High-resolution fungal plasma membrane H+-ATPase profiling combined with hyphenated HPLC-HRMS-SPE-NMR for identification of antifungal constituents in plants

AbstractXenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

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High-resolution freeze-etching replica images of the disk and the plasma membrane surfaces in purified bovine rod outer segments

Journal of Electron Microscopy ◽

10.1093/oxfordjournals.jmicro.a023860 ◽

2000 ◽

Vol 49 (5) ◽

pp. 691-697 ◽

Cited By ~ 14

Author(s):

N. Kajimura ◽

Y. Harada ◽

J. Usukura

Keyword(s):

Plasma Membrane ◽

High Resolution ◽

Rod Outer Segments ◽

Outer Segments ◽

Membrane Surfaces ◽

Freeze Etching

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High-Resolution Imaging of the Distributions of Cholesterol, Sphingolipids, and Specific Proteins in the Plasma Membrane with Secondary Ion Mass Spectrometry

Microscopy and Microanalysis ◽

10.1017/s1431927615012763 ◽

2015 ◽

Vol 21 (S3) ◽

pp. 2397-2398 ◽

Cited By ~ 1

Author(s):

Mary L. Kraft ◽

Peter K. Weber ◽

Jessica F. Frisz ◽

Haley A. Klitzing ◽

Robert Wilson ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasma Membrane ◽

High Resolution ◽

Secondary Ion Mass Spectrometry ◽

High Resolution Imaging ◽

Ion Mass Spectrometry ◽

Specific Proteins ◽

Resolution Imaging ◽

Secondary Ion

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9. Imaging the distributions of lipids and proteins in the plasma membrane with high-resolution secondary ion mass spectrometry

Characterization of Biological Membranes ◽

10.1515/9783110544657-009 ◽

2019 ◽

pp. 287-322 ◽

Cited By ~ 2

Keyword(s):

Mass Spectrometry ◽

Plasma Membrane ◽

High Resolution ◽

Secondary Ion Mass Spectrometry ◽

Ion Mass Spectrometry ◽

Secondary Ion

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Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

Journal of Biological Chemistry ◽

10.1074/jbc.m113.473207 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16855-16861 ◽

Cited By ~ 102

Author(s):

Jessica F. Frisz ◽

Haley A. Klitzing ◽

Kaiyan Lou ◽

Ian D. Hutcheon ◽

Peter K. Weber ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasma Membrane ◽

High Resolution ◽

Mammalian Cells ◽

Plasma Membranes ◽

Secondary Ion Mass Spectrometry ◽

Cholesterol Depletion ◽

Fibroblast Cells ◽

Secondary Ion ◽

Rule Out

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

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