Biology subjects

Somerville, E.

Publications and source records attributed to Somerville, E..

8 recordsLinked to original sources

bioRxiv2026

Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

cell biology↗

bioRxiv2025

Tau seeding in neurons enabled by transient endolysosomal perforations are confined within endolysosomes

Pathogenic tau assemblies propagate by templated seeding. For endocytosed fibrils to initiate aggregation of endogenous tau, a breach must occur in the limiting membrane of an endosome or lysosome. To study the route by which internalized tau seeds access cytosolic monomers and to identify the site of aggregate growth, we imaged live human iPSC-derived neurons (iNs) expressing tau P301L-eGFP after exposure to recombinant tau pre-formed fibrils (PFFs) or Alzheimers disease (AD) brain-derived oligomers or fibrils. We detected seeded tau P301L- eGFP aggregation within late endosomes/lysosomes of iNs but not in undifferentiated iPSCs. Colocalization with a Dextran pH biosensor showed that the aggregates remained within the lumen of an intact, low-pH compartment. Reporters of endolysosomal injury and repair (endolysosomal recruitment cytosolic galectin-3 and the ESCRT-III component IST1) did not change during seeding. Volume focused-ion-beam scanning electron microscopy showed fibrillar material exclusively inside membrane-bounded endolysosomes, with no membrane discontinuities in the fibril-containing compartments and with no evidence of cytosolic aggregates. Because tau and -synuclein can cross-seed, we adapted a HaloTag pulse-chase assay to test for the persistence of trans-membrane access. AD fiber-containing endolysosomes progressively recruited cytosolic -synuclein-Halo over days, with heterogeneous incorporation histories consistent with recurrent, self-limited access events rather than persistent rupture or terminal sealing. Pharmacologic inhibition of the endolysosomal lipid kinase PIKfyve with apilimod suppressed seeded tau aggregation and prevented neuronal toxicity. These data indicate that templated conversion proceeds within acidic, membrane-intact endolysosomes; tau seeding in neurons is enabled by transient, self-limited endolysosomal perforations yet remains confined to the endolysosomal lumen, and it requires PIKfyve- dependent PI(3,5)P2.

neuroscience↗

bioRxiv2025

Close-Up of vesicular ER Exit Sites by Volume Electron Imaging using FIB-SEM

Volume electron microscopy by high-pressure freeze substitution combined with block-face focused ion beam scanning electron microscopy (FIB-SEM) provides comprehensive 3D views of subcellular architecture, essential for understanding cellular activity in context. Aided by Automated Segmentation in Electron Microscopy (ASEM)--a 3D-Unet convolutional neural network trained with sparse annotations--we characterized the spatial organization of endoplasmic reticulum exit sites (ERES), the initial locations for membrane remodeling in protein secretion, in cells not overexpressing secretory cargo. The ASEM model, trained on 50-70 nm in diameter COPI vesicles, successfully identified vesicles adjacent to the Golgi apparatus of HeLa, SVG-A, and iPSC-derived neurons. It also revealed abundant vesicles of similar appearance, often within a group of [~]5-40 vesicles clustered within a 250 nm3 region adjacent to flattened endoplasmic reticulum (ER) domains, forming what we propose are COPII-mediated vesicular ER-exit sites. Elsewhere, similar assemblies appeared alongside tubular ER networks emerging from similarly enlarged ER domains previously reported as the only ERES in HeLa cells. These findings underscore the power of large-scale volume electron microscopy to resolve contradictions regarding membrane organization in vesicular trafficking. We encourage the scientific community to use our publicly accessible repository, containing open-source code, trained models, annotations, predictions, and FIB- SEM datasets, to facilitate continued advances in automated segmentation methods. SummaryNair et. al. show abundance of vesicular ERES in mammalian cells imaged by volume focused ion beam electron microscopy.

cell biology↗

bioRxiv2024

Depletion of endomembrane reservoirs drives phagocytic appetite exhaustion in macrophages

