High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS.
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 with15N-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.
Department of Medicine, University of California, Los Angeles, CA 90095.
Cuiwen
C
He
Department of Medicine, University of California, Los Angeles, CA 90095.
Xuchen
X
Hu
Department of Medicine, University of California, Los Angeles, CA 90095.
Rachel S
RS
Jung
Department of Medicine, University of California, Los Angeles, CA 90095.
Thomas A
TA
Weston
Department of Medicine, University of California, Los Angeles, CA 90095.
Norma P
NP
Sandoval
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095.|Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095.
Peter
P
Tontonoz
Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth 6009, Australia.
Matthew R
MR
Kilburn
Department of Medicine, University of California, Los Angeles, CA 90095.
Loren G
LG
Fong
Department of Medicine, University of California, Los Angeles, CA 90095; sgyoung@mednet.ucla.edu haibo.jiang@uwa.edu.au.|Department of Human Genetics, University of California, Los Angeles, CA 90095.
Stephen G
SG
Young
Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth 6009, Australia; sgyoung@mednet.ucla.edu haibo.jiang@uwa.edu.au.
Haibo
H
Jiang
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002784:Cholesterol / Q000378:metabolism*; D003412:Cricetulus; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D008562:Membrane Glycoproteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D043942:Nanotubes / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry; D011485:Protein Binding; D018629:Spectrometry, Mass, Secondary Ion; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008562:Membrane Glycoproteins; D009587:Nitrogen Isotopes; D013109:Sphingomyelins; C498000:anthrolysin O, Bacillus anthracis; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase
NanoSIMS; anthrolysin O; cholesterol; microvilli; perfringolysin O