Segregation of PIP2 and PIP3 into distinct nanoscale regions within the plasma membrane

Abstract

PIP(2) and PIP(3) are implicated in a wide variety of cellular signaling pathways at the plasma membrane. We have used STORM imaging to localize clusters of PIP(2) and PIP(3) to distinct nanoscale regions within the plasma membrane of PC12 cells. With anti-phospholipid antibodies directly conjugated with AlexaFluor 647, we found that PIP(2) clusters in membrane domains of 64.5±27.558 nm, while PIP(3) clusters had a size of 125.6±22.408 nm. With two color direct STORM imaging we show that >99% of phospholipid clusters have only one or other phospholipid present. These results indicate that lipid nano-domains can be readily identified using super-resolution imaging techniques, and that the lipid composition and size of clusters is tightly regulated.

Keywords: Lipid raft; PC12 cell; PIP2; PIP3.

Fig. 1.. PIP₂ localizes to 64 nm clusters in the plasma membrane.

(A) A representative image of a PC12 cell stained with anti-PIP₂ antibody, and an Alexa Fluor 568 anti-mouse secondary. The image was obtained as a 5 micron Z-stack with 100 nm sections, and then deconvolved with a constrained iterative algorithm (SoftWorX from Applied Precision). This illustrates the diffraction barrier for conventional optical imaging. (B) A different PC12 cell imaged using dSTORM with anti-PIP₂ antibody directly conjugated to Alexa Fluor 647. A and B are at the same scale, scale bar 1 µm. (C) Size distribution of labeled clusters analyzed as Full Width Half Maximum (FWHM) of X and Y Gaussian fits, (n = 1348 rafts). (D) 3D intensity plot of a bright object with amongst the smallest diameter, to illustrate instrument resolution. (E) 3D intensity plot of a typical PIP₂ cluster.

Fig. 2.. PIP₃ localizes to 103 nm clusters in the plasma membrane.

(A) A representative image of a PC12 cell stained with anti-PIP₃ antibody, and an Alexa Fluor 568 anti-mouse secondary. The image was obtained as in Fig. 1A. (B) A different PC12 cell imaged using dSTORM with anti-PIP₃ antibody directly conjugated to Alexa Fluor 647. A and B are at the same scale, scale bar 1 µm. (C) Size distribution of labeled clusters analyzed as Full Width Half Maximum (FWHM) of X and Y Gaussian fits, (n = 796 rafts).

Fig. 3.. Testing for detection of co-localization.

(A) dSTORM image of lipid clusters labeled with anti-PIP₃- Alexa Fluor 488 and anti-PIP₃- Alexa Fluor 647. Scale bar 1 µm. (B) Size distribution of labeled clusters analyzed as Full Width Half Maximum (FWHM) of X and Y Gaussian fits, (n = 926 rafts). (C) Intensity of green and red channels plotted against each other for each raft. (D) Comparison of raft fractions labeled with Alexa Fluor 488, Alexa Fluor 647, or both.

Fig. 4.. Almost complete segregation of PIP2 and PIP3 labeling.

(A) dSTORM image of lipid clusters labeled with anti-PIP₃- Alexa Fluor 488 and anti-PIP₂- Alexa Fluor 647. Scale bar 1 mm. (B) Higher magnification image (8-fold) of a rare double-labeled raft, with intensity profiles for the two channels plotted beneath. (C) Intensity of green and red channels plotted against each other for each raft. (D) Size distribution of labeled clusters analyzed as Full Width Half Maximum (FWHM) of X and Y Gaussian fits, (n = 1433 rafts). (E) Shape distribution of PIP₂ and PIP₃ harboring domains. The distribution of PIP₂ clusters appears to be bi-modal, while that of PIP₃ clusters is broader, but apparently uni-modal. Data are from 468 (PIP₃) or 521 (PIP₂) domains.