Biology subjects

Somerville, E.

Publications and source records attributed to Somerville, E..

3 recordsLinked to original sources

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↗