Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools.

Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.

A bacterial phytochrome-based optogenetic system controllable with near-infrared light.

Light-mediated control of protein-protein interactions to regulate cellular pathways is an important application of optogenetics. Here, we report an optogenetic system based on the reversible light-induced binding between the bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1-PpsR2 interaction both in vitro and in mammalian cells and then used this interaction to translocate target proteins to specific cellular compartments, such as the plasma membrane and the nucleus. We showed light-inducible control of cell morphology that resulted in a substantial increase of the cell area. We demonstrated light-dependent gene expression with 40-fold contrast in cultured cells, 32-fold in subcutaneous mouse tissue, and 5.7-fold in deep tissues in mice. Characteristics of the BphP1-PpsR2 optogenetic system include its sensitivity to 740- to 780-nm near-infrared light, its ability to utilize an endogenous biliverdin chromophore in eukaryotes (including mammals), and its spectral compatibility with blue-light-driven optogenetic systems.

Microfluidic System for In-Flow Reversible Photoswitching of Near-Infrared Fluorescent Proteins.

We have developed a microfluidic flow cytometry system to screen reversibly photoswitchable fluorescent proteins for contrast and stability of reversible photoconversion between high- and low-fluorescent states. A two-color array of 20 excitation and deactivation beams generated with diffractive optics was combined with a serpentine microfluidic channel geometry designed to provide five cycles of photoswitching with real-time calculation of photoconversion fluorescence contrast. The characteristics of photoswitching in-flow as a function of excitation and deactivation beam fluence, flow speed, and protein concentration were studied with droplets of the bacterial phytochrome from Deinococcus radiodurans (DrBphP), which is weakly fluorescent in the near-infrared (NIR) spectral range. In agreement with measurements on stationary droplets and HeLa S3 mammalian cells expressing DrBphP, optimized operation of the flow system provided up to 50% photoconversion contrast in-flow at a droplet rate of few hertz and a coefficient of variation (CV) of up to 2% over 10 000 events. The methods for calibrating the brightness and photoswitching measurements in microfluidic flow established here provide a basis for screening of cell-based libraries of reversibly switchable NIR fluorescent proteins.

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins.

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

Biochemical characterization of small heat shock protein HspB8 (Hsp22)-Bag3 interaction.

Interaction of human Bag3 with small heat shock proteins HspB6, HspB8 and its K141E mutant was analyzed by different biochemical methods. The data of size-exclusion chromatography indicate that the wild type HspB8 forms tight complexes with Bag3. K141E mutant of HspB8 and especially HspB6 weaker interact with Bag3. The data of chemical crosslinking and analytical ultracentrifugation indicate that in vitro the stoichiometry of complexes formed by HspB8 and Bag3 is variable and is dependent on concentration of protein partners. Interaction of Bag3 and HspB8 is accompanied by increase of thermal stability measured by intrinsic tryptophan fluorescence and increased resistance to limited chymotrypsinolysis. The data of size-exclusion chromatography, analytical ultracentrifugation and limited proteolysis indicate that Bag3 belongs to the group of intrinsically disordered proteins. It is supposed that having unordered structure Bag3 might weakly interact with different small heat shock proteins which recognize unfolded proteins and this interaction is especially strong with intrinsically disordered HspB8. The complexes formed by Bag3 and HspB8 might have variable stoichiometry and can participate in different processes including clearing of the cell from improperly folded proteins.

Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase.

A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.

A near-infrared genetically encoded calcium indicator for in vivo imaging.

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep-tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared (NIR) fluorescent proteins (FPs), we engineered an NIR Förster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 µm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with resolutions of ~3 µm (lateral) and ~25-50 µm (axial). Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales.

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

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.

Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing.

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.

Focusing light inside live tissue using reversibly switchable bacterial phytochrome as a genetically encoded photochromic guide star.

