Cholesterol depletion mimics the effect of cytoskeletal destabilization on membrane dynamics of the serotonin1A receptor: A zFCS study.
Single-point fluorescence correlation spectroscopy (FCS) of membrane-bound molecules suffers from a number of limitations leading to inaccurate estimation of diffusion parameters. To overcome such problems and with the overall goal of addressing membrane heterogeneities, we performed z-scan FCS (zFCS) of the serotonin(1A) receptor. We analyzed the results according to FCS diffusion laws that provide information on the organization of the diffusing species. Analysis of our results shows that the diffusion coefficients of the receptor and a fluorescently labeled phospholipid are similar when probed at length scales approximately 210 nm. We discuss the significance of the spatiotemporal evolution of dynamics of membrane-bound molecules in the overall context of membrane domains and heterogeneity. Importantly, our results show that the serotonin(1A) receptor exhibits confinement in cell membranes, possibly due to interaction with the actin cytoskeleton. Surprisingly, depletion of membrane cholesterol appears to reduce receptor confinement in a manner similar to that observed in the case of cytoskeletal destabilization, implying possible changes in the actin cytoskeleton induced upon cholesterol depletion. These results constitute the first report on G-protein-coupled receptor dynamics utilizing a combination of zFCS and the FCS diffusion laws, and present a convenient approach to explore cell membrane heterogeneity at the submicron level.
Ganguly
Sourav
S
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.
Chattopadhyay
Amitabha
A
D000199:Actins / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds / Q000378:metabolism; D016466:CHO Cells; D002138:Calibration; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000172:deficiency* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004058:Diffusion; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000199:Actins; D001896:Boron Compounds; D011993:Recombinant Fusion Proteins; D044282:Receptor, Serotonin, 5-HT1A; D012701:Serotonin; D002784:Cholesterol
Mol Biol Cell. 1999 Apr;10(4):961-74
10198050
Annu Rev Biophys Biomol Struct. 1997;26:373-99
9241424
Annu Rev Biophys Biomol Struct. 2007;36:151-69
17477838
Mol Endocrinol. 2009 May;23(5):590-9
19196832
Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6775-80
10359788
Trends Cell Biol. 2001 Dec;11(12):492-6
11719055
Biochemistry. 2002 Jan 22;41(3):697-705
11790090
Nat Rev Mol Cell Biol. 2000 Oct;1(1):31-9
11413487
J Cell Biol. 2004 Jun 7;165(5):735-46
15173190
Prog Lipid Res. 2006 Jul;45(4):295-333
16616960
J Phys Chem B. 2008 Jan 10;112(1):143-9
18069820
Biophys J. 2005 Dec;89(6):4029-42
16199500
Biochemistry. 2004 Dec 21;43(50):15852-62
15595840
Anal Biochem. 1991 Nov 1;198(2):228-37
1799206
Methods. 2008 Oct;46(2):116-22
18634881
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13964-9
14612561
Biochim Biophys Acta. 2007 Mar;1768(3):655-68
17292852
EMBO J. 2006 Jul 26;25(14):3245-56
16858413
Appl Opt. 1989 Mar 15;28(6):1214-20
20548642
J Cell Biol. 2000 Mar 6;148(5):997-1008
10704449
J Cell Biol. 2002 Jun 10;157(6):1071-81
12058021
Biophys J. 2004 Feb;86(2):861-9
14747321
Biophys J. 2008 Jul;95(1):451-63
18339759
Biophys J. 2004 Aug;87(2):1034-43
15298908
Cell. 2003 Nov 14;115(4):377-88
14622593
Cell Mol Neurobiol. 2005 Jun;25(3-4):553-80
16075379
EMBO J. 2006 Aug 9;25(15):3446-57
16900097
Curr Biol. 2006 Apr 18;16(8):R272-6
16631568
Biophys J. 2005 Jan;88(1):334-47
15516525
Brain Res. 2006 Aug 9;1104(1):27-38
16828067
Drug Discov Today. 2009 Mar;14(5-6):231-40
19121411
Biophys J. 2006 Aug 1;91(3):L23-5
16751239
FEBS Lett. 1988 Jul 4;234(1):1-7
2839357
Biochim Biophys Acta. 2009 Feb;1788(2):295-302
19111523
Biochem Biophys Res Commun. 2009 Oct 2;387(4):661-5
19615977
Prog Neurobiol. 2007 Feb;81(3):133-78
17316955
Biochemistry. 1991 Feb 19;30(7):1973-80
1899622
Nature. 2009 May 21;459(7245):356-63
19458711
J Cell Biol. 1986 Jan;102(1):88-96
3001105
Annu Rev Cell Dev Biol. 2004;20:839-66
15473862
Biochim Biophys Acta. 2007 Jun;1768(6):1311-24
17493580
Biopolymers. 1974 Jan;13(1):29-61
4818131
Methods Enzymol. 2003;361:69-92
12624907
Nat Cell Biol. 2007 Jan;9(1):7-14
17199125
Trends Pharmacol Sci. 2007 Dec;28(12):637-45
18001848
Biochemistry. 2007 Sep 18;46(37):10484-97
17711354
Biophys J. 2005 May;88(5):3659-80
15681644
Biochim Biophys Acta. 2008 Jun;1778(6):1508-16
18381059
J Cell Biol. 1991 Jul;114(1):53-60
1646825
EMBO J. 2005 Dec 7;24(23):4106-14
16292347
Biophys J. 2007 Feb 1;92(3):913-9
17085499