Rational design of true monomeric and bright photoactivatable fluorescent proteins

Zhang, Mingshu; Chang, Hao; Zhang, Yongdeng; Yu, Junwei; Wu, Lijie; Ji, Wei; Chen, Juanjuan; Liu, Bei; Lu, Jingze; Liu, Yingfang; Zhang, Junlong; Xu, Pingyong; Xu, Tao

doi:10.1038/nmeth.2021

Cited by 439 publications

(417 citation statements)

References 30 publications

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“…Each mEos3.2 molecule can be turned on and off more than once before being irreversibly photobleached (32). This intrinsic blinking behavior of mEos3.2 results in each tagged molecule being localized more than once.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, each localized singlemolecule emitter contains spatial and temporal information that provides quantitative data that surpass those obtained from other super-resolution techniques. We took advantage of recent developments in instrumentation and data analysis methods (26)(27)(28)(29)(30)(31)(32) to perform FPALM super-resolution microscopy at ∼35-nm resolution (localization precision, σ loc , ∼14 nm). These methods enabled us to observe that nodes are uniform units with stoichiometric ratios and distinct distributions of constituent proteins, and provided the quantitative data necessary to build a molecular model of the node.…”

mentioning

confidence: 99%

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Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Laplante

Huang

Tebbs

et al. 2016

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

123

282

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Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/ Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.T he mechanism of cell division by a contractile ring of actin and myosin II appeared in the common ancestor of amoebas, fungi, and animals (1). Although much is known about the protein composition of contractile rings, relatively little is known about the 3D organization of these proteins. This information is required to formulate computer models and simulations to test ideas regarding the mechanisms of contractile ring function.Electron microscopy has shown actin filaments in contractile rings of animal cells (2, 3) and yeast cells (4,5). Myosin II concentrates in cleavage furrow (6) and is the main motor for constricting contractile rings (7,8). In animal cells, electron microscopy has revealed rods the size of myosin II minifilaments in contractile rings (2, 9), and structured-illumination fluorescence microscopy has shown that this myosin II is organized in bipolar assemblies (10). Contractile rings contain other structural and regulatory proteins, including anillin (11), IQ motif containing GTPase-activating proteins (IQGAP) (12), formins (13), alphaactinin (14), and Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) proteins (15), but how these proteins are anchored to the plasma membrane or organized into functional complexes in animal cells is unknown.Our understanding of the cytokinetic apparatus is most advanced in fission yeast (16). Only in fission yeast do we know the concentrations of the major cytokinesis proteins (17) and the time course of events leading to cellular division (18,19). Molecularly explicit computer models can account for both the ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Laplante

Huang

Tebbs

et al. 2016

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

123

282

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“…We first took inspiration from the two mutations that make mEos3.2 more monomeric than mEos2: I102N and Y189A (15). Based on a sequence alignment between mEos2 and mMaple, we made the comparable mutations, I111N and Y198A, in mMaple.…”

Section: New Pafps With High Signaling Efficiency and Low Dimerizationmentioning

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.

320

381

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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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“…Previous studies have shown that the high accumulation of PIP2 is required for receptor protein syntaxin-1A clustering [15]. To test whether PIP2 was involved in the EGFR clustering, we labeled PIP2 in COS-7 cell membrane with a PIP2 probe consisting of the Pleckstrin homology (PH) domain [26] of phospholipase C delta fused to a photoactivatable fluorescent protein mEos3.2 [27] (PH-PLCδ-mEos3.2) and analyzed the spatial distribution of PIP2 and EGFR using PALM and dSTORM, respectively. Our data showed that PIP2, similar to EGFR protein, also formed clusters in the cell membrane (Figure 2A).…”

Section: Pip2 Depletion In the Plasma Membrane Impairs The Egfr Clustmentioning

confidence: 99%

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

et al. 2014

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The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

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Rational design of true monomeric and bright photoactivatable fluorescent proteins

Cited by 439 publications

References 30 publications

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

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

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

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