Focusing light deep by engineering wavefronts toward guide stars inside scattering media has potential biomedical applications in imaging, manipulation, stimulation, and therapy. However, the lack of endogenous guide stars in biological tissue hinders its translations to in vivo applications. Here, we use a reversibly switchable bacterial phytochrome protein as a genetically encoded photochromic guide star (GePGS) in living tissue to tag photons at targeted locations, achieving light focusing inside the tissue by wavefront shaping. As bacterial phytochrome-based GePGS absorbs light differently upon far-red and near-infrared illumination, a large dynamic absorption contrast can be created to tag photons inside tissue. By modulating the GePGS at a distinctive frequency, we suppressed the competition between GePGS and tissue motions and formed tight foci inside mouse tumors in vivo and acute mouse brain tissue, thus improving light delivery efficiency and specificity. Spectral multiplexing of GePGS proteins with different colors is an attractive possibility.

Structure, properties, and functions of the human small heat-shock protein HSP22 (HspB8, H11, E2IG1): a critical review.

The recently described human HSP22 belongs to the superfamily of small heat-shock proteins containing a conservative alpha-crystallin domain. HSP22 seems to be involved in regulation of cell proliferation, cardiac hypertrophy, apoptosis, and carcinogenesis, and expression of point mutants of HSP22 correlates with development of different neuromuscular diseases. Therefore, an investigation of the structure and properties of HSP22 is desirable for understanding its multiple functions. HSP22 seems to belong to the group of so-called intrinsically disordered proteins and possesses a highly flexible structure. HSP22 tends to form small-molecular-mass oligomers and interacts with biological membranes and many different proteins, among them glycolytic enzymes and different protein kinases. HSP22 possesses chaperonelike activity and prevents aggregation of denatured proteins both in vitro and in vivo. Depending on the cell type and its expression, HSP22 might have either pro- or anti-apoptotic effects. Chaperonelike activity seems to be important for antiapoptotic effects, whereas interaction with and regulation of certain protein kinases might be important for the proapoptotic effects of HSP22. Expression of K141N or K141E mutants of HSP22 correlates with development of distal hereditary motor neuropathy and/or Charcot-Marie-Tooth disease. These mutations destabilize the structure of HSP22, affect its interaction with other small heat-shock proteins, and decrease its chaperonelike activity. HSP22 decreases or prevents aggregation of Huntingtin fragments and amyloid-beta peptide 1-40 of the Dutch type. Thus, HSP22 seems to play an important role in the nervous system, and further investigations are needed to understand the molecular mechanisms of its functioning.

Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo.

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells.

Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. For instance, in common HeLa cells miRFP703, mIFP, and smURFP were 2-, 9-, and 53-fold dimmer than miRFP670. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. mIFP showed intermediate, while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range.

Obatoclax kills anaplastic thyroid cancer cells by inducing lysosome neutralization and necrosis.

Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited.We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo.We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members.Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.

Molecular interaction of proteins and peptides with nanoparticles.

The interaction of proteins in living cells is one of the key processes in the maintenance of their homeostasis. Introduction of additional agents into the chain of these interactions may influence homeostatic processes. Recent advances in nanotechnologies have led to a wide use of nanoparticles (NPs) in industrial and biomedical applications. NPs are small enough to enter almost all compartments of the body, including cells and organelles, and to complicate the pattern of protein interactions. In some cases, interaction of nanoscale objects with proteins leads to hazardous consequences, such as abnormal conformational changes leading to exposure of cryptic peptide epitopes or the appearance of abnormal functions caused by structural modifications. In addition, the high local protein concentration resulting from protein adsorption on NPs may provoke avidity effects arising from close spatial repetition of the same protein. Finally, the interaction of NPs with proteins is known to induce cooperative effects, such as promotion or inhibition of protein fibrillation or self-assembling of NPs on macromolecules serving as a template. It is obvious that better understanding of the molecular mechanisms of nano-bio interactions is crucial for further advances in all nanotechnological applications. This review summarizes recent progress in understanding the molecular mechanisms of the interactions between proteins or peptides and NPs in order to predict the structural, functional, and/or nanotoxic consequences of these interactions.