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bioRxiv · 10.1101/2020.09.12.294801

Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

Vlassakis, J.·Hansen, L. L.·Higuchi-Sanabria, R.·Zhou, Y.·Tsui, C. K.·Dillin, A.·Huang, H.·Herr, A. E.
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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 100s of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock dysregulates F- and G-actin correlation. The assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

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Vlassakis, J., Hansen, L. L., Higuchi-Sanabria, R., Zhou, Y., Tsui, C. K., Dillin, A., Huang, H., Herr, A. E.. 2020-09-13. Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation. https://doi.org/10.1101/2020.09.12.294801

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