Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.Peer reviewe

Neurotrophin receptor tyrosine kinases regulated with near-infrared light

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.Peer reviewe

Mobility of edge dislocations in stressed iron crystals during irradiation

The behavior of a/2(111){110} edge dislocations in iron in shear loading and irradiation conditions was studied by means of molecular dynamics simulation. Edge dislocations were exposed to shock waves formed by atomic displacement cascades of different energies. It was shown that starting from a certain threshold amplitude shock waves cause displacement of edge dislocations in the loaded samples. Calculations showed that the larger the shear load and the amplitude of the shock wave, the greater the displacement of dislocations in the crystallite

Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation by generating reactive oxygen species, degFusionRed undergoes proteasomal degradation upon illumination with 567 nm light. Similarly to the parent protein, degFusionRed has minimal absorbance at 450 nm and above 650 nm, making it spectrally compatible with blue and near-infrared-light-controlled optogenetic tools. The autocatalytically formed chromophore provides degFusionRed with an additional advantage over most optogenetic tools that require the binding of the exogenous chromophores, the amount of which varies in different cells. The degFusionRed efficiently performed in the engineered light-controlled transcription factor and in the targeted photodegradation of the protein of interest, demonstrating its versatility as the optogenetic module of choice for spectral multiplexed interrogation of various cellular processes

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

Polyamine Sharing between Tubulin Dimers Favours Microtubule Nucleation and Elongation via Facilitated Diffusion

We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation by generating reactive oxygen species, degFusionRed undergoes proteasomal degradation upon illumination with 567 nm light. Similarly to the parent protein, degFusionRed has minimal absorbance at 450 nm and above 650 nm, making it spectrally compatible with blue and near-infrared-light-controlled optogenetic tools. The autocatalytically formed chromophore provides degFusionRed with an additional advantage over most optogenetic tools that require the binding of the exogenous chromophores, the amount of which varies in different cells. The degFusionRed efficiently performed in the engineered light-controlled transcription factor and in the targeted photodegradation of the protein of interest, demonstrating its versatility as the optogenetic module of choice for spectral multiplexed interrogation of various cellular processes

Mobility of edge dislocations in stressed iron crystals during irradiation

The behavior of a/2(111){110} edge dislocations in iron in shear loading and irradiation conditions was studied by means of molecular dynamics simulation. Edge dislocations were exposed to shock waves formed by atomic displacement cascades of different energies. It was shown that starting from a certain threshold amplitude shock waves cause displacement of edge dislocations in the loaded samples. Calculations showed that the larger the shear load and the amplitude of the shock wave, the greater the displacement of dislocations in the crystallite