern techniques. These abandoned systems offer outstanding opportunities for teaching modern light and electron microscopy techniques, thus providing students with opportunities to create new knowledge in cell biology. In addition, the authors of the early work are interesting subjects for storytelling, which can increase student engagement with a research topic and identification as a scientist. Here we report on the design of a course-based research class built on several foundational works including the early works of Hermann Henking, Michael J.D. White, Edmund Beecher Wilson, Sally Hughes-Schrader, and others that provide students with the same technical training they previously received in the cookbook lab. Students are trained to image both living and fixed spermatocytes, to prepare, section, and image specimens for electron microscopy, and to contribute to the publication of the research that is combined with the training. In the multiple iterations of the class, students also explored chromosome alignment during cell division and other cytological features such as mitochondrial structure and fusion during spermatogenesis. Students note that they value learning the techniques and scientific principles, in addition to exploring the unknown and creating new scientific knowledge.

How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology ^1–11 . Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2-to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation ^12,13 with large-scale electron tomography ¹⁴ , we find that central spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size ^{12,13,15–17} . Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces elongation dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo ¹⁸ , spindle-length scaling across nematode species spanning ~100 million years of divergence ¹⁷ , and spindle rotation in human cells ¹⁹ . Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species.

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as an additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime separation, in live‐cell imaging using open‐source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we separated the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six‐dimensional datasets to investigate cell division in the developing early embryo. Second, we separated fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open‐source plugin napari‐flim‐phasor‐plotter to implement conversion, visualisation, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime unmixing, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we unmixed the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we unmixed fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-FLIM-phasor-plotter to implement conversion, visualization, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

Ribosomes, discovered in 1955 by George Palade, were initially described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, extensive research has focused on both the structure and function of ribosomes. However, a fundamental question – how many ribosomes are present within whole cells – has remained largely unaddressed. In this study, we developed a microscopic method to quantify the total number of ribosomes in hTERT‐RPE‐1 cells and in nematode cells from various tissues of Caenorhabditis elegans hermaphrodites. Using electron tomography of high‐pressure frozen, freeze‐substituted and resin‐embedded samples, we determined that the ribosome number in hTERT‐RPE‐1 cells is in the same order of magnitude as biochemical measurements obtained via RNA capillary electrophoresis. As expected, control worms exhibited a higher number of ribosomes compared to RNA polymerase I A subunit (RPOA‐1)‐depleted worms in two out of three analysed tissue types. Our imaging‐based approach complements established biochemical methods by enabling direct quantification of ribosome numbers in specific samples. This method offers a powerful tool for advancing our understanding of ribosome localisation and distribution in cells and tissues across diverse model systems.