December 2011

New protein sensors to quantify phosphoinositides in situ


Figure 1. The epsin1 ENTH domain (PDB 1HOA) was used to engineer a high-affinity PI(4,5)P2 binding reporter. Methionine 10 (magenta) was mutated to cysteine to attach the environmentally sensitive probe, while serine 4 (green) was mutated to tryptophan to increase membrane affinity. Inositol(1,4,5)P3 bound to the ENTH domain is shown in red.

Cellular membranes harbor receptors, ion channels, lipid domains, lipid signals and scaffolding complexes that function to maintain cellular growth, metabolism and homeostasis (1). Abnormalities in lipid metabolism attributed to genetic changes, among other causes, are associated with a host of diseases (2). Thus, there is a need to understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development.

The lipid bilayer has a highly polarized structure that consists of a central hydrocarbon core and two flanking interfacial regions that are highly dynamic and could contain thousands of different lipids (1). This dynamic variety of glycerolipids, sphingolipids and sterols in membrane organelles provide spatial and temporal cues to direct signaling processes through target proteins (3). However, there remains a large gap in our understanding of the spatial and temporal dynamics of the lipids that produce these bioactive signals.

Given that nearly half of all proteins are located in or on membranes, it is not surprising that there are a variety of conserved lipid-binding domains in eukaryotes. Some of these domain families rank in the top 15 modular domains in the human genome and are most often found in signal-transduction and membrane-trafficking proteins (4). To date, fluorescently tagged lipid-binding domains (such as the PH domain) that harbor high specificity and affinity for phosphoinositides (PIs) have most often served to study PI dynamics and localization (5). While the overall spatial distribution of lipids such as PI(3)P and PI(4,5)P2 (5) is well appreciated, the actual concentration, distribution and spatiotemporal dynamics have not been determined quantitatively. Thus, real-time lipid sensors that could provide high sensitivity for a specific PI to quantify its role in a cellular-signaling cascade would be a great advantage to researchers.

Recently, Wonhwa Cho and colleagues developed such an approach to quantify PI(4,5)P2 using a chemically modified lipid-binding domain (6).

The probe was first engineered for optimal lipid-binding properties and minimal affinity for cellular proteins. Through the introduction of an environmentally sensitive chemical probe on a free cysteine, the engineered domain serves as a turn-on sensor that undergoes a large increase in fluorescence upon lipid binding.



Figure 2. A. The engineered PI(4,5)P2 sensor undergoes an increase and blue shift in fluorescence upon binding PI(4,5)P2-containing membranes.

B. The fluorescence shift and increase observed with PI(4,5)P2 binding can be quantified to determine the concentration of PI(4,5)P2 in cellular membranes.

In addition, the probe undergoes a blue shift upon PI(4,5)P2-dependent membrane binding, which allows ratiometric detection of PI(4,5)P2 in vitro and in cells. The ratiometric approach will allow researchers to overcome obstacles associated with fluorescently tagged domains, such as photobleaching.

The probe’s successful microinjection, or liposome-mediated delivery, into multiple cell lines further demonstrated its applicability. Ultimately, Cho and colleagues were able to use the probe to investigate the threshold level of PI(4,5)P2 required to trigger phagocytosis in immune cells. Taken together, environmentally sensitive lipid probes will be applicable to studying the quantitative role of lipids in signal transduction, membrane trafficking, apoptosis and cell migration and may serve as readout assays for therapeutic efficacy and potency.

The approach designed by Cho and colleagues will be of much use, as structural and functional knowledge of lipid-binding domains, including the C1, C2, PH and PX domains (4), are available and should allow the engineering of lipid probes for diacylglycerol, phosphatidylserine and PIs. While it may now be difficult to sense both sides of membrane organelles in an unbiased manner using this chemical approach, this is a significant leap forward in studying real-time lipid signaling.

  1. 1. Van Meer, G., Voelker, D.R., and Feigenson, G.W. Membrane lipids: where they are and how they behave. (2008) Nat. Rev. Mol. Cell Biol. 9, 112 – 124.
  2. 2. Sudhahar, C.G., Haney, R.M., Xue, Y., and Stahelin, R.V. Cellular membranes and lipid-binding domains as attractive targets for drug development. (2008) Curr Drug Targets. 9, 603 – 613.
  3. 3. Cho, W., Stahelin, R.V. Membrane-protein interactions in cell signaling and membrane trafficking. (2005) Annu. Rev. Biophys. Biomol. Struct. 34, 119 – 151.
  4. 4. Stahelin, R.V. Lipid binding domains: more than simple lipid effectors. (2009) J. Lipid Res. 50, S299 – S304.
  5. 5. Varnai, P. and Balla, T. Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. (2006) Biochim. Biophys. Acta. 8, 957 – 967.
  6. 6. Yoon, Y., Lee, P.J., Kurilova, S., and Cho, W. In situ quantitative imaging of cellular lipids using molecular sensors. (2011) Nat. Chem. 11, 868 – 874.


Lipid_News_StahelinRobert V. Stahelin ( is an assistant professor at the Indiana University School of Medicine-South Bend and a concurrent assistant professor at the University of Notre Dame.

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