JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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생물학

Immunofluorescens Analys av endogena och exogena centromer-kinetochore proteiner

Published: March 03, 2016
doi:
10.3791/53732
,
1Center for Childhood Cancer and Blood Diseases,Nationwide Children’s Hospital

Summary

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Abstract

“Centromeres” and “kinetochores” refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Introduction

"centromerer" var klassiskt definieras som områden av undertryckt meiotiska rekombination i genetik och senare erkänd som den primära sammandragning av mitotiska kromosomer, som spelar en viktig roll i exakt kromosomsegregation under mitos. "kinetokorer" beskrevs som de flerskiktade strukturer som binder till mikrotubuli vid ytan av centromerer, såsom avslöjas genom elektronmikroskopi; "kinetokorer" senare definieras som makromolekylära komplex som lokaliserar vid centromeren mitotiska kromosomer. Trots en dramatisk avvikelse av centro DNA-sekvenser bland ryggradsdjur, kinetochor struktur och sammansättning är mycket konserverad. En dynamisk interaktion mellan spindel mikrotubuli och kinetochor krävs för trogen segregering av kromosomer under mitos och defekter i centromer-kinetochor funktion leder till aneuploidi och därmed cancer.

Centromeren i de flesta eukaryoterhar ingen definierad DNA-sekvens, men består av stora matriser (0,3-5 MB) repetitiva alphoid DNA bestående av 171 bp α-satellit-DNA. Utom i knoppande jäst, är centromeridentitet uppnås inte av DNA-sekvensen men genom närvaro av en speciell nukleosomen som innehåller histon H3 varianten CenH3 (centromer Protein A [CENP-A] hos människa). 5 CENP-A nukleosomer lokalisera till inre platta av däggdjurs kinetokorer 7 och binder till 171 bp α-satellit-DNA. Aktiva centromerer kräva CENP-A-innehållande nukleosomer för att rikta rekryteringen av en konstitutiv centromer-associerat nätverk (Ccan) och kinetochor proteiner, som tillsammans reglera vidhäftningen av kromosomer till den mitotiska spolen och efterföljande cykelprogression genom spindeln checkpoint.

Mot bakgrund av ovanstående bevis har CENP-A har föreslagits vara den epigenetiska märke centromeren 8; emellertid den process genom vilken CENP-A är inkorporerad into centro DNA och de faktorer som ligger bakom denna integration har ännu inte väl karakteriserade. En kort centromer-targeting domain (CATD) befinner sig i histon faldig regionen av CENP-A, och ersättning av motsvarande region i H3 med CATD är tillräcklig för att direkt H3 till centromeren. 9 Flera studier föreslog funktionella roller för posttranslationell modifiering (PTM) av CENP-A 16/12; Men, de molekylära mekanismerna bakom dessa PTMs av CENP-A i rekryteringen till centromerer har ännu inte klarlagts. Vi rapporterade tidigare att CUL4A-RBX1-COPS8 E3-ligas-aktivitet krävs för CENP-A K124 ubiquitylation och lokalisering av CENP-A till centromerer. 17

Upptäckten och karakteriseringen av kinetokoren proteiner har lett till nya insikter om kromosomsegregation. 18 Mer än 100 kinetokoren komponenter har identifierats i celler från ryggradsdjur av olika metoder. 19,20 En understående av hur kinetokorer monterar och funktion kommer också från karakterisering av de cellulära funktionerna hos varje centromer-kinetochore proteiner och protein-protein nätverk inom celler. 19 Direkt visualisering och avancerad avbildningsmetoder i fluorescensmikroskopi ger anmärkningsvärd upplösning av centromer-kinetochore komponenter och tillåta direkt observation av specifika molekylära komponenter i centromerer och kinetokorer. Dessutom, metoder för indirekt immunofluorescerande (IIF) färgning med användning av specifika antikroppar är avgörande för dessa observationer. Men trots många rapporter om IIF protokoll, få diskuteras i detalj problem med specifika centromer-kinetochore proteiner. 1-4 är således mycket viktigt att utveckla och rapportering metoder för IIF-färgning och en kvantitativ IIF analys för att specifikt analysera varje centromer-kinetochore protein. I IIF-färgning, bör man fortsätta med färgningsprotokollet för att undvika förlust av proteinet av intresse ellerresten av cellen. Emellertid förstör fixering antigena ställen ibland, och olika antikropp-antigen kombinationer fungerar dåligt med ett fixativ, men mycket väl med en annan, 21 och val av fixativ beror till stor del på protein (er) av intresse. Därför olika fästmetoder är avgörande för IIF-färgning av centromer-kinetochore proteiner.

