Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells.
Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae.
Wüstner
Daniel
D
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark. wuestner@bmb.sdu.dk
Faergeman
Nils J
NJ
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D021941:Caveolae / Q000378:metabolism; D022461:Caveolins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002782:Cholestenes / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D022461:Caveolins; D002782:Cholestenes; D011993:Recombinant Fusion Proteins; C010905:dehydroergosterol; C025498:cholesta-5,7,9-trien-3 beta-ol; D004875:Ergosterol
Biochim Biophys Acta. 1989 Jul 24;983(1):109-12
2758045
Biophys J. 2005 May;88(5):3625-34
15722423
J Clin Invest. 2002 Oct;110(7):891-8
12370264
J Biol Chem. 2004 Oct 22;279(43):45226-34
15316012
IEEE Trans Image Process. 2005 Sep;14(9):1372-83
16190472
J Biol Chem. 1996 Nov 15;271(46):29427-35
8910609
J Lipid Res. 2008 Sep;49(9):2023-37
18523240
Mol Biol Cell. 2002 Jul;13(7):2421-35
12134080
Cell. 1984 Jul;37(3):789-800
6204769
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10339-43
7479780
J Struct Biol. 1991 Aug;107(1):6-14
1817611
Mol Biol Cell. 1995 Jul;6(7):929-44
7579703
J Cell Biol. 1998 Jan 12;140(1):39-47
9425152
Biochim Biophys Acta. 2007 Sep;1768(9):2182-94
17588529
Annu Rev Biophys Biomol Struct. 1997;26:373-99
9241424
J Biol Chem. 2002 Jan 4;277(1):609-17
11682487
J Cell Biol. 1985 Aug;101(2):446-53
4040520
Biochem J. 2004 Sep 1;382(Pt 2):451-61
15149285
J Pharmacol Exp Ther. 2007 May;321(2):423-30
17142645
Nature. 1983 Jun 16-22;303(5918):637-8
6855909
Mol Biol Cell. 2004 Aug;15(8):3542-52
15146059
J Microsc. 2005 Oct;220(Pt 1):47-64
16269063
J Cell Biol. 1984 Jul;99(1 Pt 1):315-9
6145719
Biophys J. 1999 Apr;76(4):2262-71
10096921
Traffic. 2000 Mar;1(3):212-7
11208104
Traffic. 2003 Nov;4(11):724-38
14617356
J Biol Chem. 2000 Aug 18;275(33):25595-9
10833523
J Cell Biol. 1983 May;96(5):1273-8
6132922
Biophys J. 1999 Nov;77(5):2887-95
10545386
Traffic. 2005 May;6(5):396-412
15813750
J Cell Biol. 2001 Oct 15;155(2):291-300
11604424
Nature. 1979 Jul 26;280(5720):328-9
460406
J Cell Sci. 1981 Oct;51:95-107
7320089
Biophys J. 2003 Jan;84(1):356-66
12524289
Eur J Biochem. 2004 May;271(10):2028-36
15128312
Nat Cell Biol. 2001 Jun;3(6):567-72
11389441
J Biol Chem. 2002 Nov 29;277(48):46769-78
12356772
J Cell Sci. 1994 Aug;107 ( Pt 8):2177-89
7983176
J Biol Chem. 2002 Aug 16;277(33):30325-36
12050151
Biochemistry. 2005 Apr 19;44(15):5816-26
15823040
Cell Metab. 2008 Jun;7(6):508-19
18522832
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6391-5
293728
Biochemistry. 2005 Mar 1;44(8):2864-84
15723530
Biochemistry. 2006 Oct 3;45(39):12100-16
17002310
J Cell Biol. 1989 Nov;109(5):2097-104
2681226
Annu Rev Cell Dev Biol. 2004;20:839-66
15473862
Physiol Rev. 1997 Jul;77(3):759-803
9234965
Chem Phys Lipids. 2007 Mar;146(1):1-25
17241621
Cytometry A. 2008 Aug;73(8):727-44
18561197
Cell Motil Cytoskeleton. 2002 Aug;52(4):221-30
12112136
Mol Biol Cell. 2002 Jan;13(1):238-50
11809836
Biophys J. 2004 Nov;87(5):3518-24
15339818
Biochim Biophys Acta. 1990 Mar 30;1023(1):25-33
2317494
Nature. 1997 Jun 5;387(6633):569-72
9177342
J Biol Chem. 2003 Nov 14;278(46):45563-9
12947110
Biophys J. 2002 Nov;83(5):2693-701
12414701
Mol Biol Cell. 2007 Jan;18(1):211-28
17065557
Mol Biol Cell. 2004 May;15(5):2253-63
15004223