Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Out of the blue: Phototropins of the leaf vascular bundle sheath mediate the regulation of leaf hydraulic conductance by blue light.

The Arabidopsis (Arabidopsis thaliana) leaf veins bundle-sheath cells (BSCs)-a selective barrier to water and solutes entering the mesophyll-increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase,  AHA2. Based on this and on the BSCs' expression of phototropins PHOT1 and PHOT2, and the known blue light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs' phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5 phot2-1, and aha2-4 was lower than that of the wild-type (WT). BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light-ineffective in the phot1-5 mutant-acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts' water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.

Blue-light receptor phototropin 1 suppresses immunity to promote Phytophthora infestans infection.

Blue-light (BL) phototropin receptors (phot1 and phot2) regulate plant growth by activating NPH3/RPT2-like (NRL) family members. Little is known about roles for BL and phots in regulating plant immunity. We showed previously that Phytophthora infestans RXLR effector Pi02860 targets potato (St)NRL1, promoting its ability to enhance susceptibility by facilitating proteasome-mediated degradation of the immune regulator StSWAP70. This raises the question: do BL and phots negatively regulate immunity? We employed coimmunoprecipitation, virus-induced gene silencing, transient overexpression and targeted mutation to investigate contributions of phots to regulating immunity. Whereas transient overexpression of Stphot1 and Stphot2 enhances P. infestans colonization of Nicotiana benthamiana, silencing endogenous Nbphot1 or Nbphot2 reduces infection. Stphot1, but not Stphot2, suppressed the INF1-triggered cell death (ICD) immune response in a BL- and NRL1-dependent manner. Stphot1, when coexpressed with StNRL1, promotes degradation of StSWAP70, whereas Stphot2 does not. Kinase-dead Stphot1 fails to suppress ICD, enhance P. infestans colonization or promote StSWAP70 degradation. Critically, BL enhances P. infestans infection, which probably involves phots but not other BL receptors such as cryptochromes and F-box proteins ZTL1 and FKF1. We demonstrate that Stphot1 and Stphot2 play different roles in promoting susceptibility, and Stphot1 kinase activity is required for BL- and StNRL1-mediated immune suppression.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

A BLUS1 kinase signal and a decrease in intercellular CO2 concentration are necessary for stomatal opening in response to blue light.

Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening.

New Light on the Mechanism of Phototransduction in Phototropin.

Phototropins are photoreceptor proteins that regulate blue light-dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (light-oxygen-voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism that leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from C. reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- to millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. In particular, the change in conformation and the associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the ß-sheet, ultimately driving a change in the electrostatic potential on the protein surface. On the basis of the change in the electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from C. reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

Conformational properties of LOV2 domain and its C450A variant within broad pH region.

LOV2 (Light-Oxygen-Voltage) domain from Avena sativa phototropin 1 (AsLOV2) belongs to the superfamily of PAS (Per-Arnt-Sim) domains, members of which function as signaling sensors. AsLOV2 undergoes a conformational change upon blue-light absorption by its FMN cofactor. AsLOV2 wild type (wt) is intensively studied as a photo-switchable element in conjugation with various proteins. On the other hand, its variant AsLOV2 with replaced cysteinyl residue C450, which is critical for the forming a covalent adduct with FMN upon irradiation, forms a precursor for some recently developed genetically encoded photosensitizers. In the presented work, we investigated conformational properties of AsLOV2 wt and its variant C450A by circular dichroism, tryptophan and FMN fluorescence, and differential scanning calorimetry in dependence on pH and temperature. We show that both variants are similarly sensitive towards pH of solvent. On the other hand, the mutation C450A leads to a more stable AsLOV2 variant in comparison with the wild type. Thermal transitions of the AsLOV2 proteins monitored by circular dichroism indicate the presence of significant residual structure in thermally-denatured states of both proteins in the pH range from 4 to 9. Both pH- and thermal- transitions of AsLOV2 are accompanied by FMN leaching to solvent. Higher stability, reversibility of thermal transitions, and efficiency of FMN rebinding in the case of C450A variant suggest that the cofactor release may be modulated by suitable mutations in combination with a suitable physicochemical perturbation. These findings can have implications for a design of genetically encoded photosensitizers.

Minimally disruptive optical control of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Measurements of Photoreaction and Kinase Activity of Phototropin, a Photoreceptor Protein for Tropic Response in Plants: Involvement of Kinase Activity in the Photosensitivity of Tropic Response.

Phototropin is a photoreceptor protein responsible for phototropic responses in plants. A phototropin molecule has two photoreceptive domains named LOV1 and LOV2 in the N-terminal region. Blue light absorbed by a chromophore in these domains triggers conformational changes in the protein moiety. The C-terminal region of phototropin forms a Ser/Thr kinase that is activated by these conformational changes. The activated phototropin kinase transmits signals downstream leading to tropic responses. The lifetime of the activated state may concern the sensitivity of the tropic responses to light. Thus, spectrophotometric and kinase activity analyses of phototropin are important to understand the light signaling processes related to the photosensitivity. The preparation of polypeptide samples of Arabidopsis phototropin and the methods of spectroscopic measurements and kinase assay of these samples are shown in this chapter.

