Acute manipulation of phosphoinositide levels in cells.
Phosphoinositides are membrane-bound signaling phospholipids that function in a myriad of cellular processes, including membrane trafficking, cytoskeletal dynamics, ion channel and transporter function, and signal transduction. In order to better understand the role of phosphoinositides in cellular processes, different approaches to study the effects of the presence or absence of these lipids must be devised. Conventional approaches of manipulating phosphoinositide levels such as over-expression or genetic ablation of lipid enzymes cause prolonged exposure of the cells to changes in lipid levels that could result in compensatory actions by the cell or downstream alterations in cell physiology. In this chapter we present an approach used recently by various laboratories, including our own, to acutely manipulate phosphoinositide levels at target locations using chemically induced dimerization (CID) that can be spatially and temporally controlled. We discuss considerations when designing expression constructs for targeting specific cellular compartment membranes and present examples from the literature on different ways of perturbing phosphoinositide levels at particular organelle membranes using CID. In addition, we provide details on image acquisition, data collection, and data interpretation. CID technology can be applied to many lipid enzymes to broaden the understanding of the role lipid signaling plays in cell physiology.
Department of Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, USA.
Belle
B
Chang-Ileto
Samuel G
SG
Frere
Gilbert
G
Di Paolo
D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D015415:Biomarkers / Q000737:chemistry; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments; D010716:Phosphatidylinositols / Q000737:chemistry*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D059010:Single-Cell Analysis; D013194:Staining and Labeling; D059008:Time-Lapse Imaging
D015415:Biomarkers; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C439615:EIN protein, E coli