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First published online 28 June 2005
doi: 10.1242/jcs.02426

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Mobility of proteins associated with the plasma membrane by interaction with inositol lipids

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

View larger version (45K): [in a new window]   Fig. 1. Subcellular distribution of GFP, GFP-GAP1IP4BP and GFP-GAP1m in HEK-293 cells under conditions of increased and decreased levels of PtdIns(3,4,5)P3. HEK-293 cells expressing GFP (A), GAP1IP4BP (B) or GAP1m (C) were cultured under conditions designed to increase (i) or decrease (ii) cellular PtdIns(3,4,5)P3. All images represent a 3 µm optical section whose position is indicated by the horizontal white bar (the y axis is in fluorescent units).

View larger version (35K): [in a new window]   Fig. 2. Photobleaching protocol. Fluorescence image series of a HEK-293 cell producing GFP-GAP1IP4BP. (A) Pre-bleaching image (i) shows the cell immediately before the photobleaching. The 3 µm ROI is indicated by the white box. (i) Pre-bleaching. (ii) Immediately after bleaching. (iii) Fluorescence recovery. (B) The fluorescence profile of the photobleaching experiment indicates fluorescence values at pre-bleaching (i) and immediately after bleaching (ii), and the fluorescence recovery (iii). Bar, 6 µm.

View larger version (17K): [in a new window]   Fig. 3. FRAP data for GFP, GFP-GAP1IP4BP and GFP-GAP1m in HEK-293 cells under conditions of increased and decreased levels of PtdIns(3,4,5)P3. Relative mobility (t; A) and mobile fractions (Mf; B) of GFP, GFP-GAP1IP4BP and GFP-GAP1m under conditions designed to increase (white bars) or decrease (black bars) cellular PtdIns(3,4,5)P3 levels. Data are presented as the means±s.e.m. of at least six experiments.

View larger version (48K): [in a new window]   Fig. 4. FRAP analysis of GFP-GAP1IP4BP in cells transfected with Ins(1,4,5)P3-3-kinase. This figure illustrates a typical experiment to explore the effect of Ins(1,3,4,5)P4 generation on the mobility of GAP1IP4BP, with this particular example being from cells transfected with Ins(1,4,5)P3-3-kinase in order to maximize Ins(1,3,4,5)P4 production. (A) Subcellular distribution of GFP-GAP1IP4BP and DsRed-Ins(1,4,5)P3-3-kinase in co-transfected HEK-293 cells. The plasma-membrane distribution of GFP-GAP1IP4BP (i) contrasts with the cytosolic distribution of the modified DsRed-Ins(1,4,5)P3-3-kinase (ii); (iii) a merged image. (B) A 3 µm ROI was subjected to three photobleaches at the times indicated. Following the first photobleach (B1), carbachol (Cch) was added 5 minutes before the second photobleach (B2). Following fluorescence recovery of the second photobleach (B2), atropine (At) was added and, 2 minutes after At addition, the ROI was bleached for a third time (B3). This protocol was repeated in the absence of Cch and At, and in HEK-293 cells co-producing a catalytically dead DsRed-Ins(1,4,5)P3-3-kinase variant or in cells transfected with only GAP1IP4BP; the pooled data are presented in Table 2. Bar, 10 µm.

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