In situ lipophile labeling in living cells

New Mexico State University, Las Cruces NM

Investigators

Abstract

With this award, the Chemical Measurement and Imaging Program in the Division of Chemistry is funding Professors Jeffrey Arterburn, Charles B. Shuster, and Tanner Schaub of New Mexico State University to develop methods for labeling unsaturated lipids in the membranes of living cells with fluorescent dyes. To meet this challenge, a combined approach involving catalytic cross-metathesis coupling chemistry, live cell imaging and ultrahigh resolution mass spectrometry will be used. The aims are to synthesize useful lipid probes for imaging, and then characterize the labeling chemistry within the biological context of the cell membrane. The interdisciplinary methodology and research environment are well suited to the education of scientists at all levels, and continue the PI's efforts to promote participation of women and underrepresented minorities in science. The methodology will be employed outside the University setting in a boot camp for science journalists. The team will develop displays featuring "Chile Chemistry" to introduce chemistry and scientific principles to elementary school students during tours of the NMSU Chile Pepper Institute. The identity and distribution of labeled lipids will be determined by ultrahigh resolution mass spectrometry and their localization and dynamic motion will be imaged using optical microscopy. The strategy of chemically modifying native unsaturated lipids by fluorescent dye attachment to produce optical imaging probes is new and complementary to existing strategies that utilize exogenous probes, bioorthogonal labeling, and direct spectroscopic methods to study membrane lipids. Aim 1 will optimize chemistry to synthesize useful lipid probes for live-cell imaging, evaluate metabolic alterations to the lipid probes, and label lipid extract mixtures to characterize the composition of unsaturated lipids as a basis for comparative analyses. Aim 2 will label specific lipids in live cells to investigate biological context of the cell membrane as a factor in selectivity. Additionally, the identity and distribution of dye-labeled lipids will be determined by ultrahigh resolution mass spectrometry and their localization and dynamic motion will be tracked by optical imaging. Aim 3 will use advanced fluorescence microscopy experiments to assess the membrane localization and the functionality of the CM-labeled lipids through short and long term observations. In the long term, the realization of this labeling methodology will provide a powerful analytical approach for fundamental studies of lipid localization, dynamics and determining the roles of discrete lipids in membrane biology.