Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
Nat Commun. 2021 Aug 17;12(1):4969. doi: 10.1038/s41467-021-25212-3.
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.
多聚体细胞骨架蛋白复合物协调正常细胞功能。然而，在应激、异质细胞群体中，蛋白质复合物的分布情况尚不清楚。细胞染色和基于邻近性的方法对内源性多聚体蛋白复合物从单个细胞中的定量具有有限的选择性和/或灵敏度。我们引入了微阵列差异去污剂分步分离，以同时检测数百个单个细胞中的蛋白质复合物。通过 60 秒的大小排阻电泳进行分步分离，使用稳定蛋白质复合物的缓冲液，最大限度地减少解聚。用大约 5 小时的免疫测定法测量蛋白质。用丝氨酸（F-actin）解聚Latrunculin A 处理的 U2OS 细胞中的细胞骨架蛋白复合物的共检测检测到一个独特的亚群（约 2%）表现出下调的 F-actin，但上调的微管。因此，一些细胞可能会上调其他细胞骨架复合物来抵消 Latrunculin A 处理的应激。我们还试图了解非化学应激对 F-actin 细胞异质性的影响。我们发现热休克可能会使丝状和球状肌动蛋白的相关性失调。在这项工作中，我们的测定方法克服了选择性限制，可用于生物化学定量分析受多种刺激影响的单细胞蛋白质复合物。
