Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion

Hur, Kwang Ho; Macdonald, Patrick J.; Berk, Serkan; Angert, Isaac; Chen, Yan; Mueller, Joachim D.

doi:10.1371/journal.pone.0097440

Cited by 26 publications

(40 citation statements)

References 35 publications

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“…For example, the obtained tetramer brightness (etetramer=3.01 ± 0.08) is closer to the theoretical trimer brightness value ( Figure 2B, white boxes). Hence, we performed a brightness correction based on a simple two-state model 11,31 , taking into account the probability that each FP subunit emits a fluorescence signal. The pf values were determined from the brightness of 2xmEGFP (edimer=1.65 ± 0.06, pf=0.65).…”

Section: Figure 1 Brightness Comparison Of Different Fps In Living Hmentioning

confidence: 99%

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Dunsing

Luckner

Zühlke

et al. 2018

Preprint

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Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing. Here, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions. * Measured at four times higher laser power (19.6 µW) than used in N&B measurements ** Average molecular brightness in N&B, interpolated to the same laser power (4.9 µW) for all red FPs

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Section: Figure 1 Brightness Comparison Of Different Fps In Living Hmentioning

confidence: 99%

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Dunsing

Luckner

Zühlke

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…To build the kinetic run, a different cell was imaged in a different field of view at each time point to avoid photobleaching and photo-damaging due to repeated illumination. To avoid distortion of the brightness by intensity changes during acquisition, the total acquisition time was within 11-22 s and time series showing fluorescence intensity changes >5% were discarded (Fukushima et al, 2018;Hur et al, 2014;Trullo et al, 2013). Control time stacks were acquired in parallel for HeLa cells expressing GPI-mEGFP or GPI-mEGFP-mEGFP to account for the intrinsic variability of the laser power during each run.…”

Section: Nandb Analysismentioning

confidence: 99%

Number and brightness analysis in live cells reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1

Zamai

Trullo

Giordano

et al. 2018

Journal of Cell Science

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Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

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“…The excitation power measured at the objective ranged from 1.0 to 1.2 mW at 1000 nm. These power levels were sufficiently low to avoid saturation and bleaching (42). Both EGFP and mCherry can be simultaneously excited with 1000 nm two-photon excitation.…”

Section: Instrumentationmentioning

confidence: 99%

Association of Endophilin B1 with Cytoplasmic Vesicles

Baryłko

Mueller

et al. 2017

Biophysical Journal

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Endophilins are SH3-and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins.

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Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion

Cited by 26 publications

References 35 publications

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Number and brightness analysis in live cells reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1

Association of Endophilin B1 with Cytoplasmic Vesicles

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