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

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

A FRET-based biosensor for measuring G alpha 13 activation in single cells

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PLCbeta isoforms differ in their subcellular location and their CT-domain dependent interaction with Galphaq

Item does not contain fulltextPhospholipase C (PLC) beta isoforms are implicated in various physiological processes and pathologies. However, mechanistic insight into the localization and activation of each of the isoforms is limited. Therefore, it is crucial to gain more in-depth knowledge as to the regulation of the different isoforms. Here we describe the subcellular location of full-length PLCbeta isozymes and their C-terminal (CT) domains. Strikingly, we found isoforms PLCbeta1 and PLCbeta4 to be enriched at the plasma membrane, contrary to isoforms PLCbeta2 and PLCbeta3. We determined that the CT domain is an inhibitor of Gq-mediated increases in intracellular calcium, the potency of its effect being dependent upon the CT domain isoform used. Furthermore, ratiometric fluorescence resonance energy transfer (FRET) imaging was used to study the kinetics of the Galphaq-CTbetax interactions. By the use of recently developed tools, which enable the on-demand activation of Galphaq, we could show that the interaction between constitutively active Galphaq and PLCbeta3 prolongs the residence time of PLCbeta3 at the plasma membrane. These findings suggest that under physiological circumstances, PLCbeta3 and Galphaq interact in a kiss-and-run fashion, likely due to the GTPase-activating activity of PLCbeta towards Galphaq