Submitted on November 26, 2007
Revised on January 28, 2008
Accepted on February 8, 2008
A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains
Sarita Hebbar, Esther Lee, Manoj Manna, Steffen Steinert, Goparaju Sravan Kumar, Markus Wenk, Thorsten Wohland, and Rachel Kraut
Insititute of Bioengineering and Nanotechnology, Singapore 138669
Corresponding Author: rkraut{at}ibn.a-star.edu.sg
We have designed a tagged probe (SBD) to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the sphingolipid binding domain of the amyloid precursor protein [1]. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in-vivo imaging. Here, we present evidence to describe the characteristics of SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol and sphingolipid dependent manner via detergent resistant microdomains. Furthermore, using fluorescence correlation spectroscopy (FCS) to assay the mobility of SBD in live cells, we show that SBD’s behavior at the plasma membrane is similar to that of the previously described raft marker Cholera Toxin B (CtxB), displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent resistant microdomains that are cholesterol dependent.
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