CUED Publications database

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.

Okumus, B and Landgraf, D and Lai, GC and Bakshi, S and Arias-Castro, JC and Yildiz, S and Huh, D and Fernandez-Lopez, R and Peterson, CN and Toprak, E and El Karoui, M and Paulsson, J (2016) Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells. Nat Commun, 7. 11641-.

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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.

Item Type: Article
Uncontrolled Keywords: Bacterial Proteins Cytoplasm DNA-Binding Proteins Diffusion Escherichia coli Escherichia coli Proteins Gene Expression Regulation, Bacterial Lab-On-A-Chip Devices Pressure Sigma Factor Transcription Factors
Subjects: UNSPECIFIED
Divisions: Div F > Control
Depositing User: Cron Job
Date Deposited: 05 Aug 2019 20:01
Last Modified: 26 Nov 2019 02:52
DOI: 10.1038/ncomms11641