Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

Abstract

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single‐cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1‐24.4.27. © 2009 by John Wiley & Sons, Inc.

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

“…To evaluate whether there was a similar correlation between actin and PIP 2 during the recovery of cortical actin density at the synapse, we cotransfected CTLs with Lifeact-mApple and Tubby-EGFP (30,31) and performed TIRF imaging on CTLs as they formed synapses on a planar activating surface. At early time points during interaction with anti-CD3-coated surfaces, we observed that the reduction in cortical actin density at the center of the synapse was concurrent with a reduction in levels of PIP 2 , consistent with our previous CTL-target conjugate imaging ( Fig.…”

“…One of the key enzymes downstream of the TCR is phospholipase C gamma 1 (PLCγ1), which cleaves PIP 2 into inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG), two critical intermediates required for T-cell activation that trigger Ca 2+ flux and cell polarization changes, respectively (2,28,29). To address whether inhibition of PLCγ1 could modify local levels of its substrate, PIP 2 , at the synapse, we cotransfected CTLs with Lifeact-mApple and a sensor expressing the PIP 2 -binding domain of Tubby fused to EGFP (Tubby-EGFP) (30,31). We then pretreated cells with either U-73122, an inhibitor of PLCγ1 activity, or U-73343, an inactive analog (32), and allowed CTL-target conjugates to form before fixation.…”

Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain

“…EGFP-lactadherin-C2, developed by the Grinstein laboratory (27), was obtained from Addgene. PLC␦1-PH-GFP was a generous gift from Tamas Balla (29,30).…”

“…Single molecule TIRF microscopy assays in live cells indicate that mutations to either the NTM or the CTM reduce the Myo1a-TH1 membrane-bound lifetime. Finally, although Myo1a-TH1 co-localizes with both the PS-binding protein, lactadherin C2 (27,28), and the PI(4,5)P 2 -binding protein, PLC␦1-PH (29,30), only lactadherin C2 is able to compete with Myo1a for targeting to microvilli. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif.…”

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

“…The PH domain of PLCδ1 (Phospholipase C δ1) that specifically binds to PI(4,5)P 2 is a well-established probe for imaging PI(4,5)P 2 sub-cellular distribution and dynamics 33,34 in the PM. We have genetically tagged this domain with two fluorescent proteins, PAmCherry1 35 and iRFP 36 to 1.…”