A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010  GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia.

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.

Mislocalization of Cancer-associated Thyroid Hormone Receptor Mutants.

The thyroid hormone receptor (TR) is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone. Nuclear localization signals (NLSs) and nuclear export signals (NESs) allow for TR transport into and out of the nucleus, respectively. Previous research suggests that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases. Here, we examined the impact of cancer-associated mutations on TR localization patterns as a way of analyzing key structural components of TR and to further explore the correlation between TR trafficking, misfolding, and disease. Through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP) and mCherry-tagged TRα1 and quantitative fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer, and are associated with NLSs and NESs of TRα1. We also investigated structural alterations of the mutants by in silico modeling. Our results show striking shifts towards a more cytoplasmic localization for many of the mutants and an increased tendency to form cytosolic and nuclear aggregates.

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 play a role in the development of fatty liver in inbred animals. The ability to perform similar studies using human tissue has been limited by the genetic variability in man. We generated human induced pluripotent stem cells (iPSCs) with controllable expression of SIRT1. By differentiating edited iPSCs into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1 knockdown iPSC-derived hepatocytes and found that the human iPSC-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype, and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically edited human liver tissue may become an important tool for investigating human liver biology and disease.

Dynamic modelling of an ACADS genotype in fatty acid oxidation - Application of cellular models for the analysis of common genetic variants.

BACKGROUND: Genome-wide association studies of common diseases or metabolite quantitative traits often identify common variants of small effect size, which may contribute to phenotypes by modulation of gene expression. Thus, there is growing demand for cellular models enabling to assess the impact of gene regulatory variants with moderate effects on gene expression. Mitochondrial fatty acid oxidation is an important energy metabolism pathway. Common noncoding acyl-CoA dehydrogenase short chain (ACADS) gene variants are associated with plasma C4-acylcarnitine levels and allele-specific modulation of ACADS expression may contribute to the observed phenotype. METHODS AND FINDINGS: We assessed ACADS expression and intracellular acylcarnitine levels in human lymphoblastoid cell lines (LCL) genotyped for a common ACADS variant associated with plasma C4-acylcarnitine and found a significant genotype-dependent decrease of ACADS mRNA and protein. Next, we modelled gradual decrease of ACADS expression using a tetracycline-regulated shRNA-knockdown of ACADS in Huh7 hepatocytes, a cell line with high fatty acid oxidation-(FAO)-capacity. Assessing acylcarnitine flux in both models, we found increased C4-acylcarnitine levels with decreased ACADS expression levels. Moreover, assessing time-dependent changes of acylcarnitine levels in shRNA-hepatocytes with altered ACADS expression levels revealed an unexpected effect on long- and medium-chain fatty acid intermediates. CONCLUSIONS: Both, genotyped LCL and regulated shRNA-knockdown are valuable tools to model moderate, gradual gene-regulatory effects of common variants on cellular phenotypes. Decreasing ACADS expression levels modulate short and surprisingly also long/medium chain acylcarnitines, and may contribute to increased plasma acylcarnitine levels.

Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation.

Cost-effective, expedited approaches for bone regeneration are urgently needed in an ageing population. Bone Morphogenetic Proteins (BMPs) stimulate osteogenesis but their efficacy is impeded by their short half-life. Delivery by genetically modified cells can overcome this problem. However, cell isolation and propagation represent significant obstacles for the translation into the clinic. Instead, complete gene activated fragments of adipose tissue hold great potential for bone repair. Here, using an in-vitro culture system, we investigated whether adenoviral transduction with human BMP-2 can promote osteogenic differentiation within adipose tissue fragments. Osteoinduction in adipose tissue fragments was evaluated by quantitative reverse transcriptase polymerase chain reaction, immunohistology and histomorphometry. BMP-2 transduced adipose tissue synthesized BMP-2 protein over 30 days peaking by day six, which significantly promoted osteogenic differentiation as indicated by increased calcium depositions, up-regulation of bone marker genes, and bone-related protein expression. Our results demonstrate that cells within adipose tissue fragments can differentiate osteogenically after BMP-2 transduction of cells on the surface of the adipose tissue. BMP-2 gene activated adipose tissue represents an advanced osteo-regenerative biomaterial that can actively contribute to osteogenesis and potentially enable the development of a novel, cost-effective, one-step surgical approach to bone repair without the need for cell isolation.

Osteoinduction within BMP-2 transduced muscle tissue fragments with and without a fascia layer: implications for bone tissue engineering.

Bone can be engineered in vivo by implantation of gene-activated muscle tissue fragments. This expedited approach may be further improved by use of muscle tissue with attached fascia. The aim of this in vitro study was to provide an in depth comparison of the osteogenic differentiation capacity of muscle alone and muscle with fascia after BMP-2 transduction. Skeletal muscle tissue from rats was cut into pieces with and without a fascia layer on the surface. Adenoviral BMP-2 or GFP vectors were used for transduction. Osteogenic differentiation within the tissue fragments was evaluated and compared by qRT-PCR, alizarin red S staining, histomorphometry and immunohistology. Transduction efficiency and level of transgene expression were higher for muscle with fascia than muscle alone. Transduction with BMP-2 led to a significant upregulation of bone marker genes, proteins, and calcium deposition in both groups. Interestingly, histological evaluation revealed that osteoinduction did not occur within the fascia layer itself. The upregulation of bone marker genes in muscle with fascia was significantly lower after 2 weeks but similar after 4 weeks of in vitro culture in comparison to muscle alone. The fascia layer led to higher transduction efficiency and enhanced BMP-2 expression. Despite fascia's lower capacity for osteogenic differentiation, muscle implants may benefit from the fascia layer by the improved ability to deliver BMP-2. The presented data may contribute to the development of a novel, cost-effective, single-surgery bone engineering technology and encourage the evaluation of the osteoregenerative potential of muscle with fascia in an animal model.

Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques.

The human adenovirus type 19a/64 (hAd19a) is a rare serotype in the human population that transduces human dendritic cells (DCs) and human muscle cells more efficiently than the well-characterized human adenovirus type 5 (hAd5). To further characterize the potential of this vector as a vaccine we designed replication deficient hAd19a, hAd5 and MVA vectors expressing a papillomavirus (PV) antigen fused to the human MHC class II associated invariant chain T cell adjuvant (hIi) and investigated their immunogenicity in vivo in mice and cynomolgus macaques. We initially showed that the hIi encoded in the hAd5 enhanced PV specific CD8+ T cell responses in mice. The T cell responses induced after hAd19a vaccination was similar to those induced by hAd5 vaccination. The hAd19a induced responses were not reduced in presence of preexisting Ad5 immunity in mice. In macaques both vaccines were equally potent at inducing CD8+ T cells after MVA boost, while the level of CD4+ T cell responses were found to be broader in hAd19a primed animals. These data demonstrate the potential of hAd19a as an alternative vector to hAd5 to elicit potent T cell responses to PV.

Gene-activated tissue grafts for sustained bone morphogenetic protein-2 delivery and bone engineering: Is muscle with fascia superior to muscle and fat?

Previously, we have presented an expedited strategy for sustained delivery of bone morphogenetic protein-2 (BMP-2) to bone lesions based on the implantation of gene-activated fat and muscle fragments. The aim of the present in vitro experiments was to evaluate the potential of muscle with fascia as a BMP-2 delivering osteo-regenerative implant in comparison to fat tissue and muscle alone. Subcutaneous fat, muscle, and muscle with fascia were harvested from Fischer 344 rats. The tissues were cut into small pieces and cultured for up to 90 days after direct transduction with adenoviral BMP-2 or green fluorescence protein vectors. Different vector doses were applied, and proliferation, long-term BMP-2 production, and osteogenic differentiation of the 3 different tissues were investigated in vitro. Muscle with fascia produced the largest amounts of BMP-2. Expression of the transgene was detected for up to 90 days. Proliferation was reduced with increased vector doses. Muscle with fascia showed a higher potential for osteogenic differentiation than fat, but it was not improved as compared to muscle alone. A dose of 4 × 108 plaque forming units of the adenoviral BMP-2 vector appeared to be the optimal dose for transduction of muscle with fascia. Because muscle with fascia produced higher amounts of BMP-2 as compared to muscle alone or fat tissue grafts, showing a high potential for osteogenic differentiation, it might represent an improved osteo-regenerative implant facilitating endogenous repair. Future studies should investigate the effect of muscle with fascia transduced with 4 × 108 plaque forming units on bone healing in vivo.

BMP-2 gene activated muscle tissue fragments for osteochondral defect regeneration in the rabbit knee.

BACKGROUND: Previously published data indicate that BMP-2 gene activated muscle tissue grafts can repair large bone defects in rats. This innovative abbreviated ex vivo gene therapy is appealing because it does not require elaborative and time-consuming extraction and expansion of cells. Hence, in the present study, we evaluated the potential of this expedited tissue engineering approach for regenerating osteochondral defects in rabbits. METHODS: Autologous muscle tissue grafts from female White New Zealand rabbits were directly transduced with an adenoviral BMP-2 vector or remained unmodified. Osteochondral defects in the medial condyle of rabbit knees were treated with either BMP-2 activated muscle tissue implants or unmodified muscle tissue or remained empty. After 13 weeks, repair of osteochondral defects was examined by biomechanical indentation testing and by histology/imunohistochemistry applying an extended O'Driscoll scoring system and histomorphometry. RESULTS: Biomechanical investigations revealed a trend towards slightly improved mechanical properties of the group receiving BMP-2 activated muscle tissue compared to unmodified muscle treatment and empty defect controls. However, a statistically significant difference was noted only between BMP-2 muscle and unmodified muscle treatment. Also, histological evaluation resulted in slightly higher histological scores and improved collagen I/II ratio without statistical significance in the BMP-2 treatment group. Histomorphometry indicated enhanced repair of subchondral bone after treatment with BMP-2 muscle, with a significantly larger bone area compared to untreated defects. CONCLUSIONS: Gene activated muscle tissue grafts showed potential for osteochondral defect repair. There is room for improvement via the use of appropriate growth factor combinations.

An expedited approach for sustained delivery of bone morphogenetic protein-7 to bone defects using gene activated fragments of subcutaneous fat.

BACKGROUND: Delivery of bone morphogenetic protein-7 (BMP-7) to bone defects can be improved by applying gene transfer methods. However, traditional ex vivo gene therapy approaches are cumbersome and costly, requiring the extraction and culturing of cells. Therefore, we evaluated a novel, expedited ex vivo BMP-7 gene transfer technology based on the use of fragments of subcutaneous fat tissue. METHODS: We created 5-mm mid-femoral bone defects in the right femora of 23 male, syngeneic Fischer 344 rats. Adipose tissue was harvested from the subcutaneous fat depot of two donor rats. Bone defects were treated with either unmodified fat (control group) or adenovirally BMP-7 transduced fat fragments (treatment group). Healing of bone defects was assessed by radiographs, microcomputed tomography (µCT) and histology at 6 weeks after the implantation of fat tissue fragments. RESULTS: Radiographs, µCT-imaging and histology revealed relevant bone formation in six out of 10 rats treated with BMP-7 activated fat grafts. Two of the defects were bridged. By contrast, femora of the control group receiving unmodified fat did not display signs of osseous healing. BMP-7 gene activated fat treatment led to a significantly higher bone volume (11.18 ± 9.48 mm(3) ) than treatment with unmodified fat grafts (3.19 ± 1.68 mm(3) ) (p = 0.008). CONCLUSIONS: Implantation of BMP-7 gene activated fat tissue fragments can elicit regeneration of large bone defects in rats and could become a clinically expeditious strategy for in vivo bone tissue engineering. However, gene expression must be improved in order to reliably induce osseous bridging of critical-size bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Gene-activated fat grafts for the repair of spinal cord injury: a pilot study.

BACKGROUND: Spinal cord injury (SCI) is a complex disease requiring a concerted multi-target approach. The most appropriate combination of therapeutic gene, cellular vehicle, and space filling scaffold still has to be determined. We present an approach that employs syngeneic adipose tissue serving as a three-dimensional biological implant, source of progenitor cells, and delivery system for therapeutic genes. In this pilot experiment, we evaluated the feasibility and short-term effects using gene-activated autologous fat grafts after SCI. METHODS: An experimental SCI model was established in syngeneic Fischer 344 rats by a T9-T10 hemimyelonectomy. Fat tissue was harvested from two donor rats. Animals were divided into four groups and treated with either (i) fat grafts activated by an adenoviral vector carrying the human NT-3 cDNA, (ii) or BDNF, (iii) or with untreated fat grafts or (iv) remained untreated. Animals were euthanized either 7 or 21 days after surgery, and spinal cord tissue was investigated by histological and immunohistochemical methods. RESULTS: NT-3 and BDNF were produced by gene-activated fat grafts for at least 21 days in vitro and in vivo. Fat tissue grafts remained stable at the site of implantation at 7 days and at 21 days. Neither BDNF-activated nor NT-3-activated fat graft had a detectable limiting effect on the neuronal degeneration. BDNF recruited microglia to perilesional site and attenuated their inflammatory response. CONCLUSIONS: Gene-activated syngeneic fat tissue serves as a three-dimensional biological material delivering therapeutic molecules to the site of SCI over an extended period of time. The BDNF-fat graft attenuated the inflammatory response. Whether these findings translate into functional recovery will require extended observation times.

The effect of BMP-7 gene activated muscle tissue implants on the repair of large segmental bone defects.

BACKGROUND: This study was conducted in order to investigate the effect of Bone Morphogenetic Protein-7 (BMP-7) transduced muscle cells on bone formation and to further develop an innovative abbreviated ex vivo gene therapy for bone repair. As conventional ex vivo gene therapy methods require an elaborative and time-consuming extraction and expansion of cells we evaluated an expedited approach. Fragments of muscle tissue were directly activated by BMP-7 cDNA and implanted into bone defects. METHODS: 25 male, syngeneic Fischer 344 rats were used in the present study. Muscle tissue was harvested from two donor rats and either transduced with an adenovirus carrying the BMP-7 cDNA or remained unmodified. 5mm osseous defects in the right femora of 23 rats were treated with either unmodified muscle tissue (control group) or BMP-7 activated muscle tissue (treatment group). Six weeks after surgery, rat femora were evaluated by radiographs, micro-computed tomography (µCT) and histology. RESULTS: Implantation of BMP-7 activated muscle grafts led to bony bridging in 5 out of 12 defects (41.7%) and to bone formation without bridging in 2 out of 12 defects. In 2 femoral defects of this group radiographs, µCT-imaging and histology did not reveal significant mineralization. Three animals of the BMP-7 treatment group had to be euthanized due to serious wound infection. The bone volume of the treatment group was significantly (p=0.007) higher compared to the control group. CONCLUSION: This study shows that BMP-7 gene activated muscle fragments have the potential to regenerate critical-size segmental bone defects in rats. However, further development of this promising expedited treatment modality is required to improve the healing rate and to investigate if the high infection rate is related to treatment with BMP-7 activated muscle grafts.

A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment.

BACKGROUND: Radiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance. METHODS: We have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation. RESULTS: Of ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds. CONCLUSIONS: For the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.

Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control.

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.

Chromophore exchange in the LOV2 domain of the plant photoreceptor phototropin1 from oat.

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.

Autophosphorylation, electrophoretic mobility and immunoreaction of oat phototropin 1 under UV and blue Light.

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

Dimerization of the plant photoreceptor phototropin is probably mediated by the LOV1 domain.

Phototropin is a membrane-bound UV-A/blue light photoreceptor of plants responsible for phototropism, chloroplast migration and stomatal opening. Characteristic are two LOV domains, each binding one flavin mononucleotide, in the N-terminal half and having a serine/threonine kinase domain in the C-terminal half of the molecule. We purified the N-terminal half of oat phototropin 1, containing LOV1 and LOV2 domains, as a soluble fusion protein with the calmodulin binding peptide (CBP) by expression in Escherichia coli. Gel chromatography showed that it was dimeric in solution. While the fusion protein CBP-LOV2 was exclusively monomeric in solution, the fusion protein CBP-LOV1 occurred as monomer and dimer. The proportion of dimer increased on prolonged incubation. We conclude that native phototropin is a dimer and that the LOV1 domain is probably responsible for dimerization.

Tissue-specific and subcellular localization of phototropin determined by immuno-blotting.

Phototropin (phot) is a UV/blue- light receptor mediating phototropic reactions of plants as a response to unilateral irradiation. Using an antiserum directed against the N-terminal part of Arabidopsis phot1, we show here cross-reaction with phototropin from Avena sativa, Eruca sativa, Glycine max, Lepidium sativum, Lycopersicon esculentum, Pisum sativum, Sinapis alba, and Zea mays. In all investigated plants, blue light irradiation led to a gel mobility shift of phototropin corresponding to an apparent increase in size of 2-3 kDa. This increase is transient: the apparent size of the phototropin band reverted back to the original size in the dark within 60-90 min. The capacity for in vitro phosphorylation increased to 350% ( A. sativa) and 200% ( L. sativum) at 90 min after a blue light pulse without an increase in the amount of phototropin protein. Starting from coleoptile tips of monocots that contained the highest concentration of phototropin, we found an exponential decrease in basipetal sections of equal size while a linear decrease was determined for dicots in basipetal sections starting from the section below the hypocotyl hook. We confirmed the membrane association of all phototropin in dark-grown seedlings; after a 2-min blue light pulse, however, 20% of phototropin was found in the cytosolic fraction and only 80% in the membrane fraction. Both fractions showed the gel mobility shift indicating light-dependent autophosphorylation. Detergent-free solubilization of phototropin with chaotropic reagents was investigated with etiolated A. sativa seedlings. Up to 95% of phototropin was solubilized with a mixture of sodium bromide and sodium diphosphate, and subsequently subjected to affinity purification using Cibachron Blue 3GA-agarose as a dinucleotide analogue. Immediately after solubilization, soluble phototropin still showed blue-light-dependent autophosphorylation but lost its activity within less than 1 h.