Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Area of Science:

• Cellular Biology
• Biochemistry
• Proteomics

Background:

• Multimeric cytoskeletal protein complexes are vital for cellular functions.
• Understanding protein-complex distributions in stressed, heterogeneous cell populations is challenging.
• Existing methods lack selectivity and sensitivity for endogenous multimeric protein complex quantification in single cells.

Purpose of the Study:

• To develop a novel assay for simultaneous detection of protein complexes in hundreds of individual cells.
• To overcome selectivity limitations in biochemically quantifying single-cell protein complexes.
• To investigate the effects of chemical and non-chemical stress on cellular heterogeneity of protein complexes.

Main Methods:

• Micro-arrayed, differential detergent fractionation coupled with size-exclusion electrophoresis.
• Protein complex stabilization using a specialized buffer during fractionation.
• High-throughput immunoassay for protein measurement within approximately 5 hours.

Main Results:

• Identified a unique subpopulation (~2%) of U2OS cells with downregulated filamentous actin (F-actin) but upregulated microtubules upon Latrunculin A treatment.
• Demonstrated that heat shock can dysregulate the correlation between filamentous and globular actin.
• Successfully quantified single-cell protein complexes under diverse stimuli, overcoming previous limitations.

Conclusions:

• The developed assay enables robust biochemical quantification of single-cell protein complexes.
• Cellular stress can induce unique compensatory responses in specific cell subpopulations.
• This method provides new insights into cellular heterogeneity and stress responses at the protein complex level.