Xiaoyu Shi

Intermediate filaments called lamins line the metazoan nuclear envelope and organize the nucleus and genome. Unlike actin and microtubules, purified intermediate filament proteins assemble into non-physiological structures, making it difficult to connect lamin functions to their assembly and regulation. To overcome this challenge and shed light on physiological lamin assembly mechanisms, we conducted biochemical studies of lamin-B3 endogenously present in Xenopus laevis egg extract, which recapitulates physiological context. When we mimicked nucleoplasm conditions that would support assembly of lamin filaments in intact cells, lamin-B3 assembled into higher-order structures resembling filamentous meshworks without accompanying nuclear assembly. This ectopic lamin assembly occurs on nuclear pore complex-containing membranes, but does not apparently recruit known nuclear lamina components, demonstrating that a lamin assembly process is partially separable from the rest of the nuclear lamina and nucleus. This assembly assay in the physiological context of cellular components opens the door to further dissecting nuclear lamina function in nuclear organization.

Landscape expansion microscopy (land-ExM) is a light microscopy technique that visualizes both the lipid and protein ultrastructural context of cells. Achieving this level of detail requires both superresolution and a high signal-to-noise ratio. Although expansion microscopy (ExM) provides superresolution, obtaining high signal-to-noise images of both proteins and lipids remains challenging. land-ExM overcomes this limitation by using self-retention trifunctional anchors to significantly enhance protein and lipid signals in expanded samples. This improvement enables the accurate visualization of diverse membrane organelles and phase separations, as well as the 3D visualization of their contact sites. As a demonstration, we revealed triple-organellar contact sites among the stress granule, the nuclear tunnel, and the nucleolus. Overall, land-ExM offers a high-contrast superresolution platform that advances our understanding of how cells spatially coordinate interactions between membrane organelles and phase separations.

The abundance and stoichiometry of membrane lipid species vary across a cell’s lifespan and metabolic state. In the retinal pigment epithelium (RPE), age-related alterations in lipid composition contribute to vision loss and diseases such as age-related macular degeneration (AMD), yet the molecular drivers of these changes remain unclear. Here, we show that age-dependent remodeling of the composition and biophysical properties of the plasma membrane compromises membrane integrity and function. Remarkably, rather than undergoing cell death, affected cells activate a lysosome-dependent plasma membrane repair program to preserve barrier integrity. While this adaptive response may protect RPE structure under metabolic stress, it also drives spatially polarized release of lysosomal contents that potentially can contribute to extracellular matrix remodeling and sub-RPE deposit formation during aging and AMD. Finally, we demonstrate that supplementation with the direct product of the aging-associated lipid elongase ELOVL2 alleviates these phenotypes, providing direct evidence for a critical role of ELOVL2-mediated PUFA elongation in healthy aging. Taken together, our results propose a model in which age-dependent decline in PUFA elongation disrupts the balance between membrane flexibility and stability, initiating a compensatory cycle of membrane stress and repair.

Landscape Expansion Microscopy (land-ExM) is a light microscopy technique that visualizes both lipid and protein ultrastructural context of cells. Achieving this level of detail requires both superresolution and a high signal-to-noise ratio. Although expansion microscopy (ExM) provides superresolution, obtaining high signal-to-noise images of both proteins and lipids remains challenging. Land-ExM overcomes this limitation by using self-retention trifunctional anchors to significantly enhance protein and lipid signals in expanded samples. This improvement enables the accurate visualization of diverse membrane organelles and phase separations, as well as the three-dimensional visualization of their contact sites. As a demonstration, we revealed triple-organellar contact sites among the stress granule, the nuclear tunnel, and the nucleolus. Overall, land-ExM offers a high-contrast superresolution platform that advances our understanding of how cells spatially coordinate interactions between membrane organelles and phase separations. eTOC Summary: Zhuang et al. introduce land-ExM, a super-resolution approach that simultaneously maps protein and lipid ultrastructure in cells with high contrast. This method visualizes 3D interactions between membrane-bound organelles and phase-separated condensates, uncovering organelle contact sites such as stress granules at nuclear tunnels adjacent to nucleoli.

Vertebrate radial glia progenitors (RGPs) balance self-renewal and differentiation through asymmetric cell division (ACD), which involves unequal centrosome inheritance. How centrosome asymmetry directs cell fate remains poorly understood. Here, we identify Pericentriolar material 1 (Pcm1) as a key player in this process. In zebrafish embryonic RGPs, Pcm1 is asymmetrically associated with Cep83, a mother centrosome marker. Using in vivo time-lapse imaging and nanoscale-resolution expansion microscopy, we detect Pcm1 on Notch ligand-containing endosomes, where it interacts–either directly or indirectly–with Par-3 and dynein. Loss of pcm1 disrupts endosome dynamics, increasing neuronal differentiation at the expense of RGP self-renewal. Mechanistically, Pcm1 facilitates the transition from Rab5b to Rab11a and promotes the assembly of Par-3 and dynein macromolecular complexes on recycling endosomes. Furthermore, we find conserved PARD3-PCM1-CEP83-RAB11 associations in human cortical brain organoids. Our findings uncover that Pcm1 links centrosome asymmetry to polarized endosome trafficking, thereby regulating RGP fate decisions.

