title: Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.
abstract: Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Burak
authors.initials: B
authors.lastname: Okumus
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Dirk
authors.initials: D
authors.lastname: Landgraf
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Ghee Chuan
authors.initials: GC
authors.lastname: Lai
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Somenath
authors.initials: S
authors.lastname: Bakshi
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.|Department of Physics, Universidad de los Andes, Bogota 4976-12340, Colombia.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Arias-Castro
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Sadik
authors.initials: S
authors.lastname: Yildiz
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Dann
authors.initials: D
authors.lastname: Huh
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Fernandez-Lopez
authors.affiliation: Department of Biology, Suffolk University, Boston, Massachusetts 02108, USA.
authors.forename: Celeste N
authors.initials: CN
authors.lastname: Peterson
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Erdal
authors.initials: E
authors.lastname: Toprak
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Meriem
authors.identifier: 0000-0003-2522-613X
authors.initials: M
authors.lastname: El Karoui
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Johan
authors.initials: J
authors.lastname: Paulsson
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology*; D003593:Cytoplasm / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004058:Diffusion; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D056656:Lab-On-A-Chip Devices; D011312:Pressure; D012808:Sigma Factor / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C098923:rssB protein, E coli; C069567:sigma factor KatF protein, Bacteria