Kurz, T. | bioRxiv Science

bioRxiv2020

In vivo identification of apoptotic and extracellular vesicle-bound live cells using image-based deep learning

The in vivo detection of dead cells remains a major challenge due to technical hurdles. Here we present a novel method, where injection of fluorescent Milk fat globule-EGF factor 8 protein (MFG-E8) in vivo combined with imaging flow cytometry and deep learning allows the identification of dead cells based on their surface exposure of phosphatidylserine (PS) and other image parameters. A convolutional autoencoder (CAE) was trained on defined pictures and successfully used to identify apoptotic cells in vivo. However, unexpectedly, these analyses also revealed that the great majority of PS+ cells were not apoptotic, but rather live cells associated with PS+ extracellular vesicles (EVs). During acute viral infection apoptotic cells increased slightly, while up to 30% of lymphocytes were decorated with PS+ EVs of Dendritic cell exosomal origin. The combination of recombinant fluorescent MFG-E8 and the CAE-method will greatly facilitate analyses of cell death and EVs in vivo.

cell biology↗

bioRxiv2020

Global proteomics of Ubqln2-based murine models of ALS

Familial forms of neurodegenerative diseases commonly involve mutation of aggregation-prone proteins or components of the protein degradation machinery that act on aberrant proteins. Ubqln2 encodes a member of the UBL/UBA family of proteasome shuttle factors that is thought to facilitate proteasomal degradation of substrates, and mutation of this gene results in a familial form of ALS/FTD in humans. How Ubqln2 dysfunction leads to neurodegeneration, however, remains uncertain. We undertook a comprehensive study to identify proteomic changes upon Ubqln2 perturbation in multiple murine models of Ubqln2-mediated neurodegenerative disease. By performing quantitative multiplexed proteomics on neural tissues of affected animals, we identified a small group of proteins whose abundance is tightly linked to UBQLN2 function: the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Further studies using cultured cells of human origin, including induced neurons, found similar changes in protein abundance upon Ubqln2 loss, and pulse-chase studies suggested that PEG10 and TRIM32 are direct clients of UBQLN2. In conclusion, our study provides a deep understanding of the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.

cell biology↗