Biology subjects

Bax, S.

Publications and source records attributed to Bax, S..

4 recordsLinked to original sources

bioRxiv2023

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

Defining the subcellular distribution of all human proteins and its remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immuno-capture coupled to mass spectrometry. We apply this proteomics workflow to a cell-wide collection of membranous and membrane-less compartments. A graph-based representation of our data reveals the subcellular localization of over 7,600 proteins, defines spatial protein networks, and uncovers interconnections between cellular compartments. We demonstrate that our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following hCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in their total abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides essential insights for the elucidation of cellular responses. Our dataset can be explored at organelles.czbiohub.org.

cell biology↗

bioRxiv2023

An open-source FACS automation system for high-throughput cell biology

Recent advances in gene editing are enabling the engineering of cells with an unprecedented level of scale. To capitalize on this opportunity, new methods are needed to accelerate the different steps required to manufacture and handle engineered cells. Here, we describe the development of an integrated software and hardware platform to automate Fluorescence-Activated Cell Sorting (FACS), a central step for the selection of cells displaying desired molecular attributes. Sorting large numbers of samples is laborious, and, to date, no automated system exists to sequentially manage FACS samples, likely owing to the need to tailor sorting conditions ("gating") to each individual sample. Our platform is built around a commercial instrument and integrates the handling and transfer of samples to and from the instrument, autonomous control of the instruments software, and the algorithmic generation of sorting gates, resulting in walkaway functionality. Automation eliminates operator errors, standardizes gating conditions by eliminating operator-to-operator variations, and reduces hands-on labor by 93%. Moreover, our strategy for automating the operation of a commercial instrument control software in the absence of an Application Program Interface (API) exemplifies a universal solution for other instruments that lack an API. Our software and hardware designs are fully open-source and include step-by-step build documentation to contribute to a growing open ecosystem of tools for high-throughput cell biology.

bioengineering↗

bioRxiv2021

mTOR controls neurotoxic lysosome exocytosis in inflammatory reactive astrocytes

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo - IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states, was upregulated in astrocytes in multiple sclerosis lesions, and preferentially co-localized with astrocytes in hypoxic-ischemic encephalopathy. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

cell biology↗

bioRxiv2021

PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation

The inter-cellular prion-like propagation of -synuclein aggregation is emerging as an important mechanism driving the progression of neurodegenerative diseases including Parkinsons disease and multiple system atrophy (MSA). To discover therapeutic strategies reducing the spread of -synuclein aggregation, we performed a genome-wide CRISPR interference screen in a human cell-based model. We discovered that inhibiting PIKfyve dramatically reduced -synuclein aggregation induced with both recombinant -synuclein fibrils and fibrils isolated from MSA patient brain. While PIKfyve inhibition did not affect fibril uptake or -synuclein clearance or secretion, it reduced -synuclein trafficking from the early endosome to the lysosome, thereby limiting fibril escape from the lysosome and reducing the amount of fibrils that reach cytosolic -synuclein to induce aggregation. These findings point to the endolysosomal transport of fibrils as a critical step in the propagation of -synuclein aggregation and a potential therapeutic target.

cell biology↗