The RNA polymerase II‐associated factor 1 complex (PAF1C) is a protein complex that consists of LEO1, RTF1, PAF1, CDC73, and CTR9, and has been shown to be involved in RNA polymerase II‐mediated ...transcriptional and chromatin regulation. Although it has been shown to regulate a variety of biological processes, the precise role of the PAF1C during germ line development has not been clarified. In this study, we found that reduction in the function of the PAF1C components, LEO‐1, RTFO‐1, PAFO‐1, CDC‐73, and CTR‐9, in Caenorhabditis elegans affects oogenesis. Defects in oogenesis were also confirmed using an oocyte maturation marker, OMA‐1::GFP. While four to five OMA‐1::GFP‐positive oocytes were observed in wild‐type animals, their numbers were significantly decreased in pafo‐1 mutant and leo‐1(RNAi), pafo‐1(RNAi), and cdc‐73(RNAi) animals. Expression of a functional PAFO‐1::mCherry transgene in the germline significantly rescued the oogenesis‐defective phenotype of the pafo‐1 mutants, suggesting that expression of the PAF1C in germ cells is required for oogenesis. Notably, overexpression of OMA‐1::GFP partially rescued the oogenesis defect in the pafo‐1 mutants. Based on our findings, we propose that the PAF1C promotes oogenesis in a cell‐autonomous manner by positively regulating the expression of genes involved in oocyte maturation.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection ...is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.
Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a ...potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration (O2) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous O2 within the intracellular environments. The mitochondrial O2 at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.
Access of adeno-associated virus (AAV) to ganglion cells following intravitreal injection for gene therapy is impeded by the internal limiting membrane of the retina. As an alternative, one could ...transduce ganglion cells via retrograde transport after virus injection into a retinal target nucleus. It is unknown if recombinant AAV2-retro (rAAV2-retro), a variant of AAV2 developed specifically for retrograde transport, is capable of transducing retinal ganglion cells. To address this issue, equal volumes of rAAV2-retro-hSyn-EGFP and rAAV2-retro-hSyn-mCherry were mixed in a micropipette and injected into the rat superior colliculus. The time-course of viral transduction was tracked by performing serial in vivo fundus imaging. Cells that were labeled by the fluorophores within the first week remained consistent in distribution and relative signal strength on follow-up imaging. Most transduced cells were double-labeled, but some were labeled by only EGFP or mCherry. Fundus images were later aligned with retinal wholemounts. Ganglion cells in the wholemounts matched precisely the cells imaged by fundus photography. As seen in the fundus images, ganglion cells in wholemounts were sometimes labeled by only EGFP or mCherry. Overall, there was detectable label in 32–41% of ganglion cells. Analysis of the number of cells labeled by 0, 1, or 2 fluorophores, based on Poisson statistics, yielded an average of 0.66 virions transducing each ganglion cell. Although this represents a low number relative to the quantity of virus injected into the superior colliculus, the ganglion cells showed sustained and robust fluorescent labeling. In the primate, injection of rAAV2-retro into the lateral geniculate nucleus might provide a viable approach for the transduction of ganglion cells, bypassing the obstacles that have prevented effective gene delivery via intravitreal injection.
•rAAV2-retro vector transduces 32–41% rat retinal ganglion cells retrogradely.•In vivo gene expression in ganglion cells can be monitored by serial fundus imaging.•Vectors encoding different fluorophores label only partially overlapping populations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS ...processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both O2 and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular O2 sensor Myoglobin‐mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and O2. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making O2 sensing available to a wider group of laboratories.
Metmyoglobin and oxygen detection using fluorescence lifetime imaging of EYFP‐Myoglobin‐mCherry
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Perfectly defined, monodisperse fusion protein block copolymers of a thermoresponsive coil‐like protein, ELP, and a globular protein, mCherry, are demonstrated to act as fully biosynthetic analogues ...to protein‐polymer conjugates that can self‐assemble into biofunctional nanostructures such as hexagonal and lamellar phases in concentrated solutions. The phase behavior of two mCherry‐ELP fusions, E10‐mCherry‐E10 and E20‐mCherry, is investigated to compare linear and bola fusion self‐assembly both in diluted and concentrated aqueous solution. In dilute solution, the molecular topology impacts the stability of micelles formed above the thermal transition temperature of the ELP block, with the diblock forming micelles and the bola forming unstable aggregates. Despite the chemical similarity of the two protein blocks, the materials order into block copolymer‐like nanostructures across a wide range of concentrations at 30 wt% and above, with the bola fusion having a lower order‐disorder transition concentration than the diblock fusion. The topology of the molecule has a large impact on the type of nanostructure formed, with the two fusions forming phases in the opposite order as a function of temperature and concentration. This new system provides a rich landscape to explore the capabilities of fusion architecture to control supramolecular assemblies for bioactive materials.
Self‐assembly of fusion proteins containing a folded globular protein into solid nanomaterials is demonstrated for the first time. Using model molecules containing ELP and mCherry, the ability to tune nanostructure is demonstrated through control over the fusion topology. As fully biosynthetic analogues to protein‐polymer conjugates, the mCherry‐ELP fusions provide an efficient route to introduce complex biological functionality into supramolecular assemblies.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that ...remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector‐induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di‐mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD‐actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq‐mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.
We examined the ultrastructural localization of the DREADDs hM4Di and hM3Dq fused to different tags in monkey and mouse neurons. Poor plasma membrane expression was observed with hM4Di‐mCherry in monkeys. In contrast, strong plasma membrane localization was found with HA‐hM4Di in mice and monkeys, hM4Di‐mCherry in mice and hM3Dq‐mCherry in monkeys. Our results suggest that ultrastructural expression of DREADDs depends on the species, and the DREADD/tag combination.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
•G. diazotrophicus mCherry-tagged colonizing elephant grass.•The strain LP343 quickly attached itself to the roots.•The xylem vessels of PCEA were colonized by the strain LP343.
Gluconacetobacter ...diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In the central nervous system, the A6 noradrenaline (NA) and the B3 serotonin (5-HT) cell groups are well-recognized players in the descending antinociceptive system, while other NA/5-HT cell groups ...are not well characterized. A5/A7 NA and B2 5-HT cells project to the spinal horn and form descending pathways. We recorded G-CaMP6 green fluorescence signal intensities in the A5/A7 NA and the B2 5-HT cell groups of awake mice in response to acute tail pinch stimuli, acute heat stimuli, and in the context of a non-noxious control test, using fiber photometry with a calcium imaging system. We first introduced G-CaMP6 in the A5/A7 NA or B2 5-HT neuronal soma, using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamine β-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus (AAV-TetO(3G)-G-CaMP6). After confirming the specific expression patterns of G-CaMP6, we recorded G-CaMP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli. G-CaMP6 fluorescence intensity in the A5, A7, and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after, it returned to baseline fluorescence intensity. This was not observed in the non-noxious control test. The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B2 5-HT neurons but the non-noxious stimuli do not. The present study suggests that A5/A7 NA or B2 5-HT neurons play important roles in nociceptive processing in the central nervous system. We suggest that A5/A7/B2 neurons may be new therapeutic targets. All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University (MD17105) on February 22, 2018.
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