Quantitative analysis of protein translocations by microfluidic total internal reflection fluorescence flow cytometry.
Protein translocation, or the change in a protein's location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations.
Wang
Jun
J
Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana 47907, USA.
Fei
Bei
B
Geahlen
Robert L
RL
Lu
Chang
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D016328:NF-kappa B / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*
D016328:NF-kappa B
Oncogene. 1999 Nov 22;18(49):6938-47
10602468
J Immunol. 2006 Nov 1;177(9):5829-39
17056507
J Res Natl Inst Stand Technol. 2002 Aug 01;107(4):339-53
27446735
Traffic. 2001 Nov;2(11):764-74
11733042
J Immunol. 2005 Dec 15;175(12):7800-4
16339514
Nature. 1995 Feb 16;373(6515):573-80
7531822
Anal Chem. 2008 Dec 15;80(24):9840-4
19007249
Lab Chip. 2004 Apr;4(2):125-30
15052352
Nat Rev Cancer. 2004 Feb;4(2):106-17
14732865
Science. 2002 Mar 8;295(5561):1910-2
11884760
Anal Chem. 2007 Jan 15;79(2):682-7
17222037
Pharm Res. 2007 Jan;24(1):17-27
16969692
Oncogene. 2006 Oct 30;25(51):6706-16
17072323
J Biol Chem. 2002 Oct 4;277(40):37702-10
12149258
J Immunol. 2001 Feb 1;166(3):1507-16
11160190
J Biol Chem. 1997 Apr 18;272(16):10377-81
9099676
J Immunol. 1998 Nov 15;161(10):5276-83
9820500
Nature. 2003 Mar 13;422(6928):176-80
12634788
Cell. 1999 Dec 23;99(7):677-90
10619422
Science. 2002 Nov 8;298(5596):1241-5
12424381
Anal Chem. 2007 Dec 15;79(24):9321-30
18004819
Nature. 2007 Nov 29;450(7170):663-9
18046402
Adv Cancer Res. 2005;94:29-86
16095999
Science. 2000 Apr 7;288(5463):113-6
10753110
Nature. 1993 Oct 21;365(6448):767-70
7692309
Anal Chem. 2006 Dec 1;78(23):7918-25
17186633
Science. 2001 Aug 31;293(5535):1653-7
11533489
Nat Biotechnol. 2004 Mar;22(3):306-12
14990952
Immunol Rev. 2006 Apr;210:171-86
16623771
J Biol Chem. 1986 Nov 25;261(33):15637-43
3782080
EMBO J. 1994 Mar 15;13(6):1341-9
8137818
Annu Rev Cell Dev Biol. 1999;15:607-60
10611974
Cytometry. 1998 Nov 1;33(3):376-82
9822350
Anal Chem. 2008 Feb 15;80(4):1087-93
18154306
Nat Rev Mol Cell Biol. 2001 Apr;2(4):268-75
11283724
Annu Rev Immunol. 1998;16:225-60
9597130
Science. 2001 Nov 30;294(5548):1929-32
11729319
Nature. 1997 Apr 24;386(6627):779-87
9126736
Circ Res. 2003 May 16;92(9):962-8
12750306
J Biol Chem. 1988 Dec 15;263(35):19195-202
3198621
Science. 2005 Dec 2;310(5753):1452-6
16322447
J Biol Chem. 2001 Nov 30;276(48):45225-35
11571291
J Biol Chem. 2006 Apr 28;281(17):11712-20
16522635
New J Phys. 2009;11(31):75017
19809528
Biochem J. 2000 Aug 15;350 Pt 1:1-18
10926821
Nat Biotechnol. 2005 Oct;23(10):1308-14
16155565
Science. 1998 May 8;280(5365):895-8
9572732
Nature. 1995 Nov 16;378(6554):298-302
7477352
Science. 2003 Apr 4;300(5616):82-6
12677057
Nat Rev Immunol. 2007 Nov;7(11):897-904
17917673
Curr Opin Cell Biol. 2002 Apr;14(2):140-8
11891111
Science. 2004 Oct 22;306(5696):704-8
15499023
Lab Chip. 2006 Nov;6(11):1445-9
17066168