Evaluation of Pulsed-FRAP and Conventional-FRAP for Determination of Protein Mobility in Prokaryotic Cells Jacek T. Mika, Victor Krasnikov, Geert van den Bogaart, Foppe de Haan, Bert Poolman* Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands Abstract Background: Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP). Throughout literature a wide range of diffusion coefficients for GFP in the cytoplasm of Escherichia coli (3 to 14 mm2/s) is reported using FRAP-based approaches. In this study, we have evaluated two of these methods: pulsed-FRAP and ‘‘conventional’’-FRAP. Principal Findings: To address the question whether the apparent discrepancy in the diffusion data stems from methodological differences or biological variation, we have implemented and compared the two techniques on bacteria grown and handled in the same way. The GFP diffusion coefficients obtained under normal osmotic conditions and upon osmotic upshift were very similar for the different techniques. Conclusions: Our analyses indicate that the wide range of values reported for the diffusion coefficient of GFP in live cells are due to experimental conditions and/or biological variation rather than methodological differences. Citation: Mika JT, Krasnikov V, van den Bogaart G, de Haan F, Poolman B (2011) Evaluation of Pulsed-FRAP and Conventional-FRAP for Determination of Protein Mobility in Prokaryotic Cells. PLoS ONE 6(9): e25664. doi:10.1371/journal.pone.0025664 Editor: Marek Cebecauer, J. Heyrovsky Institute of Physical Chemistry, Czech Republic Received August 24, 2011; Accepted September 7, 2011; Published September 28, 2011 Copyright: 2011 Mika et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge financial support from SysMo via the Biotechnology and Biological Sciences Research Council (BBSRC)-funded KosmoBac programme coordinated by Ian R. Booth (Aberdeen) and Nederlandse Wetenschappelijke Organisatie (NWO) (top-subsidy grant 700.56.302). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: b.poolman@rug.nl Introduction In 1999 Elowitz and co workers [1] published a pioneering study on the mobility of proteins inside live E. coli cells, using Fluorescence Recovery After Photobleaching (FRAP). Although FRAP has been a well established tool for studying macromolecule mobility inside living cells since the 1970’s [2], the attempts to use FRAP in bacteria have proven to be difficult mainly due to their small size. For example an E.coli cell is typically 163 mm, while human fibroblasts can easily reach dimensions over 100 mm. This property renders bacterial cells difficult to study as their dimension are only a few times larger than the diffraction limit of optical microscopy. Moreover, traditional FRAP protocols include a relatively large photo-bleaching spot and high laser intensity, both of which are not amenable for the small volume of bacteria. Elowitz et al. [1] have modified the FRAP protocol by using a smaller bleaching spot and weaker laser power, so that sufficient non-photobleached GFP is left to measure fluorescence recovery. This approach has been successful implemented by others [3,4,5,6]. Subsequently, other methods have been tailored to probe macromolecule diffusion in bacteria, including FRAP related techniques like pulsed–FRAP [7] and continuous photobleaching, using total internal reflection microscopy [8,9], single molecule tracking [10] and fluorescence correlation spectroscopy [11]. For an overview of those techniques, we refer to Mika and Poolman [12]. There are now several studies that report the mobility of GFP and related proteins like YFP and mEos2, yielding diffusion coefficients for these proteins in the E.coli cytoplasm that range from 3 mm2/s [7,13] through 6–7 mm2/s [1,6] up to 14 mm2/s [5,10]. The question arises whether this relatively wide range of values reflects differences in the methods used or biological and/or experimental variations? We have now compared ‘conventional FRAP’ as initially used by Elowitz [1] and pulsed-FRAP, developed in our laboratory [7], to determine the diffusion of GFP in E. coli under well-defined conditions. We demonstrate that both techniques yield very similar distributions of diffusion coefficients of GFP in the cytoplasm under normal and osmotic stress conditions. We conclude that the different D values reported for GFP(-like) proteins in the literature [1,5,6,7,10,13,14] are a result of different handling of the cells and true biological variations rather than differences in the FRAP methods used. Materials and Methods Strains, growth and preparation of bacterial cells for microscopy Escherichia coli K-12 strain MG1655 harboring pGFPCR [7] was grown as described previously [13]. Briefly, the cells were grown from single colonies in Luria Broth (10 g/L Bacto Tryptone (Becton Dickinson), 5 g/L Yeast extract (Becton Dickinson) plus 10 g/L NaCl (Merck)) supplemented with 100 mg/mL ampicillin (Sigma) at 37uC with vigorous shaking until the culture had reached an OD600 of 0.3–0.4. Leaky expression of GFP from the PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25664