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

Klitzing, Haley Ann

Permalink

https://hdl.handle.net/2142/88182 Copy

Description

Title

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

Author(s)

Klitzing, Haley Ann

Issue Date

2015-07-13

Director of Research (if dissertation) or Advisor (if thesis)

Kraft, Mary

Doctoral Committee Chair(s)

Kraft, Mary

Committee Member(s)

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

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

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

Abstract

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.

Graduation Semester

2015-8

Type of Resource

text

Permalink

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

Copyright and License Information

Copyright 2015 Haley A. Klitzing

Owning Collections