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High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS
- Source :
- Proceedings of the National Academy of Sciences of the United States of America, vol 114, iss 8
- Publication Year :
- 2017
- Publisher :
- eScholarship, University of California, 2017.
-
Abstract
- Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is "accessible" to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with 15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-β-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.
- Subjects :
- 0301 basic medicine
Cell Culture Techniques
Spectrometry, Mass, Secondary Ion
chemistry.chemical_compound
Hemolysin Proteins
0302 clinical medicine
NanoSIMS
perfringolysin O
microvilli
Microscopy
Multidisciplinary
Microscopy, Confocal
Membrane Glycoproteins
Nanotubes
Membrane cholesterol
Microvilli
Chinese hamster ovary cell
beta-Cyclodextrins
Biological Sciences
Sphingomyelins
Molecular Imaging
Membrane
Cholesterol
Sphingomyelin Phosphodiesterase
Biochemistry
Confocal
Isotope Labeling
lipids (amino acids, peptides, and proteins)
Sphingomyelin
anthrolysin O
Protein Binding
Secondary Ion
Bacterial Toxins
CHO Cells
Biology
03 medical and health sciences
Cricetulus
Bacterial Proteins
Animals
High resolution imaging
Nitrogen Isotopes
Spectrometry
Cell Membrane
cholesterol
Plasma
Mass
030104 developmental biology
chemistry
Biophysics
Cytolysin
Generic health relevance
030217 neurology & neurosurgery
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America, vol 114, iss 8
- Accession number :
- edsair.doi.dedup.....80ebfb591f9356cc8545f3f5300e9a41