To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding linear HA epitope with high affinity and specificity vivo. The resulting probe, called frankenbody, can light up multiple colors HA-tagged nuclear, cytoplasmic, membrane, mitochondrial proteins diverse cell types. frankenbody also enables state-of-the-art single-molecule experiments which demonstrate by tracking single histones U2OS cells mRNA translation dynamics both neurons....

ABSTRACT Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique monitor the changes in histone levels zygotic genome activation (ZGA) living zebrafish embryos. Among various modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating two nuclear foci 64- 1k-cell-stage The elongating form of RNA...

Abstract The localization of transcriptional activity in specialized transcription bodies is a hallmark gene expression eukaryotic cells. It remains unclear, however, if and how affect expression. Here we disrupted the formation two prominent endogenous that mark onset zygotic zebrafish embryos analysed effect on using enriched SLAM-seq live-cell imaging. We find disruption results misregulation hundreds genes. focus genes are upregulated. These have accessible chromatin poised to be...

The Xenopus oocyte is known to accumulate filamentous or F‐actin in the nucleus, but it currently unknown whether also accumulates embryo nuclei. Using fluorescence‐labeled actin reporters, we examined distribution embryonic cells and found that nuclei during blastula stage not gastrula stage. To further investigate nuclear F‐actin, devised a egg extract reproduces formation of which accumulates. this extract, primarily at subnuclear membranous region essential maintain chromatin binding...

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including migration, stress fiber formation, and cytokinesis. Recent studies have shown that filaments form nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated dynamics nuclear zebrafish (Danio rerio) embryos. early embryos up to around high stage, UtrCH-sfGFP increasingly accumulated nuclei...

14C wiggle-matching was applied to two wood samples closely related geological and archaeological events with associated dendrochronological dates, demonstrate the accuracy of dating accelerator mass spectrometry (AMS). Wiggle-matching on charred bark, excavated from a pyroclastic mud-flow deposited by huge 10th Century eruption Baitoushan Volcano, revealed age as cal A.D. 935 8/−5 95% confidence. This date is consistent 912 972 estimated dendrochronology wooden boards that had clear...

Transcription factor (TF) clusters have been suggested to facilitate transcription. The mechanisms driving the formation of TF and their impact on transcription, however, remain largely unclear. This is mostly due lack a tractable system. Here, we exploit transcriptional activation mir430 in zebrafish embryos simultaneously follow dynamic large Nanog cluster, underlying DNA, transcription output by live imaging at high temporal spatial resolution. We find that cluster can support requires...

Whether or not nutritionally-regulated foreign gene expression in vivo is achievable was examined mouse liver after transfer by electroporation (EP). Electric pulses were applied to a left lobe immediately injection of luciferase reporter driven the liver-type phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cooling treatments especially with solid carbon dioxide transfection site prior EP increased factor 100. Body bioluminescence imaging also confirmed strong transferred transfected...

Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which a noncollagenous protein found in bone matrix involved mineralization calcium ion homeostasis, one of the major morphogenetic markers used evaluation osteoblast maturation osteogenic activation. We established transgenic mouse line expressing luciferase under control 10-kb osteocalcin enhancer/promoter sequence. Using these mice, we evaluated active forms vitamins D2 D3 their function by...

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped microviscosity. However, involvement charge-separated species fluorescence process potentially limits quantitative evaluation viscosity. Herein, we developed viscosity-responsive for cell imaging that not dependent on...

ABSTRACT To expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds classic linear HA epitope with high affinity and specificity vivo . The resulting probe, which call frankenbody, is capable lighting up multiple colors HA-tagged nuclear, cytoplasmic, membrane proteins diverse cell types. frankenbody also enables state-of-the-art single-molecule experiments, demonstrate by tracking mRNA translation dynamics U2OS cells neurons....

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped microviscosity. However, involvement charge-separated species fluorescence process potentially limits quantitative evaluation viscosity. Herein we developed viscosity-responsive for cell imaging that not dependent on...

Abstract The cell nucleus is a dynamic structure repeating disassembly and reformation during mitosis. Reformation of the essential for proliferation, therefore, factors required nuclear are fundamental eukaryotes. Although various have been identified in vitro systems using frog egg extracts vivo imaging somatic cells, little known about formation functional structures living mouse eggs. To identify such factors, we used reconstruction approach to construct an artificial around DNA T4 phage...

Abstract In eukaryotes, DNA is housed within the cell nucleus. Molecules required for formation of a nucleus have been identified using in vitro systems with frog egg extracts and vivo imaging somatic cells. However, little known about physicochemical factors conditions nuclear mouse oocytes. this study, reconstitution approach purified DNA, we aimed to determine factors, such as amount timing introduction, nuclei transport activity T4 phage (~166 kbp) was microinjected into...

ABSTRACT The localization of transcriptional activity in specialized transcription bodies is a hallmark gene expression eukaryotic cells. How proteins the machinery come together to form such bodies, however, unclear. Here, we take advantage two large, isolated, and long-lived that reproducibly during early zebrafish embryogenesis, characterize dynamics body formation. Once formed, these are enriched for initiating elongating RNA polymerase II, as well factors Nanog Sox19b. Analyzing events...

Abstract Histone posttranslational modifications (PTMs) are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We describe an approach that images fluorescent antibody fragments quantify PTM enrichment and active transcription at defined loci zygotic genome activation in living zebrafish embryos. The technique reveals a drastic increase histone H3 Lys27 acetylation (H3K27ac) before both specific locus globally.