title: Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.
abstract: Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Chaoxiang
authors.initials: C
authors.lastname: Chen
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Shuyue
authors.initials: S
authors.lastname: Zhang
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Shaobin
authors.initials: S
authors.lastname: Zhu
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Xu
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Jinyan
authors.initials: J
authors.lastname: Han
authors.affiliation: School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, PR China.
authors.forename: Jin-Zhang
authors.initials: JZ
authors.lastname: Zeng
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China. Electronic address: xmyan@xmu.edu.cn.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Yan
mesh_terms: D005434:Flow Cytometry / Q000295:instrumentation*; D018628:Gene Dosage / Q000235:genetics*; D020869:Gene Expression Profiling / Q000295:instrumentation; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008928:Mitochondria / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation
chemical_list: D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2
keywords: Apoptosis; Bcl-2 family protein; Flow cytometry; Immunofluorescence; Protein copy number; Single-mitochondria analysis