van Engelenburg, S. B. | Search bioRxiv

bioRxiv2020

Isotropic 3D electron microscopy reference library of whole cells and tissues

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) demonstrated the ability to image cellular samples at 4-nm isotropic voxels with rather limited imageable volume. Here, we present 3D EM images of whole cells and tissues with two orders of magnitude increases in imageable volume at 4-nm voxels. Such data with a combined fine resolution scale and large sample size do not currently exist, and are enabled by the advances in higher precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning. More importantly, we have generated a volume EM atlas encompassing ten diverse datasets of whole cells and tissues, from cancer cells to immune cells, and from mouse pancreatic islets to Drosophila neural tissues. These open-access data (via OpenOrganelle) represent a foundation to nucleate a new field of high-resolution whole-cell volume EM and subsequent analyses, and invite biologists to explore this new paradigm and pose fundamentally new questions.

cell biology↗

bioRxiv2020

Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail

HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study we investigated the viral and cellular factors that contribute to the permissivity of MT-4 to gp41 CT truncation. We found that the kinetics of HIV-1 production are faster in MT-4 than in the other T-cell lines tested, but MT-4 express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 express higher levels of plasma-membrane-associated Env than non-permissive cells and Env internalization from the plasma membrane is slower compared to another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4, two-fold less Env is incorporated into virus particles in MT-4 compared to SupT1. Cell-to-cell transmission between co-cultured 293T and MT-4 is higher than in co-cultures of 293T with most other T-cell lines tested, indicating that MT-4 are highly susceptible to this mode of infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.Importance The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 transmission. This study reveals that multiple factors contribute to the unique ability of a gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a requirement for the gp41 CT in MT-4 is associated with the combined effects of rapid HIV-1 protein production, high levels of cell-surface Env expression, and increased susceptibility to cell-to-cell transmission compared to non-permissive cells.List of abbreviationsCTcytoplasmic tailVSvirological synapseC-Ccell-to-cellEnvenvelope glycoproteinSTRshort tandem repeatMFImedian fluorescence intensityPMplasma membraneRadio-IPradio-immunoprecipitationhPBMChuman peripheral blood mononuclear cellsRTreverse transcriptaseVSV-GVesicular stomatitis virus glycoproteinPBSphosphate buffered salineFBSfetal bovine serumWTwild typeHRPhorseradish peroxidaseERendoplasmic reticulumMAHIV-1 Matrix proteinEBVEpstein-Barr virusHTLVHuman T-cell leukemia virusALLAcute lymphocytic leukemiaATLAdult T-cell lymphomaATCCAmerican Type Culture CollectionNIH ARPNIH AIDS Reagent Programn.s.not statistically significantINHIV-1 Integrase proteinCAHIV-1 Capsid proteinRINRNA Integrity NumberView Full Text

microbiology↗