Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane

Abstract

Background: Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Results: Superresolution imaging reveals homogeneous distribution of PI(4,5)P 2 , PI4P, and PI(3,4,5)P 3 in the major area of the PM. Conclusion: Phosphatidylinositides detected by PH domains are uniformly distributed in the major regions of the PM, with limited concentration gradients. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale.

Gradients Of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling

“…These lipid nanoclusters might be segregated (Ji et al, 2015), and their diameters are 70 nm for PIP2 and 120 nm for PIP3. The spatial distribution of PIP3 and PIP2 nanoclusters was not addressed here, but other studies reported nonoverlapping distributions of PIP3 and PIP2 at the cellular scale (Petrie et al, 2009).…”

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

“…The localization uncertainty (σ) of our super-resolution system is 14.73 nm 23 . Direct comparisons between TIRF and PALM images demonstrated a significant improvement of spatial resolution.…”

“…However, our live PALM imaging shows similar relatively homogeneous distribution of PI(4,5)P 2 , supporting the results observed with membrane sheet samples. Second, as we discussed in our previous work 23 , SMLM requires imaging expertise and extra attention to avoid imaging artifacts that might arise from different processes, including the probes used, sample preparation and fixation, image sampling and reconstruction. Lastly, though the PH domain based probes and antibodies have been widely used in phosphoinositide studies 46,47 it remains possible that not all PI(4,5)P 2 in the membrane can be detected by this approach.…”

“…Moreover, nanoscale PI(4,5)P 2 organization in membrane sheets may differ in live and fixed cells. Our recent work investigated this issue in both fixed and live INS-1 cells using single-molecule localization microscopy (SMLM) 23 .…”

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins

“…Notably, however, it was suggested that PtdIns(4,5)P2 is found in rafts (59), which is where CAAX domains with polybasic sequences would be enriched. The distribution of PtdIns(4,5)P 2 or its protein binding partners to specialized PM domains could not be substantiated by several credible efforts (60,61). This topic is worth mentioning because of the significant literature on the distribution of the various Ras proteins into specialized PM domains designated as rafts (62)(63)(64).…”