Visualizing cellular phosphoinositide pools with GFP-fused protein-modules.
Inositol phospholipids are well known for their pivotal role in calcium signaling as precursors of important second messengers generated in response to various stimuli. However, over the last 10 years, inositides have also emerged as universal signaling components present in virtually every membrane of eukaryotic cells. These lipids are locally produced and degraded by the numerous inositide kinase and phosphatase enzymes, to control the recruitment and activity of protein signaling complexes in specific membrane compartments. The spatial and temporal constraints imposed on changes in cellular inositides pose new challenges in finding experimental techniques through which such changes can be examined. Taking advantage of the protein domains selected by evolution to recognize cellular phosphoinositides, we have created fluorescent molecules by fusing these domains to the improved version of green fluorescent protein (EGFP); the distribution of these fusion proteins can be followed within live cells, thereby reporting on changes in phosphoinositides. Although this technique is one of the few that provide information on phosphoinositide dynamics in live cells with subcellular resolution and has rapidly gained popularity, it also has limitations that need to be taken into account when interpreting the data. Here, we summarize our experience in designing and using these constructs and review our position concerning the interpretation of the data obtained by this technique.
Balla
Tamas
T
Endocrinology and Reproduction Research Branch, National Institute of Child Health & Human Development, National Institutes of Health, Bethesda, MD 20892, USA. tambal@box-t.nih.gov
Várnai
Péter
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D004735:Energy Transfer; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry / Q000379:methods; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008845:Microinjections / Q000379:methods; D008856:Microscopy, Fluorescence; D010716:Phosphatidylinositols / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection / Q000379:methods
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins