Measuring expression heterogeneity of single-cell cytoskeletal protein complexes
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Aug 17, 2021
- Source ID
- 10.1038/s41467-021-25212-3
Entities
People
- Amy E Herr
- Andrew Dillin
- C Kimberly Tsui
- Haiyan Huang
- Julea Vlassakis
- Louise L. Hansen
- Ryo Higuchi-sanabria
- Yun Zhou
Organizations
- Howard Hughes Medical Institute
- National Institutes of Health
- National Science Foundation
- United States Department of Defense
- United States Department of Health and Human Services