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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein—catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 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.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a \"double-layer particle\": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca 2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca 2+ -stabilized trimer; loss of Ca 2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca 2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions (\"triple-layer particles\": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca 2+ \"leak\". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a \"double-layer particle\": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca.sup.2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca.sup.2+ -stabilized trimer; loss of Ca.sup.2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca.sup.2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions (\"triple-layer particles\": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca.sup.2+ \"leak\". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Subcellular diffusion in living systems reflects cellular processes and interactions. Recent advances in optical microscopy allow the tracking of this nanoscale diffusion of individual objects with unprecedented precision. However, the agnostic and automated extraction of functional information from the diffusion of molecules and organelles within the subcellular environment is labor intensive and poses a significant challenge. Here we introduce DeepSPT, a deep learning framework integrated in an analysis software, to interpret the diffusional two- or three-dimensional temporal behavior of objects in a rapid and efficient manner, agnostically. Demonstrating its versatility, we have applied DeepSPT to automated mapping of the early events of viral infections, identifying endosomal organelles, clathrin-coated pits and vesicles among others with F1 scores of 81%, 82% and 95%, respectively, and within seconds instead of weeks. The fact that DeepSPT effectively extracts biological information from diffusion alone illustrates that besides structure, motion encodes function at the molecular and subcellular level. DeepSPT is a deep learning framework for the automated temporal analysis of behavior in 2D and 3D single-particle tracking. After extensive validation, DeepSPT was shown to work on diverse subcellular tracking, mapping and classification applications.

Quantitative, time-resolved 3D fluorescence microscopy can reveal complex cellular dynamics in living cells and tissues. Broader use remains limited by the difficulty of identifying, segmenting, and tracking objects of different size and shape in crowded intracellular environments in low-contrast, anisotropic, monochromatic image volumes. Objects overlap, deform, appear and disappear, and span wide ranges of size and intensity. Classical segmentation pipelines typically require high signal-to-noise data and rely on intensity heuristics with hand-tuned postprocessing that generalize poorly. Supervised deep learning methods require extensive voxel-level annotations that are costly, inconsistent across phenotypes, and rapidly become obsolete as imaging conditions change. We introduce SpatialDINO, a fully automated self-supervised method that trains a native 3D vision transformer, based on a modified version of DINOv2. SpatialDINO yields robust semantic feature maps from single channels of multi-channel microscopy that, irrespective of object shape, support object detection and segmentation directly from naïve 3D images across z-spacings and numbers of planes and different imaging modalities, without retraining or voxel annotations. We trained SpatialDINO on a small set of confocal volumes acquired by live-cell fluorescent 3D lattice light-sheet microscopy, spanning targets of different size and shape located in crowded cellular environments, from diffraction-limited clathrin coated pits and clathrin coated vesicles to bigger structures including endosomes and lysosomes, and endosomes and lysosomes pharmacologically enlarged to highlight endosomal membrane profiles. Post-processing of the features generated by SpatialDINO allows detection and unique object identification of these objects in naïve 3D images. It also enables detection of significantly different previously unseen object classes, such as cellular plasma membranes and nuclei and even tumors in MRI scans. Finally, we illustrate its value by tracking endosomes in 3D time series, combining SpatialDINO-derived feature similarity with spatial proximity to improve association through occlusion, abrupt appearance changes, and dense packing - all conditions that have been challenging for existing methods. SpatialDINO therefore lowers a major barrier to quantitative analysis of heterogeneous, monochromatic objects in crowded 3D cellular environments.

Enveloped viruses enter cells by membrane fusion. The viral membrane fuses with a host membrane, either at the cell surface or within endocytic compartments. For endocytic entry, fusion is typically triggered by low pH and often requires proteolytic priming by compartment-specific host proteases, which together define the site and mechanism of fusion and shape viral tropism. Inhibition of the lipid kinase PIKfyve, which generates PI(5)P and PI(3,5)P in late endosomes and lysosomes, swells those compartments and blocks infection by a subset of enveloped viruses, including Ebola virus, Marburg virus, coronaviruses (SARS-CoV-2), and VSV chimeras bearing Ebola, SARS-CoV-2, or Lassa glycoproteins, while showing minor effects on H1N1 influenza and no effect on VSV or VSV-rabies chimeras. In the work reported here, we have determined the basis for this selectivity. We show that swelling of late endosomes/lysosomes, independent of changes in lipid composition or altered virion trafficking, is sufficient to block virus-endosome fusion and genome release, even when endosomal acidity is preserved. Acute PIKfyve inhibition with apilimod or brief hypotonic treatment produced endosomal swelling and impaired infection by interrupting a late endosomal entry step. Imaging by live-cell 3D lattice light-sheet fluorescence microscopy tracked fluorescent virions accumulating and arresting in late endosomes prior to fusion, and single-cell, single-round assays confirmed loss of infectivity. These data support a simple biophysical mechanism: endo-lysosomal swelling, likely increasing endosomal membrane tension, creates an energy barrier to fusion and genome release. Inducing such swelling may offer a general strategy to inhibit viruses that depend on late endosomal entry.

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 -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 -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.

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 Alzheimer's 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 α-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 α-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)P .

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 α-synuclein (α-syn) in Parkinson's 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 α-syn, seeded by internalized pre-formed fibrils of α-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 α-syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated α-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 α-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 α-syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.

Sub-cellular diffusion in living systems reflects cellular processes and interactions. Recent advances in optical microscopy allow the tracking of this nanoscale diffusion of individual objects with an unprecedented level of precision. However, the agnostic and automated extraction of functional information from the diffusion of molecules and organelles within the sub-cellular environment, is labor-intensive and poses a significant challenge. Here we introduce DeepSPT, a deep learning framework to interpret the diffusional 2D or 3D temporal behavior of objects in a rapid and efficient manner, agnostically. Demonstrating its versatility, we have applied DeepSPT to automated mapping of the early events of viral infections, identifying distinct types of endosomal organelles, and clathrin-coated pits and vesicles with up to 95% accuracy and within seconds instead of weeks. The fact that DeepSPT effectively extracts biological information from diffusion alone indicates that besides structure, motion encodes function at the molecular and subcellular level.