Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein-protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.

The cytoplasmic messenger ribonucleoprotein particles of mammalian somatic cells contain the protein YB-1, also called p50, as a major core component. YB-1 is multifunctional and involved in regulation of mRNA transcription and translation. Our previous studies demonstrated that YB-1 stimulates initiation of translation in vitro at a low YB-1/mRNA ratio, whereas an increase of YB-1 bound to mRNA resulted in inhibition of protein synthesis in vitro and in vivo. Here we show that YB-1-mediated translation inhibition in a rabbit reticulocyte cell-free system is followed by a decay of polysomes, which is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it affects neither elongation nor termination of polypeptide chains and only occurs at the stage of initiation. YB-1 induces accumulation of mRNA in the form of free messenger ribonucleoprotein particles, i.e. it blocks mRNA association with the small ribosomal subunit. The accumulation is accompanied by eukaryotic initiation factor eIF4G dissociation from mRNA. The C-terminal domain of YB-1 is responsible for inhibition of translation as well as the disruption of mRNA interaction with eIF4G.All mRNAs in eukaryotic cells are associated with proteins and form messenger ribonucleoprotein particles (mRNPs) 1 (1-6). Some mRNA-associated proteins exhibit specificity for certain mRNA(s); others are universal. To date, two universal major proteins of cytoplasmic mRNPs tightly bound to mRNA are well characterized. One of them is poly(A)-binding protein that stabilizes mRNA (7, 8) and promotes protein synthesis at the initiation stage (9, 10). It is suggested that the protein biosynthesis promotion occurs due to binding of multimerized poly(A)-binding protein associated with poly(A) to eIF4G and 1) mRNA cyclization and facilitation of ribosomal recycling (11, 12) and/or 2) a conformational change in eIF4F and an increase in affinity of eIF4E for the 5Ј-cap (13).The other common mRNP component is YB-1, a 36-kDa protein with abnormal mobility in SDS gel electrophoresis that is typical for this 50-kDa protein (14 -16). According to its primary structure, YB-1 from rabbit reticulocytes was identified as a member of the Y-box (YB) transcription factor family: it was virtually identical (ϳ98% identity) to human YB-1. YB-1 has been shown to participate in different steps of mRNA biogenesis, including mRNA transcription, processing, and transport from the nucleus into the cytoplasm (5, 17, 18) where it can regulate mRNA localization, translation, and mRNA stability (15,16,19). YB-1 displays DNA and RNA melting, annealing, and strand exchange activities, which probably underlies many functions of the YB protein family (20,21).The characteristic feature of these proteins is a central, highly conserved, cold shock domain (CSD) that consists of 80 amino acid residues and exhibits 43% identity to the major Escherichia coli cold shock protein CspA (4, 5). CSD comprises a five-stranded ␤-barrel with RNP ...

Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only ϳ10 -20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.

Protein kinases are involved in the regulation of many cellular processes including cell differentiation, survival, migration, axon guidance and neuronal plasticity. A growing set of optogenetic tools, termed opto-kinases, allows activation and inhibition of different protein kinases with light. The optogenetic regulation enables fast, reversible and non-invasive manipulation of protein kinase activities, complementing traditional methods, such as treatment with growth factors, protein kinase inhibitors or chemical dimerizers. In this review, we summarize the properties of the existing optogenetic tools for controlling tyrosine kinases and serine-threonine kinases. We discuss how the opto-kinases can be applied for studies of spatial and temporal aspects of protein kinase signaling in cells and organisms. We compare approaches for chemical and optogenetic regulation of protein kinase activity and present guidelines for selection of opto-kinases and equipment to control them with light. We also describe strategies to engineer novel opto-kinases on the basis of various photoreceptors.

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.

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau Microtubules are involved in a number of critical cellular processes, such as the determination of cell shape, chromosome segregation, intracellular transport of vesicles and organelles, and cell migration. Microtubules consist mainly of ␣␤-tubulin heterodimers organized head-to-tail into protofilaments whose parallel self-association gives rise to microtubules (1-4). ␣-and ␤-tubulin monomers have each a molecular mass of 50 kDa and are organized in three domains, namely the N-terminal domain (amino acid residues 1-205) involved in nucleotide binding, the intermediate domain (amino acid residues 206 -384), and the C-terminal domain (amino acid residue 385 to the C terminus) (5). The C-terminal domain of tubulin represents a critical part of the binding site of different tubulin/microtubules partners, such as MAPs, 4 which are major regulators of microtubule dynamics (6 -8), or polycations, which promote tubulin assembly in vitro in different polymeric forms (9). The C-terminal domain comprises a highly negatively charged tail of about 20 amino acid residues (named herein the C-terminal tail (CTT)), which protrudes from the surface of microtubules. In agreement with its participation in the regulation of microtubule assembly through interactions with partners, the CTT is also the most divergent part of tubulin, and variations among tubulin isotypes (10) may explain the modulation of the dynamics of microtubule assembly in specific tissues or cytoplasmic regions.Different structure information has been obtained regarding the C-terminal domain of tubulin by using either fulllength tubulin or peptide fragments. Electron crystallography of zinc-induced tubulin sheets showed the presence of two anti-parallel ␣-helices (helix H11 (amino acid residues 385-397) and helix H12 (amino acid residues 418 -433)) lying at the outer surface of tubulin. The regions corresponding to the CTTs of either ␣-or ␤-tubulin were however not observed, probably due to the flexibility of this part of the protein (5). These observations were confirmed by x-ray diffraction analyses of crystal complexes formed between tubulin and the RB3-stathmin-like domain (11-13). Other structural data were obtained with peptides from the C-terminal region of tubulin studied either when free in solution or in interaction with different partners. NMR structure investigations on ␣-and ␤-tubulin C-terminal peptides showed that both ␣ (residues 404 -451) and ␤ (residues 394 -445) peptides have no defined secondary structure in aqueous solution but contain a well □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental 4 The abbreviations used are: MAP, microtubule-associated protein; ␣Tub410C, amino acid residues 410 -451 from ␣1a-tubulin; CTT, tubulin C-terminal tail; ITC, isothermal titration ...

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.