Segregation of molecules at cell division reveals native protein localization

Landgraf, Dirk; Okumuş, Burak; Chien, Peter; Baker, Tania A.; Paulsson, Johan

doi:10.1038/nmeth.1955

Cited by 300 publications

(304 citation statements)

References 19 publications

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“…The results on Dendra2, Dronpa, and mEos2 are consistent with a previous report (26). ‡ The number of HU-PAFP localizations per E. coli cell.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinssupporting

confidence: 91%

“…Here, instead of measuring the dimerization affinity of PAFPs in vitro, we directly probed whether the PAFPs could cause aggregation of a target protein using a previously reported method (26). In this approach, the fluorescent proteins are fused to the Escherichia coli protease ClpP, which itself oligomerizes to form a tetradecameric complex.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinsmentioning

confidence: 99%

“…In this approach, the fluorescent proteins are fused to the Escherichia coli protease ClpP, which itself oligomerizes to form a tetradecameric complex. It has been suggested that ClpP proteins tend to aggregate and form a single visible punctum in E. coli when fused to a fluorescent protein with a substantial dimerization tendency, whereas fusion to a fluorescent protein with a weak or no dimerization tendency tends to display a diffuse localization pattern (26). We thus fused E. coli codon-optimized PAFP sequences to the chromosomal copy of clpP, and report whether cells exhibit the single-punctum phenotype (Table 1 and Fig.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinsmentioning

confidence: 99%

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Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

332

382

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Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

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“…The results on Dendra2, Dronpa, and mEos2 are consistent with a previous report (26). ‡ The number of HU-PAFP localizations per E. coli cell.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinssupporting

confidence: 91%

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinsmentioning

confidence: 99%

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

332

382

View full text Add to dashboard Cite

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“…Because mCherry has recently been shown to stimulate aggregation when fused to proteins, we sought to find a better probe for live-cell studies by replacing the mCherry in the sandwich fusion with nine different fluorescent proteins, six of which have been shown to cause the least amount of aggregation (24). The majority of the fluorescent proteins tested were not able to restore rod shape in place of the sandwich fusion (Fig.…”

Section: Mreb Msfgfp Is Minimally Perturbativementioning

confidence: 99%

MreB Orientation Correlates with Cell Diameter in Escherichia coli

Ouzounov

Nguyen

Bratton

et al. 2016

Biophysical Journal

127

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Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a given population. The bacterial actin homolog, MreB, is necessary for establishment and maintenance of rod shape although the detailed properties of MreB that are important for shape control remained unknown. In this study, we perturb MreB in two ways: by treating cells with the polymerization-inhibiting drug A22 and by creating point mutants in mreB. These perturbations modify the steady-state diameter of cells over a wide range, from 790 5 30 nm to 1700 5 20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently tagged MreB polymers with cell diameter by simultaneously analyzing three-dimensional images of MreB and cell shape. Our results indicate that the helical pitch angle of MreB inversely correlates with the cell diameter of Escherichia coli. Other correlations between MreB and cell diameter are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB organizes the cell wall growth machinery to produce a chiral cell wall structure and dictate cell diameter.

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“…Fluorescent proteins have proved tremendously useful but are limited to quantifying only a few proteins per cell and sometimes introduce artifacts 4 . Multiple methods for quantifying proteins in single cells have been recently developed, including single-cell Western blots 5 , CyTOF 6 , and Proseek Multiplex, an immunoassay readout by RT-PCR 7 .…”

mentioning

confidence: 99%

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Budnik

Levy

Slavov

2017

Preprint

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Cellular heterogeneity is important to biological processes, including cancer 1,2 and development 3 . However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem Quantification of TMT-labeled peptides relies on reporter ions (RI) whose levels reflect both peptide abundances and noise contributions, such as coisolation interference and background noise 8,9 .To evaluate the contribution of background noise to single-cell RI quantification, we estimated the signal-to-noise ratio (SNR), Extended Data Fig. 1. The estimates indicated that RI intensities are proportional to the amount of labeled single-cell proteomes, and very low for channels left empty.These data suggest that the signal measured in single cells exceeds the background noise by 10-fold or more. As an added SNR control for every TMT set, SCoPE-MS leaves the 130N channel empty, thus simultaneously avoiding isotopic cross-contamination from the carrier cells in channel 131 and having a channel that reflects the background noise.To evaluate the ability of SCoPE-MS to distinguish different cell types, we prepared two labelswapped and interlaced TMT sets with alternating single Jurkat and U-937 cells, two blood cancer cell lines with average cell diameter of only 11 µm (Fig. 1b). The levels of all 583 proteins quantified in single cells were projected onto their principle components (PC). The three-dimensional projections of single-cell proteomes clustered by cell type (Fig. 1c), suggesting that SCoPE-MS can identify cell types based on their proteomes. Next, we identified proteins whose levels vary less within a cell type than between cell types. Among the 117 proteins showing such trends at FDR < 2%, we plotted the distributions for seven in Fig. 1d. Some of these proteins are expected to be cell type specific, such as the higher abundance of Complement C3 in the U-937 cells, which are myeloid lineage precursors for macrophages. The consistency of protein fold-changes between Jurkat and U-937 cells is also reflected in the positive correlations among fold-changes estimated from different cells and TMT channels, Extended Data Fig. 2.Next, we quantified single-cell proteome heterogeneity and dynamics during ES cell differentiation. To initiate differentiation, we withdrew leukemia inhibitor factor (LIF) from ES cell cultures and transitioned to suspension culture; LIF withdrawal results in complex and highly heterogeneous differentiation of epiblast lineages in embryoid bodies (EB). We used SCoPE-MS to quantify over a thousand proteins at FDR = 1 % (Extended Data Fig. 3a) and their pair-wise cor- relations (averaging across single cells) in days 3, 5, and 8 after LIF withdrawal (Fig. ...

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Segregation of molecules at cell division reveals native protein localization

Cited by 300 publications

References 19 publications

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

MreB Orientation Correlates with Cell Diameter in Escherichia coli

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

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