Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Durisic, Nela; Laparra-Cuervo, Lara; Sandoval-Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

doi:10.1038/nmeth.2784

Cited by 236 publications

(335 citation statements)

References 43 publications

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“…The actual number of needle complexes, however, is likely to be higher as some clusters may be away from the focal plane and may escape detection. In addition, cluster detection would require the imaging of a minimum number of photon-switching events (five in our case), and although there are 24 mEos3.2-PrgH molecules in each needle complex that could be potentially imaged, it is likely that some needle complexes may not reach the minimum detection threshold because it has been reported that only 42% of mEos3.2 can be photoactivated to a fluorescently detectable form (44).…”

Section: Resultsmentioning

confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

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

103

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Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.bacterial nanomachines | protein secretion | superresolution microscopy | bacterial pathogenesis | Salmonella virulence T ype III secretion systems are remarkable molecular machines with the capacity to inject multiple bacterial proteins into eukaryotic cells (1, 2). These machines play an essential role in the pathogenic or symbiotic relationships between the bacteria that encode them and their respective hosts. Consequently, type III secretion systems are rapidly emerging as targets for the development of novel therapeutic and prevention strategies with the potential to combat several important infectious diseases (3-5). The components of type III secretion machines are highly conserved across bacterial species although the effector proteins that they deliver are customized for the specific bacteria that harbor them (1, 6, 7). The entire type III secretion machine or injectisome is ∼40 nm in width and ∼150 nm in length and is organized in defined substructures (Fig. 1A). The bestcharacterized substructure is the needle complex, which is a multi-ring cylindrical structure embedded in the bacterial envelope with a needle-like extension that projects several nanometers from the bacterial surface. The needle complex is traversed by a narrow channel that serves as the conduit for the proteins that travel this secretion pathway (8). The needle is capped at its end by the tip complex, which is thought to coordinate the activation of the secretion machine upon contact with target cells (9, 10). Because the needle complex can be isolated in a manner suitable for single-particle cryo-electron microscopy, its organization at the quasi-atomic level is known (11-18). Passage of the secreted proteins through the inner membrane requires the export apparatus, which is composed of a group of poly-topic inner membrane proteins located at the center of the needle complex base (19). Finally, several cytoplasmic proteins form a large complex known as the...

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Section: Resultsmentioning

confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

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

103

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“…The number of switching cycles is another important parameter to consider. For quantifying the stoichiometry of the target proteins, it is desirable to have PAFPs that can be switched on only once, although almost all PAFPs tend to blink more than once (31). However, for mapping of protein spatial organization, a larger number of switching cycles per molecule would allow the target structure to be sampled more times, which is advantageous when it comes to time-lapsed imaging for probing dynamics.…”

Section: Discussionmentioning

confidence: 99%

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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“…This accounted for repeated emission and potential short-term blinking, but not potential long-term blinking, which was, however, expected to be minimal for PAmCherry and PAGFP (Durisic et al, 2014). This analysis was able to eliminate localization multi-appearances as evident from the pair-correlation function (Puchner et al, 2013) (Fig.…”

Section: Palm Image Analysismentioning

confidence: 99%

Ligand-induced growth and compaction of CD36 nanoclusters enriched in Fyn induces Fyn signaling

Githaka

Vega

Baird

et al. 2016

Journal of Cell Science

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Nanoclustering is an emerging organizational principle for membraneassociated proteins. The functional consequences of nanoclustering for receptor signaling remain largely unknown. Here, we applied quantitative multi-channel high-and super-resolution imaging to analyze the endothelial cell surface receptor CD36, the clustering of which upon binding to multivalent ligands, such as the anti-angiogenic factor thrombospondin-1 (TSP-1), is thought to be crucial for signaling. We found that a substantial fraction of unligated CD36 exists in nanoclusters, which not only promote TSP-1 binding but are also enriched with the downstream effector Fyn. Exposure to multivalent ligands (TSP-1 or anti-CD36 IgM) that result in larger and denser CD36 clusters activates Fyn. Conversely, pharmacological perturbations that prevent the enhancement of CD36 clustering by TSP-1 abrogate Fyn activation. In both cases, there is no detectable change in Fyn enrichment at CD36 nanoclusters. These observations reveal a crucial role for the basal organization of a receptor into nanoclusters that are enriched with the signal-transducing downstream effectors of that receptor, such that enhancement of clustering by multivalent ligands is necessary and sufficient to activate the downstream effector without the need for its de novo recruitment.

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Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Cited by 236 publications

References 43 publications

Visualization and characterization of individual type III protein secretion machines in live bacteria

Visualization and characterization of individual type III protein secretion machines in live bacteria

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

Ligand-induced growth and compaction of CD36 nanoclusters enriched in Fyn induces Fyn signaling

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