DNA template strand segregation in developing zebrafish.

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light sheet microscopy to track embryonic DNA in whole zebrafish. Larval development results in rapid depletion of older DNA template strands from stem cell niches in the retina, brain, and intestine.

A Bioorthogonal Chemical Reporter of Viral Infection.

Pathogen-selective labeling was achieved by using the novel gemcitabine metabolite analogue 2'-deoxy-2',2'-difluoro-5-ethynyluridine (dF-EdU) and click chemistry. Cells infected with Herpes Simplex Virus-1 (HSV-1), but not uninfected cells, exhibit nuclear staining upon the addition of dF-EdU and a fluorescent azide. The incorporation of the dF-EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK).

A Bioorthogonal Chemical Reporter of Viral Infection.

Pathogen-selective labeling was achieved by using the novel gemcitabine metabolite analogue 2'-deoxy-2',2'-difluoro-5-ethynyluridine (dF-EdU) and click chemistry. Cells infected with Herpes Simplex Virus-1 (HSV-1), but not uninfected cells, exhibit nuclear staining upon the addition of dF-EdU and a fluorescent azide. The incorporation of the dF-EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK).

An azide-modified nucleoside for metabolic labeling of DNA.

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) "click" reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5-azido-2'-deoxyuridine (AdU) exhibits a 4 h half-life in water, and it gives little or no detectable labeling of cellular DNA.

A SCARECROW-RETINOBLASTOMA protein network controls protective quiescence in the Arabidopsis root stem cell organizer.

Quiescent long-term somatic stem cells reside in plant and animal stem cell niches. Within the Arabidopsis root stem cell population, the Quiescent Centre (QC), which contains slowly dividing cells, maintains surrounding short-term stem cells and may act as a long-term reservoir for stem cells. The RETINOBLASTOMA-RELATED (RBR) protein cell-autonomously reinforces mitotic quiescence in the QC.

Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides.

Commonly used metabolic labels for DNA, including 5-ethynyl-2'-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosyl-ethynyluracil derivatives.

Imaging lipids in living cells.

The investigation of lipids in living cells is one of the underdeveloped areas in cell biology. Although it is possible to analyze the global lipid composition of a cell type, fractionation of the various types of membranes from cells is extraordinarily difficult, mainly because most membranes appear to be in contact with each other. Therefore, we know the lipid components, but we have a difficult time finding out their exact position, how dynamically they change location, and how rapidly they are metabolized.

Labeling lipids for imaging in live cells.

Fluorescently tagged lipid-binding domains have become a popular tool to image lipids that are involved in intracellular signaling processes. The readout usually involves the translocation of the lipid-binding domain from the cytosol or nucleosol to the membrane of interest, or vice versa. Unfortunately, this method seems to work predominantly for lipids in the plasma membrane, whereas lipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) are not recognized in the membranes of the endoplasmic reticulum or the Golgi.

Labeling lipids for imaging in fixed cells.

Fluorescently tagged lipid-binding domains have become a popular tool to image lipids that are involved in intracellular signaling processes. The readout usually involves the translocation of the lipid-binding domain from the cytosol or nucleosol to the membrane of interest, or vice versa. Unfortunately, this method seems to work predominantly for lipids in the plasma membrane, whereas lipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) are not recognized in the membranes of the endoplasmic reticulum or the Golgi.

Transfection of cells with DNA encoding a visible fluorescent protein-tagged lipid-binding domain.

Visualization of a certain lipid species can be achieved by producing a fusion protein between a lipid-binding domain and a visible fluorescent protein (VFP). After a DNA construct for a VFP-tagged lipid-binding domain has been prepared, the desired cell line is transfected with the DNA and visualized using fluorescence microscopy, as described here. The DNA encoding a VFP-tagged lipid-binding domain is isolated from Escherichia coli, and the cells to be transfected are grown on glass-bottom dishes or on coverslips.

Selective fluorescence labeling of lipids in living cells.

Click chemistry in vivo: Three phosphatidic acid derivatives with alkyne groups in their fatty acid chains were synthesized and incorporated into mammalian cell membranes. Copper(I)-catalyzed and strain-promoted azide-alkyne cycloaddition reactions were used for their visualization (see schematic representation and fluorescence microscopic image).