Red Light-Induced Phosphorylation of Plasma Membrane H+-ATPase in Stomatal Guard Cells.

Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane (PM) H+-ATPase by phosphorylating its penultimate residue, threonine, via a blue light photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated PM H+-ATPase promotes the accumulation of osmolytes and, thus, the osmotic influx of water into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, how red light induces stomatal opening and whether PM H+-ATPase is involved in this process have remained unclear. In this study, we established an immunohistochemical technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces PM H+-ATPase phosphorylation in whole leaves. Red light-induced PM H+-ATPase phosphorylation in whole leaves was correlated with stomatal opening under red light and was inhibited by the plant hormone abscisic acid. In aha1-9, a knockout mutant of one of the major isoforms of PM H+-ATPase in guard cells, red light-dependent stomatal opening was delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited red light-induced PM H+-ATPase phosphorylation as well as red light-induced stomatal opening in whole leaves. Our results indicate that red light-induced PM H+-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.

Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini "Singlet Oxygen Generator" (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen (1O2), the contribution of superoxide (O2•-) to miniSOG-generated ROS remains unclear. We measured the light-dependent O2•- production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1O2 and establish that DHE is a suitable indicator to measure O2•- production in a system that produces both 1O2 and O2•-. We report that miniSOG produces both 1O2 and O2•-, as can its free chromophore, flavin mononucleotide. miniSOG produced O2•- at a rate of ~4.0µmol O2•-/min/µmol photosensitizer for an excitation fluence rate of 5.9mW/mm2 at 470 ± 20nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•- to miniSOG phenotypes should be considered.

Dimeric Structure of the Blue Light Sensor Protein Photozipper in the Active State.

The light oxygen voltage-sensing (LOV) domain plays a crucial role in blue light (BL) sensing in plants and microorganisms. LOV domains are usually associated with the effector domains and regulate the activities of effector domains in a BL-dependent manner. Photozipper (PZ) is monomeric in the dark state. BL induces reversible dimerization of PZ and subsequently increases its affinity for the target DNA sequence. In this study, we report the analyses of PZ by pulsed electron-electron double resonance (PELDOR). The neutral flavin radical was formed by BL illumination in the presence of dithiothreitol in the LOV-C254S (without the bZIP domain) and PZ-C254S mutants, where the cysteine residue responsible for adduct formation was replaced with serine. The magnetic dipole interactions of 3 MHz between the neutral radicals were detected in both LOV-C254S and PZ-C254S, indicating that these mutants are dimeric in the radical state. The PELDOR simulation showed that the distance between the radical pair is close to that estimated from the dimeric crystal structure in the "light state" [Heintz, U., and Schlichting, I. (2016) eLife 5, e11860], suggesting that in the radical state, LOV domains in PZ-C254S form a dimer similar to that of LOV-C254S, which lacks the bZIP domain.

Switching from adduct formation to electron transfer in a light-oxygen-voltage domain containing the reactive cysteine.

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin-cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing.

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio-temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore-assisted light inactivation (CALI) of proteins, as well as for photo-induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini-antibodies or selective scaffold proteins and photo-induced cytotoxicity of miniSOG, which is particularly promising for selective non-invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin.

The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.

[Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.

Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatal opening.

In Arabidopsis thaliana, phototropins (phot1 and phot2), light-activated receptor kinases, redundantly regulate various photoresponses such as phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. However, it is still unclear how phot1 and phot2 signals are integrated into a common target and regulate physiological responses. In the present study, we provide evidence that phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening. Biochemical analysis revealed that the recombinant phot2 protein directly phosphorylated BLUS1 in vitro in a blue light-dependent manner, as reported for phot1. BLUS1 phosphorylation was observed in both phot1 and phot2 mutants, and phot2 mutant exhibited higher phosphorylation of BLUS1 than did phot1 mutant. Transgenic plants expressing phot1-GFP (P1G) and phot2-GFP (P2G) at a similar level under the PHOT2 promoter demonstrated that P1G initiated higher phosphorylation of BLUS1 than P2G, suggesting that phot1 phosphorylates BLUS1 more efficiently. Similarly, P1G mediated a higher activation of the plasma membrane H(+)-ATPase and stomatal opening than P2G, indicating that the phosphorylation status of BLUS1 is a key determinant of physiological response. Together, these findings provide insights into the signal integration and different properties of phot1 and phot2 signaling.

Auxin and chloroplast movements.

Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG.

Cytotoxic effects of a new targeted phototoxin DARPin-miniSOG and mechanism of its action were investigated in vitro. It was determined that DARPin-miniSOG causes light-induced death of HER2/neu-positive cancer cells (IC50 0.8 µM). Treatment of the cells with DARPin-miniSOG in the presence of ascorbic acid eliminated the light-induced cytotoxic action of the protein. This observation suggests the involvement of oxidative stress in the mechanism of the phototoxin action. DNA fragmentation analysis, caspase-3 activity assay and PI-staining of HER2/neu-positive cancer cells treated with DARPin-miniSOG indicated that phototoxin induces necrotic cell death under blue light illumination. Co-localization analysis showed that DARPin-miniSOG accumulates mostly in endosomes and lysosomes.