Biology subjects

Weber, C. M.

Publications and source records attributed to Weber, C. M..

3 recordsLinked to original sources

bioRxiv2022

Mechanical Stimuli such as Shear Stress and Piezo1 Stimulation Generate Red Blood Cell Extracellular Vesicles

Circulating red blood cell extracellular vesicles (RBC-EVs) are a promising biomarker for vascular health. However, generating, isolating, and characterizing physiologically relevant RBC-EVs with sufficient yield and purity for biological studies is non-trivial. Here, we present and rigorously characterize an in vitro model to mimic RBC-EV production during shear stress via mechanosensitive piezo1 ion channel stimulation. We optimize our RBC-EV isolation protocol to minimize hemolysis, maximize RBC-EV yield and purity, and improve the ease of EV characterization. RBC-EV purity was measured by quantifying protein (e.g., particles/g), large particle (e.g., protein aggregates), and platelet EV contamination. This study compared RBC-EV isolation performance using membrane-based affinity (e.g., exoEasy), ultrafiltration (e.g., Amicon Ultra-15), and ultracentrifugation, with and without size exclusion chromatography purification. We found that treating 6% hematocrit with 10 M piezo1-agonist yoda1 for 30 minutes and isolating RBC-EVs using ultracentrifugation minimized RBC hemolysis and maximized RBC-EV yield (~1012 particles/mL) and purity, provided the most consistent RBC-EV preparations, and improved ease of RBC-EV characterization. Our pressure myography experiments suggest that co-isolated protein contaminants, but not piezo1 RBC-EVs, induce rapid mouse carotid artery vasodilation. These results underscore the importance of characterizing EV purity for biological experiments. The standardized methods outlined here enable mechanistic studies of how RBC-EVs generated in physiological flow affect vascular response.

bioengineering↗

bioRxiv2021

mSWI/SNF interacts with the ribosome and its inhibition/mutations alter translation and sensitize to mTOR/PI3K inhibitors

The chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) subunits are mutated, deleted or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancers alterations will lead to path toward new targeted therapies. Canonically, mSWI/SNF complexes regulate the structure of chromatin, however they likely have additional functions which could be relevant in carcinogenesis. Here, we highlight the substantial alteration of mSWI/SNF subunits expression in both the nucleus and cytoplasm in breast cancer cases. We demonstrate mSWI/SNF cytoplasmic localization and interaction with the translation initiation machinery. Short-term inhibition and depletion of specific subunits alter protein synthesis, implicating a direct role for these factors in translation. Inhibition and depletion of specific subunits increase sensitivity to mTOR-PI3K inhibitors, suggesting a potential therapeutic opportunity for diseases harboring mutations in these complexes. Indeed, SMARCA4 pathogenic mutations decrease protein synthesis. Furthermore, taking advantage of the DepMap studies, we demonstrate cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are particularly sensitive to translation pathway inhibitors. In conclusion, we report an unexpected cytoplasmic role for mSWI/SNF in protein synthesis, suggesting potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in its subunits. Statement of significanceThis study establishes direct functions for mSWI/SNF in protein synthesis. mSWI/SNF inhibition, depletion and cancer mutations alter translation and increase sensitivity to translation pathway inhibitors, illustrating the potential for new therapeutic strategies.

cancer biology↗

bioRxiv2020

mSWI/SNF promotes distal repression by titrating polycomb dosage

The mammalian SWI/SNF, or BAF complex, has a conserved and direct role in antagonizing polycomb-mediated repression. Yet, BAF appears to also promote repression by polycomb in stem cells and cancer. How BAF both antagonizes and promotes polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-polycomb axis in embryonic stem cells on the timescale of hours. We report that rapid BAF depletion redistributes both PRC1 and PRC2 complexes from highly occupied domains, like Hox clusters, to weakly occupied sites that are normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features, and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 & PRC2 we identify both a conventional role for BAF in polycomb-mediated repression and a second mechanism acting by global redistribution of polycomb. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

molecular biology↗