GFP Localization Research Articles

Membrane-Targeted palGFP Predominantly Localizes to the Plasma Membrane but not to Neurosecretory Vesicle Membranes in Rat Oxytocin Neurons.

Recent advances in viral vector technology, specifically using adeno-associated virus (AAV) vectors, have significantly expanded possibilities in neuronal tracing. We have utilized the Cre/loxP system in combination with AAV techniques in rats to explore the subcellular localization of palmitoylation signal-tagged GFP (palGFP) in oxytocin-producing neurosecretory neurons. A distinctive branching pattern of single axons was observed at the level of the terminals in the posterior pituitary. Despite challenges in detecting palGFP signals by fluorescent microscopy, immunoelectron microscopy demonstrated predominant localization on the plasma membrane, with a minor presence on the neurosecretory vesicle membrane. These findings suggest that membrane-anchored palGFP may undergo exocytosis, translocating from the plasma membrane to the neurosecretory vesicle membrane. In this study, we observed characteristic axon terminal structures in the posterior pituitary of oxytocin neurons. This study indicates the importance of understanding the plasma membrane-specific sorting system in neuronal membrane migration and encourages future studies on the underlying mechanisms.

Notch Signaling Is Required for Extramedullary Hematopoiesis in the Adult Liver

During embryonic development, hematopoietic stem cells (HSC) in the fetal liver undergo expansion and self-renewal. Fetal HSCs migrate to the bone marrow (BM) niche towards birth and remain there during adulthood. Extramedullary hematopoiesis (EMH), or hematopoiesis outside of the BM, predominantly occurs in the fetal liver or when the adult BM niche ceases to be a site for functional hematopoiesis. The mechanism by which the adult liver becomes reactivated as a site of EMH for itinerant HSCs is unknown. Our project is investigating EMH in the adult liver by inducing acute hemolytic anemia in adult mice that induces egress and homing of hematopoietic progenitors to the liver and spleen. Our work demonstrates that long-term HSCs isolated from the portal vein in the adult liver can serially reconstitute lethally irradiated mice. Thus, the adult liver serves as a potential reservoir for long-term, functional HSCs in the presence of a damaged BM niche. In pursuit of a mechanism, we focused on Notch-signaling which is an evolutionarily conserved pathway that has been shown to play a critical role in HSC emergence and fetal liver development. When comparing WT liver donors to Notch-deficient (Notch1 +/ΔTAD) or Notch knockout (Notch1 ffVavCre +) liver donors, hematopoietic transplants reveal that Notch mutant progenitors fail to efficiently reconstitute irradiated recipients. Using an HSC-specific a-catulin-GFPmouse model, we are able to track the expression of GFP + liver-originating HSCs after hematopoietic reconstitution. We show that mice reconstituted by EMH-activated a-catulin-GFP liver HSCs are able to provide long-term reconstitution in lethally irradiated recipients and resume residence in the bone marrow niche of recipients. Additionally, we see distinct localization of GFP + HSCs localized near sinusoids throughout the EMH liver niche using whole-mount imaging. We have validated receptor-ligand binding interactions of the Notch1 receptor with ligand-expressing hematopoietic cells in the adult liver. Using intracellular flow cytometry after EMH in the adult liver, we see a 3-fold increase in mean fluorescent intensity of GATA2 in HSPCs (hematopoietic stem cell progenitors) when compared to our bone marrow and isotype control mice. We demonstrate that Notch-driven GATA2 expression is a crucial regulator of self-renewal and functional maintenance of stemness during EMH of HSCs in the adult liver. Our findings reveal a developmentally conserved Notch signaling mechanism is reactivated in the adult liver to retain the functional potential and maintenance of HSCs as they adapt to inhospitable conditions in the bone marrow niche.

Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0mg/l), NAA (0.2mg/l), TDZ (2.0mg/l) and GA3 (0.1mg/l); spectinomycin 50mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

LGR5-Positive Supporting Cells Survive Ototoxic Trauma in the Adult Mouse Cochlea.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters’ cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

Characterizing temporal and spatial recruitment of systemically administered RPE65-programmed bone marrow-derived cells to the retina in a mouse model of age-related macular degeneration

PurposePreviously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer.MethodsC57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP+ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP+RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP+ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections.ResultsGFP+RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP+ BMDC into the RPE layer and that these GFP+ human RPE65 expressing cells co-localized with murine RPE65. No GFP+ cells were observed in the neural retina or RPE layer of normal uninjured control eyes.ConclusionsOur study shows that systemically administered GFP+RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

α‐Arrestin regulation of protein trafficking: Using evolutionary rate covariation to define protein trafficking regulatory networks

α-Arrestins, a novel class of protein trafficking adaptors, help cells survive environmental changes by controlling membrane protein trafficking. One hurdle to understanding α-arrestins is that few α-arrestin-cargo pairs have been identified. It is technically challenging to identify membrane cargos trafficked by a-arrestins due to their transient associations and their biochemical nature. To identify α-arrestin-regulated cargos, we used Evolutionary Rate Covariation (ERC), which employs sequence-based signatures to identify genes with similar evolutionary histories. We compared ERC values for α-arrestins with proteins across 18 yeast species. Among the top co-evolving proteins were those previously defined as α-arrestin cargos. We are determining if membrane proteins, not yet identified as α-arrestin cargos, with the highest ERC values (>0.5), interact with α-arrestins by assessing their localization and relative protein abundances in wild-type cells versus those lacking the specific α-arrestins. Fluorescence intensity was used to quantify the abundance and/or subcellular distribution of GFP tagged proteins. Statistically significant changes in GFP abundance or localization between wild-type cells and those lacking α-arrestins demonstrate dependence on these protein trafficking adaptors. This makes them good candidates as new α-arrestin-dependent cargos, which we are confirming by co-localization, bimolecular fluorescence complementation, and biochemical approaches. Using ERC and our fluorescence imaging pipeline, we have quantitatively confirmed that 36 integral membrane proteins, previously unassociated with α-arrestins, display α-arrestin-dependent localization changes. This greatly expands the repertoire of α-arrestin cargo within cells, and raises important functional implications for this family. In conclusion, the ERC approach is a powerful new tool that is able to define protein trafficking regulatory networks, which will undoubtedly be of interest to the cell biology community.

An Arabidopsis Oxalyl-CoA Decarboxylase, AtOXC, Is Important for Oxalate Catabolism in Plants.

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism.

Anoxia-induced mitophagy in the yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is a thermotolerant, ethanol-producing yeast that requires oxygen for efficient ethanol fermentation. Under anaerobic conditions, glucose consumption and ethanol production are retarded, suggesting that oxygen affects the metabolic state of K. marxianus. Mitochondria require oxygen to function, and their forms and number vary according to environmental conditions. In this study, the effect of anoxia on mitochondrial behavior in K. marxianus was examined. Under aerobic growth conditions, mitochondria-targeted GFP exhibited a tubular and dotted localization, representing a typical mitochondrial morphology, but under anaerobic conditions, GFP localized in vacuoles, suggesting that mitophagy occurs under anaerobic conditions. To confirm mitophagy induction, the ATG32, ATG8, ATG11 and ATG19 genes were disrupted. Vacuolar localization of mitochondria-targeted GFP under anaerobic conditions was interrupted in the Δatg32 and Δatg8 strains but not the Δatg11 and Δatg19 strains. Electron microscopy revealed mitochondria-like membrane components in the vacuoles of wild-type cells grown under anaerobic conditions. Quantitative analyses using mitochondria-targeted Pho8 demonstrated that mitophagy was induced in K. marxianus by anoxia but not nitrogen starvation. To the best of our knowledge, this is the first demonstration of anoxia-induced mitophagy in yeasts.

Molecular identification and acquisition of interacting partners of a novel wheat F-box/Kelch gene TaFBK

F-box proteins constitute one of the largest protein families and play many important roles in plant. To date, less is known about functions of wheat F-box protein. A Kelch-type F-box gene TaFBK was isolated from wheat TcLr15. TaFBK transcripts were more abundant in pistil and leaf tissues, and accumulated more in the susceptible combination than that in the resistant one. The treatment of SA, MeJA, NaCl and PEG caused a slight up-regulation of TaFBK. GFP localization studies showed that TaFBK accumulated in the nucleus and cytoplasm. Moreover, TaFBK bound TaSkp1 by the F-box domain and recognized TaSLY1 via the Kelch domain.

Antistress and anti-aging activities of Caenorhabditis elegans were enhanced by Momordica saponin extract.

Momordica saponin extract (MSE) was found to not only improve longevity and neuroprotection but also alleviate fat accumulation in Caenorhabditis elegans in our previous study. However, the lipid-lowering activity of MSE alone could not fully explain its ability to improve health, so the antistress effects of MSE were further studied. Using C. elegans as an in vivo animal, the lifespan of MSE-treated C. elegans under various stressors (H2O2, paraquat and heat) and normal conditions was studied. Furthermore, the antioxidant activities of MSE were discussed. To study the underlying mechanisms, the expression of stress resistance genes and the resistance of related mutants to H2O2 stress were tested. MSE significantly improved the lifespan of C. elegans under stress and normal conditions. Meanwhile, the mobility of C. elegans was also improved. Moreover, the activities of SOD and CAT and the ratio of GSH/GSSG were elevated. Consistently, the levels of ROS and lipid oxidation (the NEFA and MDA content) were reduced. Furthermore, MSE treatment upregulated the expression of the sod-3, sod-5, clt-1, clt-2, hsp-16.1 and hsp-16.2 genes. All biomarkers indicated that the antistress and anti-aging activities of MSE were due to its strong antioxidant activities. Finally, MSE induced nuclear DAF-16::GFP localization. Studies with mutants revealed that skn-1 and hsf-1 were involved in the activity of MSE, which might upregulate the expression of downstream stress-responsive genes. Therefore, in addition to its lipid-lowering property, the ability of MSE to improve healthspan was also attributed to the stress resistance effect. Together, MSE might serve as a lead nutraceutical in geriatric research.

Disc-associated proteins mediate the unusual hyperstability of the ventral disc in Giardia lamblia.

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The 'cup-like' disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains - DAP5188KD and DAP6751KD -with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

Study of subcellular localization of Glycine max γ-tocopherol methyl transferase isoforms in N. benthamiana.

Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc-the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of γ-TMT1 and γ-TMT3 were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, Glycine max-γ-TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in Nicotiana benthamiana using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress.

Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter.

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes.

MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Coexpression of MYO19898-970 -GFP with mchr-Miro2 enhanced MYO19898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane-insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin- and microtubule-based mitochondrial activities.

Function of Atg11 in non-selective autophagy and selective autophagy of Candida albicans

Candida albicans is an important opportunistic pathogenic fungus in the human body. It is a common microbe inhabiting on the mucosa surfaces of healthy individuals, but may cause infections when the host immune system is weak. Autophagy is a “self-eating” process in eukaryotes, which can recover and utilize damaged organelles and misfolded proteins. Here we investigated the role of the autophagy-related protein Atg11 in C. albicans. Deletion of ATG11 led to the defect in growth under the nitrogen starvation condition. Western blotting and GFP localization further revealed that the transport and degradation of Atg8 was blocked in the atg11Δ/Δ mutant under both the nitrogen starvation and hypha-inducing conditions. Moreover, degradation of both Lap41 (the indicator of the cytoplasm-to-vacuole pathway) and Csp37 (the indicator of mitophagy) was also thoroughly suppressed in this mutant under nitrogen starvation. These results indicated that Atg11 plays an essential role in both non-selective and selective autophagy in C. albicans.

Identification and Characterization of Protein Phosphatase Methylesterase 1 (Ppme1) in Skeletal Muscle

Skeletal muscle atrophy results from a range of physiological conditions, including immobilization, denervation, glucocorticoid treatment, cancer, and aging. To provide a better understanding of the pathway of skeletal muscle atrophy, a previous study conducted a microarray analysis of muscle from mice undergoing neurogenic atrophy in response to denervation as well as dexamethasone exposure. The data generated from this microarray identified a suite of several hundred predicted genes that displayed differential expression under conditions of muscle wasting, including protein phosphatase methylesterase‐1 (Ppme1). The Ppme1 gene was observed to be transcriptionally upregulated in response to neurogenic skeletal muscle atrophy (i.e. denervation). To confirm that Ppme1 is expressed in muscle, qPCR was performed using RNA isolated from cultured muscle cells at different stages of proliferation and differentiation and expression was found to be relatively constant in both proliferating and differentiated cells. In addition, Ppme1 protein levels were analyzed by Western blot analysis and found to be expressed at a constant level at different time points throughout muscle cell proliferation and differentiation. Additionally, the cDNA of Ppme1 was fused in frame with GFP and subcellular localization was analyzed by confocal fluorescent microscopy in proliferating myoblasts. To assess the transcriptional activity of the Ppme1 gene, fragments of the promoter were cloned, fused to a reporter gene, transfected into C2C12 cells, and found to be highly transcriptionally active. Interestingly, the Ppme1 reporter gene constructs were not significantly affected by ectopic expression of myogenic regulatory facts such as MyoD1 and myogenin. Finally, mouse myoblast cells treated with an inhibitor of Ppme1 displayed reduced AP‐1 reporter activity as well as delayed muscle cell differentiation. The results presented here help contribute new insights into the molecular mechanisms of muscle atrophy and may provide a better understanding of the genome‐wide transcriptional changes and cellular events that occur during skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a novel bipartite nuclear localization signal in the DNA polymerase of the betabaculovirus Pieris rapae granulovirus.

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. Previous results showed that the DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) can localize in the nucleus. However, it is not clear how the DNApol is transported into the nucleus. Bioinformatic and GFP localization analysis showed that PiraGV DNApol contains a nuclear localization signal (NLS) at aa 4-25 (LFKRKLDEPPTDHTLVKAIKLS) of the N-terminus that does not match either the classical monopartite or the bipartite NLS consensus sequence. Multiple-point-substitution analysis confirmed that the NLS is required for transport of PiraGV DNApol into the nucleus. We also substituted the NLS of the PiraGV DNApol for that of the alphabaculovirus Spodoptera litura nuclear polyhedrosis virus (SpltNPV) DNApol. A viral growth curve and quantitative real-time PCR revealed that the substitution impaired viral DNA replication and resulted in a reduction in virus production. Together, our results show that PiraGV contains a novel NLS and that the NLS cannot efficiently replace that of SpltNPV DNApol for viral DNA synthesis and virus production.

Radiation Enhances the Delivery of Antisense Morphalino Oligonucleotides across the Blood-Brain Barrier

We previously demonstrated that non-ablative ionizing radiation enhanced the delivery of intravenously administered unmodified antisense morpholino oligonucleotides (aONs) to subcutaneous tumor sites and results in 50% reduction in the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT), compared to non-radiated subcutaneous tumor sites in the same animal. Delivery of aONs across the blood-brain barrier (BBB) remains a major challenge. Ionizing radiation is well known to alter BBB permeability. We hypothesized that ionizing radiation will facilitate the delivery of IV aONs across the BBB. Long Evans rats received either 5 Gy whole brain radiation, or no radiation (n=6 per group); 24 h later, half of each group received GFP-tagged nonspecific unmodified morpholino aON (10.5 mg/kg IV). Brains were harvested after perfusion with saline 24h after aON administration. GFP localization was assessed in 50 μm sections using a laser-scanning confocal microscope. We observed increased delivery of the morpholino aONs into the brain parenchyma only in the pre-radiated cohort. Rats that received radiation followed by IV GFP-conjugated aONs showed a punctate green fluorescent signal intracellularly in perivascular cells. The punctate staining pattern detected is suggestive that radiation enhanced the endocytosis of aONs and uptake into endosomes and lysosomes. Radiation enhances the targeted delivery of unmodified aONs across the BBB and uptake by perivascular cells. This observation is logical as radiation is known to enhance cellular endocytosis. Morpholino aONs are stable at physiologic pH and can be tailored to silence many key cancer proteins. This approach holds significant translational potential since radiation is already an integral part of brain tumor therapies. Follow-up studies will investigate if radiation enhances the targeted delivery of aONs in a rodent MGMT expressing-brain tumor model to inhibit MGMT expression and function as a chemo-radiation sensitizer.