Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody.

Xue Jiang,Liansheng Zhong,Guanghu Wang,Erhard Bieberich,Haiyan Qin,Sanjib Karki,Ahmed Elsherbini,Stefanka D Spassieva,Zhihui Zhu,Priyanka Tripathi,Wenbo Zhi,Simone M Crivelli

doi:10.3389/fcell.2019.00166

Abstract

Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.

Highlights

The function of ceramide as precursor and intermediate in sphingolipid metabolism is well investigated for many decades
PLA1 shows the subcellular localization of a particular ceramide-associated proteins (CAPs) that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide
One of the greatest challenges in determining the function of Ceramide-rich platforms (CRPs) is to visualize the interaction of ceramide with proteins in these contact sites

Summary

Introduction

The function of ceramide as precursor and intermediate in sphingolipid metabolism is well investigated for many decades. Novel probes were developed to identify and visualize these ceramide-associated proteins (CAPs). These probes are ceramide analogs that are termed bioorthogonal because labeling of CAPs is achieved in living cells (Walter et al, 2016; Fink and Seibel, 2018). They are bifunctional in that these ceramide analogs are: (1) cross-linked to CAPs by photoactivation; and (2) can be covalently attached to fluorophores and other functional groups using click chemistry. To understand which of these proteins are CAPs mediating the biological function of ceramide, it is critical to develop methods that verify the interaction of the pacFACer cross-linked proteins with endogenous ceramide

Methods

Results

Conclusion