SUMMARY Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways exist that limit the prevalence of such cells? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome mis-segregation leads to further genomic instability that ultimately causes cell cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance.

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

SummaryUnderstanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studies

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Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer–promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

Resolving the distribution of specific proteins at the nanoscale in the structural context of the cell is a major challenge in fluorescence microscopy. Here we present a new concept that decrowds the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins. This combination makes labeling of the proteome efficient enough that local protein densities are revealed and the cellular nanoarchitecture can be visualized by standard light microscopy.

Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.Alterations in the gravity vector and exposure to ionizing radiation can disrupt skeletal homeostasis in mice 1-3 . There are multiple stressors associated with spaceflight, including microgravity and radiation which are known to cause bone loss 4-6 . Decrements in bone mineral density (BMD) have been observed in astronauts from the Mir missions as well as missions to the International Space Station (ISS) 7-9 . While much research has focused on the detrimental effects of microgravity on skeletal tissue, less is known about the impact of spaceflight radiation. Crewed missions have, to this point, primarily remained within low-Earth orbit (LEO). While sources of ionizing space radiation within LEO include galactic cosmic radiation and charged particles from unpredictable solar particle events (SPE) 10,11 , the presence of the Earth's magnetosphere reduces exposure to ionizing space radiation. Missions beyond LEO pose the greatest risk of radiation exposure and is of significant concern for crew health 12-14 . Spaceflight-relevant radiation includes a mix of low-linear energy transfer (LET) species such as protons and helium ions as well as high-LET species such as iron 15,16 . Beyond LEO, for example, astronauts may be exposed to up to 0.7 Sv of ionizing radiation 12,15,17 during a multi-year mission to the Moon or Mars 14,15,18 .On Earth, bone homeostasis is effectively maintained by the controlled remodeling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. However, exposure to low-LET radiation ( 137 Cs or X-ray, 1-2 Gy) leads to a transient increase in the number of osteoclasts, accompanied by an increase in trabecular separation (Tb.Sp) and decrease in trabecular thickness (Tb.Th), overall leading to a reduction in bone volume fraction (BV/TV) [19][20][21][22] . Together, this early increase in bone resorption and decrease in bone formation due to radiation exposure can result in a state of osteopenia, potentially leading to an increased risk of bone fracture 16,23,24 . A possible mechanism of action responsible for these changes in bone homeostasis is the generation o...