Intermediate filaments called lamins line the metazoan nuclear envelope and organize the nucleus and genome. Unlike actin and microtubules, purified intermediate filament proteins assemble into non-physiological structures, making it difficult to connect lamin functions to their assembly and regulation. To overcome this challenge and shed light on physiological lamin assembly mechanisms, we conducted biochemical studies of lamin-B3 endogenously present in Xenopus laevis egg extracts, which recapitulate physiological context. When we mimicked nucleoplasm conditions, which would support assembly of lamin filaments in in-tact cells, lamin-B3 assembled into higher-order structures resembling filamentous meshworks without accompanying nuclear assembly. This ectopic lamin assembly occurs on nuclear pore complex-containing membranes, but does not apparently recruit known nuclear lamina components, demonstrating that a lamin assembly process is partially separable from the rest of the nuclear lamina and nucleus. This assembly assay in the physiological context of cellular components opens the door to further dissecting nuclear lamina function in nuclear organization. Summary: Mimicking nucleoplasm conditions in Xenopus laevis egg extracts triggers a lamin assembly reaction independent of nucleus assembly, giving new insights into potential lamin assembly mechanisms.

The zeta inhibitory peptide (ZIP) interferes with memory maintenance and long-term potentiation (LTP)¹ when administered to mice. However, mice lacking its putative target, protein kinase PKMζ, exhibit normal learning and memory as well as LTP^2,3, making the mechanism of ZIP unclear. Here we show that ZIP disrupts LTP by removing surface AMPA receptors through its cationic charge alone. This effect requires endophilin-A2-mediated endocytosis and is fully blocked by drugs suppressing macropinocytosis. ZIP and other cationic peptides remove newly inserted AMPA receptor nanoclusters at potentiated synapses, providing a mechanism by which these peptides erase memories without altering basal synaptic function. When delivered in vivo, cationic peptides can modulate memories on local and brain-wide scales, and these mechanisms can be leveraged to prevent memory loss in a model of traumatic brain injury. Our findings uncover a previously unknown synaptic mechanism by which memories are maintained or lost.

Proximity labeling expansion microscopy (PL-ExM) visualizes superresolution structures of interactome on widely accessible light microscopes, enabling the assessment of the precision and efficiency of proximity labeling techniques.

Severe invagination of the nuclear envelope is a hallmark of cancers, aging, neurodegeneration, and infections. However, the outcomes of nuclear invagination remain unclear. This work identified a new function of nuclear invagination: regulating ribosome biogenesis. With expansion microscopy, we observed frequent physical contact between nuclear invaginations and nucleoli. Surprisingly, the higher the invagination curvature, the more ribosomal RNA and pre-ribosomes are made in the contacted nucleolus. By growing cells on nanopillars that generate nuclear invaginations with desired curvatures, we can increase and decrease ribosome biogenesis. Based on this causation, we repressed the ribosome levels in breast cancer and progeria cells by growing cells on low-curvature nanopillars, indicating that overactivated ribosome biogenesis can be rescued by reshaping nuclei. Mechanistically, high-curvature nuclear invaginations reduce heterochromatin and enrich nuclear pore complexes, which promote ribosome biogenesis. We anticipate that our findings will serve as a foundation for further studies on nuclear deformation. Highlights Nuclear invaginations regulate ribosome biogenesis by physically contacting nucleoli. High-curvature nuclear tunnels increase ribosome biogenesis. Nanopillars reduce ribosome biogenesis by transforming high-curvature nuclear invaginations to low-curvature ones.

Expansion microscopy (ExM) is a super‐resolution imaging technique that bypasses the diffraction limit of conventional optical microscopy (∼250 nm) by enlarging samples with a swellable hydrogel. Combined with various light microscopes, ExM enables an effective resolution ranging from 5 to 70 nm. ExM has now been successfully applied to cell, tissue, and whole‐organism samples, providing biologists with a low‐cost strategy to visualize samples at the molecular level. However, fluorescence signal loss easily happens for beginners and with early versions of ExM protocols. Here, we describe a protocol called label‐retention expansion microscopy (LR‐ExM), which can preserve and enhance the signal of ExM imaging via a series of trifunctional probes. These trifunctional probes are antibody‐based and easy to prepare, and thus suit the needs of most laboratories. © 2024 Wiley Periodicals LLC. Basic Protocol : LR‐ExM using trifunctional probes for enhanced fluorescent signals