Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

Effects of Currently Used Antifouling Booster Biocides on Green Fluorescent Proteins in Anemonia Viridis

Some of the antifouling booster biocides affects the marine ecosystem negatively. The booster biocides which are resistant to degradation are accumulated in the sediment of the oceans. One of the sedentary organisms in the Mediterranean Sea is Anemonia viridis. The aim of this study is to show the toxicities of common biocides such as irgarol, seanine-211, zinc omadine, and acticide on the fluorescence by GFPs of A.viridis. The decreases in the fluorescence intensities of the GFP were measured within different booster biocide concentrations. The results show that fluorescent intensities of GFP proteins decreased more than 50 percent when they are exposed to different concentrations of irgarol, zinc omadine, acticide. In conclusion, ecosystem health should be prioritized when new antifouling paint compositions are proposed. From the results, it seems that A.viridis can be considered as a vulnerable organism and also it is sensitive to booster biocides within self-polishing antifouling paint formulations.

The Aedes aegypti (Diptera: Culicidae) hsp83 Gene Promoter Drives Strong Ubiquitous DsRed and ZsGreen Marker Expression in Transgenic Mosquitoes

Transgenic strains of the mosquito disease vector Aedes aegypti (L.) are being developed for population suppression or modification. Transgenic mosquitoes are identified using fluorescent protein genes. Here we describe DsRed and ZsGreen marker genes driven by the constitutive Ae. aegypti heat shock protein 83 (hsp83) promoter in transgenic mosquitoes. Transgenic larvae and pupae show strong full body expression of the red and green fluorescent proteins. This greatly assists in screening for transgenic individuals while making new or maintaining already established lines. Transient marker gene expression after embryo microinjection was readily visible in developing larvae allowing the separation of individuals that are more likely to produce transgenic offspring. The strongly expressed marker genes developed in this study should facilitate the detection of transgenic Ae. aegypti larvae or pupae in the field.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.

Green Fluorescence Patterns in Closely Related Symbiotic Species of Zanclea (Hydrozoa, Capitata)

Green fluorescence is a common phenomenon in marine invertebrates and is caused by green fluorescent proteins. Many hydrozoan species display fluorescence in their polyps and/or medusa stages, and in a few cases patterns of green fluorescence have been demonstrated to differ between closely related species. Hydrozoans are often characterized by the presence of cryptic species, due to the paucity of available morphological diagnostic characters. Zanclea species are not an exception, showing high genetic divergence compared to a uniform morphology. In this work, the presence of green fluorescence and the morpho-molecular diversity of six coral- and bryozoan-associated Zanclea species from the Maldivian coral reefs were investigated. Specifically, the presence of green fluorescence in polyps and newly released medusae was explored, the general morphology, as well as the cnidome and the interaction with the hosts, were characterized, and the 16S rRNA region was sequenced and analyzed. Overall, Zanclea species showed a similar morphology, with little differences in the general morphological features and in the cnidome. Three of the analyzed species did not show any fluorescence in both life stages. Three other Zanclea species, including two coral-associated cryptic species, were distinguished by species-specific fluorescence patterns in the medusae. Altogether, the results confirmed the morphological similarity despite high genetic divergence in Zanclea species and indicated that fluorescence patterns may be a promising tool in further discriminating closely related and cryptic species. Therefore, the assessment of fluorescence at a large scale in the whole Zancleidae family may be useful to shed light on the diversity of this enigmatic taxon.

A bright and high-performance genetically encoded Ca2+ indicator based on mNeonGreen fluorescent protein

Genetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost two decades of steady improvements in the Aequorea victoria GFP (avGFP)-based GCaMP series of GECIs, the performance of the most recent generation (i.e., GCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression towards ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.