Biotinylated theta-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells.
BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.
Sugii
Shigeki
S
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Reid
Patrick C
PC
Ohgami
Nobutaka
N
Shimada
Yukiko
Y
Maue
Robert A
RA
Ninomiya
Haruaki
H
Ohno-Iwashita
Yoshiko
Y
Chang
Ta-Yuan
TY
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D019921:Biotinylation; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003505:Cyclodextrins / Q000494:pharmacology; D005260:Female; D005372:Filipin / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006460:Hemolysin Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000378:metabolism*; D009154:Mutation; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000378:metabolism / Q000473:pathology; D010196:Pancreatic Elastase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D047392:beta-Cyclodextrins
D001427:Bacterial Toxins; D002352:Carrier Proteins; D003505:Cyclodextrins; D006460:Hemolysin Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D015335:Molecular Probes; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; D049452:Green Fluorescent Proteins; C020452:Clostridium perfringens theta-toxin; D005372:Filipin; D002784:Cholesterol; D010196:Pancreatic Elastase; C017130:cholesterol-binding protein; C031215:betadex