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Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information.

Hammond GR, Sim Y, Lagnado L, Irvine RF - J. Cell Biol. (2009)

Bottom Line: However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions.We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns.Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, England, UK. gruh2@cam.ac.uk

ABSTRACT
Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

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FRAP by total internal reflection. (A) Epifluorescence and TIR fluorescence micrographs of the adhesion planes (“footprints”) of HEK cells expressing either the myristoylated and palmitoylated sequence of Lyn kinase fused to YFP (PM-YFP), GFP fused to the PH domain of PLCδ1, or GFP fused to full-length GAP1IP4BP as indicated. The latter two are targeted to the plasma membrane via an interaction with PtdIns(4,5)P2. (B) The rationale behind the FRAP experiment: after bleaching by TIR (1), entry of unbleached protein into the adhesion plane via lateral diffusion through the membrane should cause fluorescence recovery from the periphery (2), whereas exchange with a cytosolic pool would lead to uniform recovery across the footprint (3). (C) Example micrographs taken at the indicated times after an 8-s bleach; residual postbleach fluorescence was subtracted from the images (see Fig. S1 for details, available at http://www.jcb.org/cgi/content/full/jcb.200809073/DC1). Stills are taken from Videos 1–3. Bar, 10 µm.
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fig1: FRAP by total internal reflection. (A) Epifluorescence and TIR fluorescence micrographs of the adhesion planes (“footprints”) of HEK cells expressing either the myristoylated and palmitoylated sequence of Lyn kinase fused to YFP (PM-YFP), GFP fused to the PH domain of PLCδ1, or GFP fused to full-length GAP1IP4BP as indicated. The latter two are targeted to the plasma membrane via an interaction with PtdIns(4,5)P2. (B) The rationale behind the FRAP experiment: after bleaching by TIR (1), entry of unbleached protein into the adhesion plane via lateral diffusion through the membrane should cause fluorescence recovery from the periphery (2), whereas exchange with a cytosolic pool would lead to uniform recovery across the footprint (3). (C) Example micrographs taken at the indicated times after an 8-s bleach; residual postbleach fluorescence was subtracted from the images (see Fig. S1 for details, available at http://www.jcb.org/cgi/content/full/jcb.200809073/DC1). Stills are taken from Videos 1–3. Bar, 10 µm.

Mentions: The central aim of this paper was to distinguish lateral diffusion of inositol lipid-bound proteins from their exchange with an unbound, cytosolic pool. Therefore, we conceived a FRAP experiment whereby the entire basal membrane could be bleached selectively by TIRF microscopy, a technique that allows the imaging of the plasma membrane adherent to a glass coverslip (Axelrod, 2001). Fig. 1 A shows HEK cells expressing GFP fused to the isolated PH domain from phospholipase Cδ1 (PLCδ1) and the PH domain–containing protein GAP1IP4BP, both of which are targeted to the plasma membrane by interaction with PtdIns(4,5)P2 (Várnai and Balla, 1998; Cozier et al., 2000). Also shown (Fig. 1 A, top) is YFP targeted to the plasma membrane by the palmitoylation and myristoylation sequence from Lyn kinase (PM-YFP). Epifluorescence images of the adhesion plane, or “footprint,” show the uniform haze from the basal plasma membrane, along with a ring of lateral membrane. Conversely, only uniform fluorescence in the footprint is seen by TIRF when the evanescent field decays with a length constant of ∼100 nm (Fig. 1 A). Consistent with a previous paper (van Rheenen et al., 2005), we saw no local enrichment of these PtdIns(4,5)P2-binding proteins in the adhesion plane.


Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information.

Hammond GR, Sim Y, Lagnado L, Irvine RF - J. Cell Biol. (2009)

FRAP by total internal reflection. (A) Epifluorescence and TIR fluorescence micrographs of the adhesion planes (“footprints”) of HEK cells expressing either the myristoylated and palmitoylated sequence of Lyn kinase fused to YFP (PM-YFP), GFP fused to the PH domain of PLCδ1, or GFP fused to full-length GAP1IP4BP as indicated. The latter two are targeted to the plasma membrane via an interaction with PtdIns(4,5)P2. (B) The rationale behind the FRAP experiment: after bleaching by TIR (1), entry of unbleached protein into the adhesion plane via lateral diffusion through the membrane should cause fluorescence recovery from the periphery (2), whereas exchange with a cytosolic pool would lead to uniform recovery across the footprint (3). (C) Example micrographs taken at the indicated times after an 8-s bleach; residual postbleach fluorescence was subtracted from the images (see Fig. S1 for details, available at http://www.jcb.org/cgi/content/full/jcb.200809073/DC1). Stills are taken from Videos 1–3. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654307&req=5

fig1: FRAP by total internal reflection. (A) Epifluorescence and TIR fluorescence micrographs of the adhesion planes (“footprints”) of HEK cells expressing either the myristoylated and palmitoylated sequence of Lyn kinase fused to YFP (PM-YFP), GFP fused to the PH domain of PLCδ1, or GFP fused to full-length GAP1IP4BP as indicated. The latter two are targeted to the plasma membrane via an interaction with PtdIns(4,5)P2. (B) The rationale behind the FRAP experiment: after bleaching by TIR (1), entry of unbleached protein into the adhesion plane via lateral diffusion through the membrane should cause fluorescence recovery from the periphery (2), whereas exchange with a cytosolic pool would lead to uniform recovery across the footprint (3). (C) Example micrographs taken at the indicated times after an 8-s bleach; residual postbleach fluorescence was subtracted from the images (see Fig. S1 for details, available at http://www.jcb.org/cgi/content/full/jcb.200809073/DC1). Stills are taken from Videos 1–3. Bar, 10 µm.
Mentions: The central aim of this paper was to distinguish lateral diffusion of inositol lipid-bound proteins from their exchange with an unbound, cytosolic pool. Therefore, we conceived a FRAP experiment whereby the entire basal membrane could be bleached selectively by TIRF microscopy, a technique that allows the imaging of the plasma membrane adherent to a glass coverslip (Axelrod, 2001). Fig. 1 A shows HEK cells expressing GFP fused to the isolated PH domain from phospholipase Cδ1 (PLCδ1) and the PH domain–containing protein GAP1IP4BP, both of which are targeted to the plasma membrane by interaction with PtdIns(4,5)P2 (Várnai and Balla, 1998; Cozier et al., 2000). Also shown (Fig. 1 A, top) is YFP targeted to the plasma membrane by the palmitoylation and myristoylation sequence from Lyn kinase (PM-YFP). Epifluorescence images of the adhesion plane, or “footprint,” show the uniform haze from the basal plasma membrane, along with a ring of lateral membrane. Conversely, only uniform fluorescence in the footprint is seen by TIRF when the evanescent field decays with a length constant of ∼100 nm (Fig. 1 A). Consistent with a previous paper (van Rheenen et al., 2005), we saw no local enrichment of these PtdIns(4,5)P2-binding proteins in the adhesion plane.

Bottom Line: However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions.We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns.Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, England, UK. gruh2@cam.ac.uk

ABSTRACT
Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

Show MeSH
Related in: MedlinePlus