December 2011

New protein sensors to quantify phosphoinositides in situ

 Lipid_News_Fig2_lg 
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.

REFERENCES
  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 (rstaheli@iupui.edu) 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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