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Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

Please always quote using this URN: urn:nbn:de:bvb:20-opus-75433
  • Background: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results: LipidBackground: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results: Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion: Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.show moreshow less

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Metadaten
Author: Jörg O. Blachutzik, Faith Demir, Ines Kreuzer, Rainer Hedrich, Gregory S. Harms
URN:urn:nbn:de:bvb:20-opus-75433
Document Type:Journal article
Faculties:Fakultät für Biologie / Julius-von-Sachs-Institut für Biowissenschaften
Language:German
Year of Completion:2012
Source:In: Plant Methods (2012) 8: 28, doi:10.1186/1746-4811-8-28
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 58 Pflanzen (Botanik) / 580 Pflanzen (Botanik)
GND Keyword:Arabidopsis thaliana
Tag:Fluorescence mi; Laurdan; Lipid polarization; Lipophilic fluorescent dyes; Liquid (dis-) ordered phase; Plasma membrane; Protoplasts; Sphingolipid
Release Date:2013/03/27
Collections:Open-Access-Publikationsfonds / Förderzeitraum 2012
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung