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

2021 ◽  
Vol 7 (7) ◽  
pp. 580
Author(s):  
Anika Groth ◽  
Carolin Schunke ◽  
Eva Johanna Reschka ◽  
Stefanie Pöggeler ◽  
Daniela Elisabeth Nordzieke
Keyword(s):  
Fluorescent Proteins ◽  
Fungal Growth ◽  
Growth Dynamics ◽  
Hyphal Growth ◽  
Polar Axis ◽  
Green Fluorescent Proteins ◽  
Polar Growth ◽  
Colletotrichum Graminicola ◽  
Growth Processes ◽  
Green Fluorescent

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.

scholarly journals Coherent Dynamics of Photoexcited Green Fluorescent Proteins

2001 ◽  
Vol 86 (15) ◽  
pp. 3439-3442 ◽  
Author(s):  
Riccardo A. G. Cinelli ◽  
Valentina Tozzini ◽  
Vittorio Pellegrini ◽  
Fabio Beltram ◽  
Giulio Cerullo ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Coherent Dynamics ◽  
Green Fluorescent

scholarly journals Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

2017 ◽  
Vol 114 (11) ◽  
pp. E2146-E2155 ◽  
Author(s):  
Chi-Yun Lin ◽  
Johan Both ◽  
Keunbong Do ◽  
Steven G. Boxer
Keyword(s):  
Quantum Yield ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Photoactive Yellow Protein ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Low Efficiency ◽  
Green Fluorescent ◽  
Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

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

2022 ◽  
Vol 23 (2) ◽  
pp. 770
Author(s):  
Mikhail Drobizhev ◽  
Rosana S. Molina ◽  
Jacob Franklin
Keyword(s):  
Chemical Structure ◽  
Fluorescent Proteins ◽  
Multiphoton Ionization ◽  
Excitation Power ◽  
Photon Absorption ◽  
Green Fluorescent Proteins ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Red Fluorescent Proteins ◽  
Green Fluorescent

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.

A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins

2018 ◽  
Vol 140 (8) ◽  
pp. 2897-2905 ◽  
Author(s):  
Martin Byrdin ◽  
Chenxi Duan ◽  
Dominique Bourgeois ◽  
Klaus Brettel
Keyword(s):  
Triplet State ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Green Fluorescent
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