During phagocytosis, a phagocytic cup grows via F-actin remodelling and localized secretion to entrap a particle within a phagosome, which then fuses with endosomes and lysosomes to digest the particle, followed by phagosome resolution. As spatially limited systems, phagocytes have a maximal phagocytic capacity, at which point further uptake must be blunted. However, the processes responsible for phagocytic appetite exhaustion as phagocytes reach their maximal phagocytic capacity are poorly defined. We found that macrophages at their capacity have lower surface levels of Fc{gamma} receptors but overexpression of these receptors did not increase their capacity, suggesting that receptor levels are not limiting. Instead, surface membrane in-folding, membrane tension, and cortical F-actin were all reduced in exhausted macrophages. While this might contribute to appetite suppression, we also found that "free" endosomes and lysosomes were severely depleted in exhausted macrophages. Consequently, focal exocytosis at sites of externally bound particles was blunted. In comparison, macrophages recovered their appetite if phagosome resolution was permitted. We propose that depletion of the endomembrane pools is a major determinant of phagocytic fatigue as macrophages reach their phagocytic capacity. Summary statementMacrophages that reach their maximal phagocytic capacity lose their appetite for further uptake. This appetite exhaustion is driven partly by depletion of endosomes and lysosomes, preventing growth of additional phagocytic cups.

cell biology↗

bioRxiv2024

Constitutive Endolysosomal Perforation in Neurons allows Induction of alpha-Synuclein Aggregation by Internalized Pre-Formed Fibrils

The endocytic pathway is both an essential route of molecular uptake in cells and a potential entry point for pathology-inducing cargo. The cell-to-cell spread of cytotoxic aggregates, such as those of -synuclein (-syn) in Parkinsons Disease (PD), exemplifies this duality. Here we used a human iPSC-derived induced neuronal model (iNs) prone to death mediated by aggregation in late endosomes and lysosomes of endogenous -syn, seeded by internalized pre-formed fibrils of -syn (PFFs). This PFF-mediated death was not observed with parental iPSCs or other non-neuronal cells. Using live-cell optical microscopy to visualize the read out of biosensors reporting endo-lysosome wounding, we discovered that up to about 10% of late endosomes and lysosomes in iNs exhibited spontaneous constitutive perforations, regardless of the presence of internalized PFFs. This wounding, absent in parental iPSCs and non-neuronal cells, corresponded to partial damage by nanopores in the limiting membranes of a subset of endolysosomes directly observed by volumetric focused ion beam scanning electron microscopy (FIB-SEM) in iNs and in CA1 pyramidal neurons from mouse brain, and not found in iPSCs or in other non-neuronal cells in culture or in mouse liver and skin. We suggest that the compromised limiting membranes in iNs and neurons in general are the primary conduit for cytosolic -syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated -syn aggregation. Significantly, eradicating the intrinsic endolysosomal perforations in iNs by inhibiting the endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase (PIKfyve kinase) using Apilimod or Vacuolin-1 markedly reduced PFF-induced -syn aggregation, despite PFFs continuing to enter the endolysosomal compartment. Crucially, this intervention also diminished iN death associated with PFF incubation. Our results reveal the surprising presence of intrinsically perforated endo-lysosomes in neurons, underscoring their crucial early involvement in the genesis of toxic -syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.

cell biology↗

bioRxiv2022

Deep neural network automated segmentation of cellular structures in volume electron microscopy

Recent advances in automated segmentation using deep neural network models allow identification of intracellular structures. This study describes a new pipeline to train a convolutional neural network for rapid and efficient detection of structures of wide range in size and complexity. AbstractThree-dimensional electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is laborious and time-consuming, however, and impairs effective use of a potentially powerful tool. Resolving this bottleneck is therefore a critical next step in frontier biomedical imaging. We describe Automated Segmentation of intracellular substructures in Electron Microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of wide range in size and complexity. We obtain for each structure a dedicated model based on a small number of sparsely annotated ground truth annotations from only one or two cells. To improve model generalization to different imaging conditions, we developed a rapid, computationally effective strategy to refine an already trained model by including a few additional annotations. We show the successful automated identification of mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin coated pits and coated vesicles in cells imaged by focused ion beam scanning electron microscopy with quasi-isotropic resolution. ASEM enabled us to uncover a wide range of membrane-nuclear pore diameters within a single cell and to derive morphological metrics from clathrin coated pits and vesicles at all stages of maturation consistent with the classical constant-growth assembly model.

cell biology↗

bioRxiv2022

Tracking infectious entry routes of SARS-CoV-2

SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity. Significance StatementInfection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement. One sentence summaryDetailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells

microbiology↗

bioRxiv2020

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as phagosome resolution. Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintain the degradative power of macrophages to handle multiple waves of phagocytosis. SummaryPhagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes fragment to reform lysosomes in a clathrin-dependent manner. This process helps maintain the degradative capacity of phagocytes for subsequent rounds of phagocytosis.

cell biology↗