Manipulation of plasma membrane phosphoinositides using photoinduced protein-protein interactions.
Phosphoinositides, the phosphorylated products of inositol phospholipids, play critical regulatory roles in cell physiology. The elucidation of their functions will greatly benefit from the methodology to manipulate their local concentrations within membranes with high spatial and temporal precision. Recently developed genetically encoded and light-regulated dimerization modules, in combination with the use of fluorescence-tagged lipid-binding domains and live-cell imaging, provide an attractive means to achieve this goal. Here we describe a protocol for blue light-dependent conversion of one phosphoinositide species into another based on the light-regulated dimerization between cryptochrome 2 (CRY2) and its ligand, CIB1. We describe the development of these tools using the dephosphorylation of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) as an example and show how they can be used to rapidly and reversibly deplete the plasma membrane of this lipid. We also provide instructions for image analysis. The CRY2-CIB1 dimerization method has also already been adapted for the acute and spatially restricted generation of PI(3,4,5)P3 in the plasma membrane. More generally, this methodology should be broadly applicable to studies of the spatiotemporal regulation of membrane lipid metabolism in many types of cells.
Department of Cell Biology, Program in Cellular Neuroscience, Neurodegeneration and Repair, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510, USA.
Olof
O
Idevall-Hagren
Pietro
P
Decamilli
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019556:COS Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism / Q000528:radiation effects*; D002522:Chlorocebus aethiops; D056931:Cryptochromes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008027:Light; D051379:Mice; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D055668:Photochemical Processes; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D029681:Arabidopsis Proteins; C103618:CRY2 protein, Arabidopsis; D056931:Cryptochromes; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; C075695:OCRL protein, human