Phosphatidylinositol 4,5 bisphosphate (PIP2
) directly regulates many processes at the inner leaflet of the plasma membrane, such as the activation of ion channels, endocytosis, and exocytosis (Di Paolo and De Camilli, 2006
). It is also the source of three second messengers: how does one lipid do so much? Many investigators have considered the possibility there are different pools of PIP2
in the plasma membrane (Hinchliffe et al., 1998
; Aikawa and Martin, 2003
; Janmey and Lindberg, 2004
). Little is known, however, about how these separate pools come about and how they are regulated. One hypothesis is that membrane-bound clusters of basic residues on peripheral (e.g., myristoylated alanine rich C kinase substrate [MARCKS], K-Ras, and gravin) and integral (e.g., epidermal growth factor receptor [EGFR] and polymeric immunoglobulin receptor [pIgR]) proteins concentrate electrostatically (sequester) a significant fraction of the PIP2
in the membrane; when the local concentration of Ca2+
rises and calcium/calmodulin (Ca/CaM) binds to these basic clusters, the sequestered PIP2
is released, increasing its local free concentration (McLaughlin and Murray, 2005
Theoretical calculations and experimental measurements on model systems support this hypothesis. Specifically, electrostatic theory predicts a membrane-bound cluster of basic residues produces a local positive potential that concentrates multivalent acidic lipids such as PIP2
(Wang et al., 2004
; McLaughlin and Murray, 2005
). Fluorescence resonance energy transfer, electron paramagnetic resonance, and phospholipase C (PLC) activity measurements confirm that basic clusters on purified proteins or peptides do indeed laterally concentrate PIP2
in phospholipid vesicles (Murray et al., 2002
; Gambhir et al., 2004
). These calculations and model studies also suggest that if the number of membrane-bound basic clusters is equal to or greater than the number of PIP2
on the inner leaflet of a plasma membrane, a significant fraction of PIP2
will be sequestered. Testing this prediction requires direct experiments on living cells, however, because the exact concentration of these basic clusters is not known with certainty for any cell type.
Studies of microvesicles released from human erythrocytes under different conditions (Hagelberg and Allan, 1990
) provided evidence that ~50% of the PIP2
is bound to cytoskeletal proteins (e.g., Band III and glycophorin) that are depleted in the microvesicles. Specifically, the microvesicles and native membranes contained identical mole fractions of most lipids, but not polyphosphoinositides (e.g., PIP2
). The higher mole fraction of PIP2
in the native membranes suggests ~50% of their PIP2
is not free to diffuse into the nascent microvesicles.
One independent way to estimate the fraction of free PIP2 in the plasma membrane is to measure directly the diffusion coefficient, D, of PIP2 on the inner leaflet in a living cell. A simple analysis (see Supplemental Material) reveals that if R is the ratio of reversibly bound or sequestered (S) to free (C) PIP2, the diffusion constant will be scaled by a factor 1/(R + 1).
Specifically, combining the diffusion equation (Fick's second law) with a linear adsorption isotherm (Henry's law) produces a diffusion equation with an apparent diffusion constant, D:
where S is the concentration of sequestered PIP2
(reversibly bound or immobilized), C is the concentration of PIP2
free to diffuse, and Dfree
is the diffusion coefficient of free PIP2
. If the total concentration of PIP2
T = S + C, then C = T/(R + 1).
If the erythrocyte result can be extrapolated to other cells, such as fibroblasts, a significant fraction of PIP2
in these plasma membranes is also bound reversibly to proteins. Equation 1
then predicts that PIP2
will diffuse less rapidly in the inner leaflet of a fibroblast plasma membrane than in a “bleb” formed on that membrane. It also predicts that PIP2
will diffuse less rapidly in the inner than in the outer leaflet of the plasma membrane and less rapidly on the inner leaflet than either neutral or monovalent lipids that are not bound to proteins.
Experiments on giant unilamellar vesicles (GUVs) showed monomers of fluorescent PIP2
can be incorporated into the outer leaflet of preformed vesicles by exposing them to micelles of PIP2
; fluorescence correlation spectroscopy (FCS) measurements showed PIP2
diffuses with a D characteristic of other lipids in the GUVs (Golebiewska et al., 2006
). Here, we report FCS measurements of the D of fluorescent PIP2
in plasma membranes after microinjecting fluorescent PIP2
micelles into fibroblasts (Rat1, Cos1, REF52, and NIH3T3) and epithelial cells (human embryonic kidney [HEK]293 and Fisher rat thyroid [FRT]).
Our most important result is that Bodipy TMR-PIP2
in the inner leaflet of plasma membranes diffuses significantly more slowly than the same lipid in blebs, in the outer leaflet of plasma membranes, or in GUVs. These observations are consistent with the Hagelberg and Allan (1990)
erythrocyte microvesicle partitioning results. Hence, both the partitioning and diffusion experiments suggest a significant fraction of the PIP2
on the inner leaflet is bound reversibly.