Probing the formation of mammalian cell lipid microdomains with nanometer-scale imaging secondary ion mass spectrometry

Over forty years ago, Singer and Nicholson began studying the organization of the plasma membranes on mammalian cells. Since then, our understanding of this membrane has shifted from that of a homogeneous to a more heterogenous environment of proteins and lipids. The complexity of living cells have made for difficulties in obtaining a complete understanding of the plasma membrane, especially that of the sphingolipids’ organization. The recent application of imaging mass spectrometry to biological samples has enabled for the chemical analysis of the plasma membrane. Through the use of a high resolution imaging secondary ion mass spectrometer (NanoSIMS 50, Cameca), we have been able to chemically map the distributions of sphingolipids on the plasma membrane of murine fibroblast cells with an astonishing sub-100 nm resolution. This required us to metabolically incorporate a rare, stable label into the molecules of interest and chemically preserving the cells in an effort to maintain close to a native organization of the membrane. The labeled lipid species could then be visualized with the NanoSIMS, providing us with a closer understanding of lipids within the plasma membrane. These experiments have revealed that sphingolipid-enriched domains exist in the plasma membrane on a length scale of ~200 nm, and they form non-random clustering on the cell surface. Disruption of the cellular cytoskeleton resulted in a loss of detectable statistically significant domains. In addition, the rapid transport of sphingolipids to the plasma membrane has made for complex interpretation of the role that vesicle transport plays in domain formation. Further studies using this methodology of chemically imaging mammalian cells, most importantly disruption of vesicle trafficking, may help to definitively identify the mechanisms behind the formation of these lipid domains as well as elucidate the full purpose they serve in the cell.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Klitzing, Haley Ann

Contributors dc:contributor

• Kraft, Mary
• Sweedler, Jonathan V.
• Harley, Brendan A.
• Bailey, Ryan

• sphingolipid
• secondary ion mass spectrometer (SIMS)
• NanoSIMS

Statement dc:rights

• Copyright 2015 Haley A. Klitzing

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/88182

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/88182