BiP availability distinguishes states of homeostasis and stress in the endoplasmic reticulum of living cells.
Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.
Lai
Chun Wei
CW
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Aronson
Deborah E
DE
Snapp
Erik Lee
EL
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000473:pathology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013312:Stress, Physiological; D014162:Transfection; D014783:Viscosity
D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
J Biol Chem. 2005 Nov 25;280(47):39002-15
16183649
Nat Cell Biol. 2000 May;2(5):288-95
10806480
Semin Cell Dev Biol. 1999 Oct;10(5):465-72
10597629
J Cell Biol. 2005 Jan 31;168(3):389-99
15684029
Trends Cell Biol. 2004 Jan;14(1):20-8
14729177
Eur J Biochem. 2004 Aug;271(15):3200-7
15265039
J Biol Chem. 1993 Sep 25;268(27):20598-605
8397210
Nature. 2003 Oct 16;425(6959):686-91
14562095
Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1164-9
18203820
Nat Cell Biol. 2005 Aug;7(8):766-72
16056268
Cell. 2000 Apr 28;101(3):249-58
10847680
J Cell Biol. 1993 Feb;120(3):647-55
8381122
J Biol Chem. 2002 May 10;277(19):16419-25
11877404
J Cell Biol. 1991 Jul;114(2):189-205
1649196
J Cell Biol. 1997 Sep 22;138(6):1193-206
9298976
J Cell Biol. 2002 Aug 5;158(3):497-506
12163472
Science. 2000 Apr 14;288(5464):331-3
10764645
J Cell Biol. 1990 Sep;111(3):857-66
1697299
J Cell Biol. 2007 Oct 8;179(1):75-86
17923530
J Chem Neuroanat. 2004 Sep;28(1-2):51-65
15363491
Exp Cell Res. 2009 Sep 10;315(15):2496-504
19538957
Cell. 2006 Dec 15;127(6):1265-81
17174899
Trends Biochem Sci. 1995 Nov;20(11):448-55
8578587
Mt Sinai J Med. 2004 Oct;71(5):289-97
15543429
Proc Natl Acad Sci U S A. 2009 Jul 14;106(28):11600-5
19553200
Annu Rev Cell Dev Biol. 2006;22:487-508
16822172
Mol Cell Biol. 2003 Nov;23(21):7448-59
14559994
J Microsc. 1995 Nov;180(Pt 2):109-16
8537958
Dev Cell. 2007 Sep;13(3):351-64
17765679
EMBO J. 1996 Dec 16;15(24):6921-30
9003768
Mol Biol Cell. 2005 Aug;16(8):3740-52
15958486
Cell. 2006 Dec 1;127(5):999-1013
17129784
Trends Biochem Sci. 2007 Oct;32(10):469-76
17920280
J Cell Biol. 1992 Aug;118(3):541-9
1353499
Nat Rev Mol Cell Biol. 2008 Dec;9(12):944-57
19002207
FEBS J. 2007 Oct;274(19):5175-87
17850331
Science. 2006 Mar 10;311(5766):1471-4
16469881
Trends Biochem Sci. 2001 Oct;26(10):597-604
11590012
Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14343-8
16973740
Proc Natl Acad Sci U S A. 2005 Dec 27;102(52):18773-84
16365312
Cell. 2008 Nov 28;135(5):933-47
19026441
Nat Cell Biol. 2000 Jun;2(6):326-32
10854322
J Biol Chem. 1990 Apr 25;265(12):6879-83
2157712
J Cell Biol. 2006 Feb 13;172(4):565-75
16461360
J Biol Chem. 1999 Feb 5;274(6):3453-60
9920890
EMBO J. 2009 Sep 2;28(17):2554-67
19680227
J Cell Biol. 1990 Sep;111(3):829-37
2118144
Dev Cell. 2002 Jul;3(1):99-111
12110171
Int Rev Cytol. 2004;237:205-77
15380669
Biophys J. 2000 Oct;79(4):1761-70
11023884
J Biol Chem. 1995 Mar 3;270(9):4697-704
7876241
EMBO J. 1992 Apr;11(4):1573-81
1563355
Genes Dev. 2004 Dec 15;18(24):3066-77
15601821
Immunity. 2001 Jul;15(1):105-14
11485742
Biophys J. 1999 May;76(5):2843-51
10233100
Experientia. 1994 Nov 30;50(11-12):1012-20
7988659
Mol Biol Cell. 2004 Oct;15(10):4512-21
15292454
Curr Opin Cell Biol. 2004 Aug;16(4):343-9
15261665
PLoS Biol. 2006 Nov;4(11):e374
17090218
EMBO J. 1993 May;12(5):2151-7
8491203
Nat Biotechnol. 2004 Dec;22(12):1567-72
15558047
Biophys J. 2005 Sep;89(3):1482-92
15951382
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9643-6
7568189
Cell. 1997 Jun 13;89(6):963-71
9200614
J Cell Biol. 2008 Jan 14;180(1):129-43
18195104
Mol Biol Cell. 2003 Aug;14(8):3437-48
12925775
J Cell Biol. 2004 Nov 8;167(3):445-56
15520230
J Cell Biol. 2003 Oct 27;163(2):257-69
14581454
Nat Cell Biol. 2008 Mar;10(3):272-82
18264092
Cell. 2010 Jan 8;140(1):13-8
20085698
Proc Natl Acad Sci U S A. 1996 May 28;93(11):5269-74
8643565
Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6536-41
16617114
J Biol Chem. 1998 Dec 11;273(50):33741-9
9837962
J Cell Sci. 2004 Jun 15;117(Pt 14):2937-49
15161941
J Biol Chem. 1997 Jan 31;272(5):3057-63
9006956
J Biol Chem. 2000 Jun 9;275(23):17556-60
10748019
J Cell Biol. 2009 Nov 16;187(4):525-36
19948500
Mol Biol Cell. 2002 Dec;13(12):4456-69
12475965
Nat Rev Mol Cell Biol. 2007 Jul;8(7):519-29
17565364
Crit Rev Biochem Mol Biol. 2005 Jul-Aug;40(4):191-228
16126486
J Cell Biol. 1986 May;102(5):1558-66
3084497
Biochemistry. 1996 Mar 12;35(10):3297-308
8605167
Trends Cell Biol. 2001 May;11(5):203-11
11316609
Nat Rev Mol Cell Biol. 2001 Jun;2(6):444-56
11389468
Mol Cell. 2003 Jan;11(1):79-90
12535523
Curr Protoc Cell Biol. 2003 Aug;Chapter 21:Unit 21.1
18228432