Limits...
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.

Show MeSH

Related in: MedlinePlus

Estimating cytoplasmic diffusion coefficients. (A) A HEK cell expressing the R40L mutant of the PH-PLCδ1-GFP; the enlarged images show the strip of the cell imaged at the indicated times after bleaching. Bar, 10 µm. (B) Fluorescence intensity profiles across the bleached spot at the indicated times after bleaching; lines are fitted from the Gaussian function described in Eq. 3. (C) Increase in the square of the Gaussian width w with time is fitted in terms of the diffusion coefficient D using Eq. 4; see Materials and methods for details.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2654307&req=5

fig3: Estimating cytoplasmic diffusion coefficients. (A) A HEK cell expressing the R40L mutant of the PH-PLCδ1-GFP; the enlarged images show the strip of the cell imaged at the indicated times after bleaching. Bar, 10 µm. (B) Fluorescence intensity profiles across the bleached spot at the indicated times after bleaching; lines are fitted from the Gaussian function described in Eq. 3. (C) Increase in the square of the Gaussian width w with time is fitted in terms of the diffusion coefficient D using Eq. 4; see Materials and methods for details.

Mentions: This model also assumes that lateral diffusion in the cytoplasm is rapid and thus does not affect the plasma membrane profile. To test this assumption, we also determined cytoplasmic diffusion coefficients, again from spot bleaching and the subsequent rate of increase of a Gaussian radius (Fig. 3), as described in Seiffert and Oppermann (2005). Cytosolic diffusion of GFP was estimated as ∼31 µm2/s (Table I), which is consistent with previous estimates; e.g., Braeckmans et al. (2007) and Brough et al. (2005).


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)

Estimating cytoplasmic diffusion coefficients. (A) A HEK cell expressing the R40L mutant of the PH-PLCδ1-GFP; the enlarged images show the strip of the cell imaged at the indicated times after bleaching. Bar, 10 µm. (B) Fluorescence intensity profiles across the bleached spot at the indicated times after bleaching; lines are fitted from the Gaussian function described in Eq. 3. (C) Increase in the square of the Gaussian width w with time is fitted in terms of the diffusion coefficient D using Eq. 4; see Materials and methods for details.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654307&req=5

fig3: Estimating cytoplasmic diffusion coefficients. (A) A HEK cell expressing the R40L mutant of the PH-PLCδ1-GFP; the enlarged images show the strip of the cell imaged at the indicated times after bleaching. Bar, 10 µm. (B) Fluorescence intensity profiles across the bleached spot at the indicated times after bleaching; lines are fitted from the Gaussian function described in Eq. 3. (C) Increase in the square of the Gaussian width w with time is fitted in terms of the diffusion coefficient D using Eq. 4; see Materials and methods for details.
Mentions: This model also assumes that lateral diffusion in the cytoplasm is rapid and thus does not affect the plasma membrane profile. To test this assumption, we also determined cytoplasmic diffusion coefficients, again from spot bleaching and the subsequent rate of increase of a Gaussian radius (Fig. 3), as described in Seiffert and Oppermann (2005). Cytosolic diffusion of GFP was estimated as ∼31 µm2/s (Table I), which is consistent with previous estimates; e.g., Braeckmans et al. (2007) and Brough et al. (2005).

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