Genetic mosaics of
            Anabaena
            filaments show that the
            hetN
            ‐dependent paracrine signal propagates from a heterocyst source resulting in the degradation of an activator of differentiation, HetR. HetN and HetR were visualized as fusion proteins to the yellow and cyan fluorescent proteins, respectively. Red autofluorescence outlines cells of the filaments. For further details readers are referred to the article by Rivers
            et al
            . on pp. 1260–1271 of this issue

doi:10.1111/mmi.12891

Impact of fluorescent protein fusions on the bacterial flagellar motor

10.1101/152595 ◽

2017 ◽

Cited By ~ 1

Author(s):

M Heo ◽

AL Nord ◽

D Chamousset ◽

E van Rijn ◽

HJE Beaumont ◽

...

Keyword(s):

Side Effects ◽

Protein Function ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Protein Complexes ◽

Switching Frequency ◽

Flagellar Motor ◽

Internal Dynamics ◽

Biologically Relevant ◽

Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

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Mutual effects of disorder and order in fusion proteins between intrinsically disordered domains and fluorescent proteins

Molecular BioSystems ◽

10.1039/c1mb05244f ◽

2012 ◽

Vol 8 (1) ◽

pp. 105-113 ◽

Cited By ~ 3

Author(s):

Marina Lotti ◽

Sonia Longhi

Keyword(s):

Fusion Proteins ◽

Fluorescent Proteins ◽

Intrinsically Disordered ◽

Effects Of Disorder

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Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0661 ◽

2019 ◽

Vol 30 (12) ◽

pp. 1369-1376 ◽

Cited By ~ 9

Author(s):

Tim N. Baldering ◽

Marina S. Dietz ◽

Karl Gatterdam ◽

Christos Karathanasis ◽

Ralph Wieneke ◽

...

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Molecular Information ◽

Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

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A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells

Biologia ◽

10.2478/s11756-007-0104-6 ◽

2007 ◽

Vol 62 (5) ◽

Cited By ~ 44

Author(s):

Wei Sun ◽

Ziyi Cao ◽

Yan Li ◽

Yanxiu Zhao ◽

Hui Zhang

Keyword(s):

Subcellular Localization ◽

Particle Bombardment ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Transformed Cells ◽

Protein Subcellular Localization ◽

Transformation Protocol ◽

Agrobacterium Mediated Transformation ◽

The Mean ◽

Onion Epidermal Cells

AbstractA modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.

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Properties of Baculovirus Particles Displaying GFP Analyzed by Fluorescence Correlation Spectroscopy

Biological Chemistry ◽

10.1515/bc.2002.218 ◽

2002 ◽

Vol 383 (12) ◽

pp. 1941-1946 ◽

Cited By ~ 14

Author(s):

J. Toivola ◽

K. Ojala ◽

P.O. Michel ◽

M. Vuento ◽

C. Oker-Blom

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Sodium Dodecyl ◽

Recombinant Baculovirus ◽

Mean Value ◽

Correlation Spectroscopy ◽

Viral Envelope ◽

Fluorescence Correlation

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

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Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

Microorganisms ◽

10.3390/microorganisms8060934 ◽

2020 ◽

Vol 8 (6) ◽

pp. 934 ◽

Cited By ~ 2

Author(s):

Peter Braun ◽

Immanuel Wolfschläger ◽

Leonie Reetz ◽

Lilia Bachstein ◽

Ana Clara Jacinto ◽

...

Keyword(s):

Bacillus Cereus ◽

Bacillus Anthracis ◽

Receptor Binding ◽

Fusion Proteins ◽

Binding Proteins ◽

Fluorescent Proteins ◽

Lambdoid Prophage ◽

Unambiguous Detection ◽

Close Genetic Relationship ◽

Bacillus Cereus Sensu Lato

Bacillus anthracis, the etiological agent of anthrax disease, is typically diagnosed by immunological and molecular methods such as polymerase chain reaction (PCR). Alternatively, mass spectrometry techniques may aid in confirming the presence of the pathogen or its toxins. However, because of the close genetic relationship between B. anthracis and other members of the Bacillus cereus sensu lato group (such as Bacillus cereus or Bacillus thuringiensis) mis- or questionable identification occurs frequently. Also, bacteriophages such as phage gamma (which is highly specific for B. anthracis) have been in use for anthrax diagnostics for many decades. Here we employed host cell-specific receptor binding proteins (RBP) of (pro)-phages, also known as tail or head fibers, to develop a microscopy-based approach for the facile, rapid and unambiguous detection of B. anthracis cells. For this, the genes of (putative) RBP from Bacillus phages gamma, Wip1, AP50c and from lambdoid prophage 03 located on the chromosome of B. anthracis were selected. Respective phage genes were heterologously expressed in Escherichia coli and purified as fusions with fluorescent proteins. B. anthracis cells incubated with either of the reporter fusion proteins were successfully surface-labeled. Binding specificity was confirmed as RBP fusion proteins did not bind to most isolates of a panel of other B. cereus s.l. species or to more distantly related bacteria. Remarkably, RBP fusions detected encapsulated B. anthracis cells, thus RBP were able to penetrate the poly-γ-d-glutamate capsule of B. anthracis. From these results we anticipate this RBP-reporter assay may be useful for rapid confirmative identification of B. anthracis.

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Model systems for studying the assembly, trafficking, and secretion of apoB lipoproteins using fluorescent fusion proteins

The Journal of Lipid Research ◽

10.1194/jlr.ra119000259 ◽

2019 ◽

Vol 61 (3) ◽

pp. 316-327

Author(s):

Meghan T. Walsh ◽

Oni M. Celestin ◽

James H. Thierer ◽

Sujith Rajan ◽

Steven A. Farber ◽

...

Keyword(s):

Fusion Proteins ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Microsomal Triglyceride Transfer Protein ◽

Model Systems ◽

C Terminus ◽

Artery Disease ◽

And Function

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.

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Folding latency of fluorescent proteins affects the mitochondrial localization of fusion proteins

10.1101/768432 ◽

2019 ◽

Author(s):

Sayaka Kashiwagi ◽

Yoichiro Fujioka ◽

Aya O. Satoh ◽

Aiko Yoshida ◽

Mari Fujioka ◽

...

Keyword(s):

Amino Acid ◽

Fusion Proteins ◽

Cell Biology ◽

Fluorescent Proteins ◽

Amino Acid Substitutions ◽

Wild Type ◽

Native Proteins ◽

Maturation Rate ◽

Similar Amino Acid ◽

Cellular Organelles

ABSTRACTThe discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

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