Changes in order parameters associated with ceramide-mediated membrane reorganization measured using pTIRFM

Download
  1. (PDF, 859 KB)
  2. Get@NRC: Changes in order parameters associated with ceramide-mediated membrane reorganization measured using pTIRFM (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1021/la403585v
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleLangmuir
ISSN0743-7463
1520-5827
Volume29
Issue51
Pages1590715918
AbstractThe enzymatic generation of ceramide has significant effects on the biophysical properties of lipid bilayers and can lead to the extensive reorganization of cell membranes. We have synthesized and characterized a headgroup-labeled fluorescent lipid probe (NBD-ceramide, NBD-Cer) and demonstrated that it can be used for polarized total internal reflection fluorescence microscopy experiments to probe changes in membrane order that result from ceramide incorporation. NBD-Cer measures significantly higher order parameters for the liquid-ordered (Lₒ) domains (⟨P₂⟩ = 0.40 ± 0.03) than for the liquid-disordered phase (Ld, fluid, ⟨P₂⟩ = 0.22 ± 0.02) of phase-separated bilayers prepared from egg sphingomyelin, dioleolyphosphatidylcholine, and cholesterol mixtures. The probe also responds to changes in packing induced by the direct incorporation of ceramide or the variation in the ionic strength of the aqueous medium. Order parameter maps obtained after enzyme treatment of bilayers with coexisting Lₒ and Ld phases show two distinct types of behavior. In regions of high enzyme activity, the initial Lₒ/Ld domains are replaced by large, dark features that have high membrane order corroborating previous hypotheses that these are ceramide-enriched regions of the membrane. In areas of low enzyme activity, the size and shape of the Lo domains are conserved, but there is an increase in the order parameter for the initial Ld phase (⟨P₂⟩ = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the Lo domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.
Publication date
PublisherAmerican Chemical Society
LanguageEnglish
AffiliationMeasurement Science and Standards; National Research Council Canada
Peer reviewedYes
NPARC number23000485
Export citationExport as RIS
Report a correctionReport a correction
Record identifierdced12ac-6d2a-41b8-b690-d52b31cb330c
Record created2016-07-25
Record modified2016-07-25
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)