| AVS 66th International Symposium & Exhibition | |
| Biomaterial Interfaces Division | Tuesday Sessions |
| Session BI+AS-TuM |
| Session: | Characterization of Biological and Biomaterial Surfaces |
| Presenter: | Shohini Sen-Britain, State University of New York, Buffalo |
| Authors: | S. Sen-Britain, State University of New York, Buffalo N. Li, State University of New York, Buffalo G.E. Atilla-Gokcumen, State University of New York, Buffalo J.A. Gardella Jr., State University of New York, Buffalo |
| Correspondent: | Click to Email |
The formation of phase segregated lipid droplets storing triacylglycerol containing polyunsaturated fatty acyl chains (PUFA-TAGs) during apoptosis, or programmed cell death, has been previously observed [1,2]. Polyunsaturated fatty acids are incorporated into PUFA-TAGs by diacylglycerol acyltransferases (DGATs) [1]. The acylation of ceramide by DGATs also produces acylceramide which has been found in lipid droplets as well [3]. The accumulation of ceramide and PUFA phospholipids sensitizes cells to cell death. Therefore, acylation of these molecules into phase segregated droplets is thought to have a protective effect [1,2].
Previous studies observing these acylated molecules in lipid droplets have utilized LC-MS of fractionated lipid droplets [1-3]. However, colocalization of these lipids has not been observed in situ. Previous imaging studies have been limited by the use of nonspecific lipid dyes, such as nile red, in observing lipid droplets.
In this study, we have utilized metal-assisted time-of-flight secondary ion mass spectrometry (ToF-SIMS) to image the colocalization of TAG (C39:0) and acylceramide (C17) in apoptotic HCT-116 human colorectal carcinoma cells. We maintained sample preparation conditions used in previous microscopy studies by cutting out squares of cell culture plates containing lyophilized apoptotic HCT-116 cells. Imaging of lipids within the cells was accomplished by milling off the top half of the cells using a focused ion beam-scanning electron microscope (FIB-SEM). Imaging of gold sputter coated samples in negative ion mode allowed for the observation of high molecular weight secondary ions (>1000 m/z) and of unique spectra of both TAG (C39:0) and AC (C17). Colocalization of endogeneous TAG and AC were observed in apoptotic cells. TAG and AC fragmentations were determined by analyzing (1) gold sputter coated TAG and AC standards on cell culture plates and (2) standard additions of TAG and AC onto milled lyophilized apoptotic cells that were also gold sputter coated.
The work accomplished in this study illustrates the potential of identifying the spatial localization of large biomolecules in cells on insulating, high topography containing samples through the use of standard additions and high mass resolution, metal-assisted ToF-SIMS. The results are also the first reported in situ observation of TAG and AC colocalization in apoptotic cells.
[1] Biochemistry 2018, 57, 72-80
[2] ACS Chemical Biology 2016, 11, 2583-2587
[3] Cell Metabolism 2017, 25, 686–697