11 research outputs found
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. RBR interacts with the stem cell transcription factor SCARECROW (SCR) through an LxCxE motif. Disruption of this interaction by point mutation in SCR or RBR promotes asymmetric divisions in the QC that renew short-term stem cells. Analysis of the in vivo role of quiescence in the root stem cell niche reveals that slow cycling within the QC is not needed for structural integrity of the niche but allows the growing root to cope with DNA damag
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 (PIP2) are not recognized in the membranes of the endoplasmic reticulum or the Golgi. Very recently, we developed an alternative way of localizing a lipid of interest by fluorescent labeling of minimally modified lipid derivatives using a single specific chemical reaction. For lipid location analyses, the method is used in fixed cells. However, for studying lipid dynamics, specific labeling in living cells is also possible. This protocol describes how to directly label lipids for imaging in living cells
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. Imaging lipids in cells seems to be the obvious solution to the problem. The most common way to image molecules is by the artificial addition of a fluorescent tag. The use of fluorescent proteins has become the mainstay of protein imaging, but this method is, of course, not suitable for small molecules such as lipids. Unfortunately, the fluorescent tag is usually as large as the lipid and is therefore likely to have a severe influence on lipid location and metabolism. To circumvent this problem, two solutions have been developed--namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell. This article describes procedures necessary to image lipids by fluorescently tagged lipid-binding domains and by labeling lipid derivatives in fixed and living cells
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. We routinely perform transfection with commercially available reagents, although, depending on the cell line, the calcium phosphate precipitation method may provide an economic alternative
Labeling lipids for imaging 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 (PIP2) are not recognized in the membranes of the endoplasmic reticulum or the Golgi. Very recently, we developed an alternative way of localizing a lipid of interest by fluorescent labeling of minimally modified lipid derivatives using a single specific chemical reaction. This protocol describes how to directly label lipids in fixed cells for lipid location analyses