Dataset Information

Simultaneous proteome localization and turnover analysis, Rep. 3

ABSTRACT: To examine the protein spatial and temporal changes upon carfilzomib-mediated proteasome inhibition in cardiac cells, we produced human iPSC-derived cardiomyocytes using a standard small molecule based protocol. Cardiomyocyte identity was confirmed by morphology, observation of contraction, and the presence of GFP tagged MLC-2a in the reporter line. We then applied the SPLAT protocol to untreated and carfilzomib-exposed iPSC-cardiomyocytes.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dr. Edward Lau

PROVIDER: PXD046649 | JPOST Repository | Fri Nov 03 00:00:00 GMT 2023

REPOSITORIES: jPOST

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			Action	DRS
	20231012_JCSPLAT_CTRL_BR3_F1.raw	Raw
	20231012_JCSPLAT_CTRL_BR3_F13.raw	Raw
	20231012_JCSPLAT_CTRL_BR3_F14.raw	Raw
	20231012_JCSPLAT_CTRL_BR3_F15.raw	Raw
	20231012_JCSPLAT_CTRL_BR3_F16.raw	Raw

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Project description:The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.

Project description:Organs are the ensemble of different cell types in a complex architectural milieu. It is well-known that progression through fate decisions sets up the complex cellular makeup and architecture of an organ, but how that same architecture may impact on cell fate is less clear. We sought to examine this by taking advantage of the unique capabilities of organoids as a tractable in vitro model to interrogate how fate and form interact during organ development. Screening methodological variations encountered in the literature revealed that common protocol adjustments, such as small molecule patterning and exposure to exogenous extracellular matrix, impacted various aspects of morphology, from macro structure to tissue architecture. We demonstrated that overall morphology is a predictor of tissue architecture and that perturbing morphology results in changes in cytoarchitecture. Vice-versa, perturbing cytoarchitecture by mechanically redistributing cells in a random spatial conformation resulted in a simplified overall morphology. We next examined the impact of these morphological perturbations on cell fate through integrated snRNA-seq and spatial transcriptomics within the phenotypic landscape. Regardless of the specific protocol, organoids with more complex morphology better mimicked in vivo human fetal brain development than organoids with simplified morphology. Further, organoids with perturbed tissue architecture profiled with scRNA-seq over a time course experiment displayed aberrant temporal progression in cell fate, with cells being intermingled in both space and time. Finally, imparting a simplified morphology through physical encapsulation led to disrupted tissue cytoarchitecture and a similar abnormal temporal progression. These data not only point to the importance of tissue morphology in organoid fidelity compared to in vivo, but also demonstrate that cells require proper spatial coordinates in order to undergo the proper temporal trajectory of events.

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Simultaneous proteome localization and turnover analysis, Rep. 3

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