Abstract
Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.
Highlights
Multimeric cytoskeletal protein complexes orchestrate normal cellular function
Do two well-studied actin-targeting drugs (Latrunculin A and Jasplakinolide) induce variation in filamentous actin (F-actin) complex-levels in single cells compared to controls? Second, as a corollary, does Latrunculin A yield cellular phenotypes distinct from controls with different organizations of other cytoskeletal protein complexes, such as microtubules and intermediate filaments? Third, what is the distribution of the F-actin ratio across a population of single cells? Fourth, how does heat shock, another cellular stress, shift the F-actin ratio distribution and coordination between F- and G-actin at the single-cell level? We show SIFTER is a versatile method for understanding cellular heterogeneity—at single-cell resolution—in protein-complex levels in response to perturbation
For the first design consideration, we focus on the F-actin filament, which is the smallest and most dynamic of the three cytoskeletal protein complexes
Summary
Multimeric cytoskeletal protein complexes orchestrate normal cellular function. protein-complex distributions in stressed, heterogeneous cell populations remain unknown. While highly multiplexed, top-down mass spectrometry currently lacks singlecell resolution for protein complexes Targeted approaches such as proximity ligation assay and FRET achieve single-cell sensitivity and can assess cellular heterogeneity with flow cytometry readout (10,000 or more cells[14]). Visualization of the actin cytoskeleton relies on fluorescently tagged actin (e.g., GFP-actin fusion or split GFPactin fusion17), GFP-fused actin-binding proteins or peptides (e.g., Lifeact, F-tractin, and Utrophin), nanobodies[18], or chemicals that directly bind actin (e.g., phalloidin) Such molecules may alter cytoskeletal dynamics both in vitro and in vivo[19,20,21]. Phalloidin competes with or is dissociated by, endogenous actinbinding proteins[22,23] and actin-targeting drugs, such as Jasplakinolide[24] Bulk ultracentrifugation overcomes these limitations while sacrificing single-cell resolution. Mild lysis in F-actin stabilization buffer solubilizes
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