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.

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 maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

Volume electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is a laborious process limiting the effective use of this potentially powerful tool. We resolved this bottleneck with automated segmentation of intracellular substructures in electron microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of a wide range in size and complexity. We obtained dedicated models for each structure based on a small number of sparsely annotated ground truth images from only one or two cells. Model generalization was improved with a rapid, computationally effective strategy to refine a trained model by including a few additional annotations. We identified mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin-coated pits, and vesicles imaged by focused ion beam scanning electron microscopy. We uncovered a wide range of membrane–nuclear pore diameters within a single cell and derived morphological metrics from clathrin-coated pits and vesicles, consistent with the classical constant-growth assembly model.

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.

Abbreviations: Arl 8: Arf-like GTPase 8, CLC-GFP: GFP-fusion of the clathrin-light chain; ConA: concanamycin A; DQ-BSA: dye-quenched bovine serum albumin; IKA: ikarugamycin; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LB: Luria-Bertani medium; Lp: Legionella pneumophila; mTORC1: mechanistic target of rapamycin complex 1; PDV: phagosome-derived vesicle; PtdIns(3)P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P 2 : phosphatidylinositol 3,5-biphosphate; PtdIns(4)P: phosphatidylinositol 4-phosphate; TFEB: transcription factor EB Summary Phagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes undergo fragmentation to reform lysosomes in a clathrin-dependent manner. This process is necessary to maintain the degradative capacity of phagocytes during subsequent phagocytosis. AbstractDuring phagocytosis, phagocytes like macrophages engulf and sequester unwanted particles like bacteria into phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, resulting in the degradation of the enclosed particle. Ultimately, phagosomes must be recycled to help recover membrane resources like lysosomes consumed during phagocytosis, 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 required cargo degradation, the actin and microtubule 3 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 showed that the 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.