Här optimerade metoder för indirekt immunofluorescerande (IIF) färgning och en analys för att ta itu med lokalisering av endogena centromer-kinetochore proteiner, innefattande CENP-A och Flag-märkta exogena CENP-A-proteiner, och kvantifiering av dessa proteiner i humana celler har utvecklats. Dessa metoder kan tillämpas på analysen av centromer-kinetochore proteiner i andra arter.

Protocol

1. Cellodling och transfektion Sätta ett täckglas (22 mm x 22 mm) i en 6-brunnars polystyrenplatta. Eventuellt belägga en täckglaset med Poly-L-lysin, 0,1% vikt / volym, i vatten (se lista över material / utrustning) för att hålla mitotiska celler på täckglaset att följa stegen nedan: Notera: Optimala förhållanden måste bestämmas för varje cellinje och tillämpning. Aseptiskt coat odlingsytan med Poly-L-lysin, 0,1% vikt / volym, i vatten (0,4 ml / brunn i en 6-brunnars polystyre…

Representative Results

Immunofluorescensanalys av endogen CENP-A stöder hypotesen att CUL4A-E3-ligas krävs för lokalisering av CENP-A till centromerer Våra nyligen gjorda studier visade att CUL4A-RBX1-COPS8 E3-ligas-aktivitet krävs för ubiquitylation av lysin 124 (K124) på CENP-A och lokalisering av CENP-A till centromerer. 17 Inledningsvis var interfas-centromer-komplex (ICEN) isoleras genom anti-CENP-A: nativ kromatin immunoprecipitation, 32-34 och det har…

Discussion

Under senare år har många studier har utvecklat olika kvantitativ mikroskopi analyser för fixerade celler. 42 Framsteg i centromer-kinetochor biologi ofta kräver en förståelse av centromer specifika eller kinetokoren-specifik funktion av proteiner som subcellulär spatial-temporal reglering återspeglar de förändrade funktioner dessa proteiner under cellcykeln. Därför, här har vi utvecklat metoder för IIF-färgning och en kvantitativ IIF analys för att särskilt analysera de relativa nivåerna av…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Detta arbete stöddes av NIH bidrag GM68418.

Materials

Lipofectamin 2000 Life Technologies/Invitrogen 11668 transfection reagent I
Lipofectamin RNAiMAX Life Technologies/Invitrogen 13778 transfection reagent II
Opti-MEM I Life Technologies/Invitrogen 31985 Reduced serum media, warm in 37 °C water bath before use
High-glucose DMEM (Dulbecco’s modified Eagle’s medium) Life Technologies/BioWhittaker 12-604 high-glucose DMEM, warm in 37 °C water bath before use
Fetal Bovine Serum, certified, heat inactivated, US origin Life Technologies/Gibco 10082 FBS (fetal bovine serum)
Poly-L-Lysine SOLUTION SIGMA-SLDRICH P 8920 Poly-L-Lysine, 0.1% w/v, in water
UltraPure Distilled Water Life Technologies/Invitrogen/Gibco 10977 Sterile tissue culture grade water 
Micro Cover glass (22 mm x 22 mm)  Surgipath 105 Cover glass (22 mm x 22 mm) 
6 Well Cell Culture Cluster Fisher/Corning Incorporated 07-200-83 6-well polystyrene plate 
Penicillin, Streptomycin; Liquid Fisher/Gibco 15-140 Penicillin-streptomycin
PAP PEN  Binding Site AD100.1 Hydrophobic barrier pen (for a water repellant barrier in immunofluorescent staining)
Paclitaxel (Taxol) SIGMA-SLDRICH T7402 Taxol for mitotic cell analysis
TN-16, microtubule inhibitor (TN16) Enzo Life Sciences BML-T120 TN16 for mitotic cell analysis
BSA (bovine serum albumin) SIGMA-SLDRICH A7906 Blocking reagent
Triton X-100 SIGMA-SLDRICH T8787 Detergent for permeabilization
Paraformaldehyde SIGMA-SLDRICH P6148 Fixation reagant
DAPI SIGMA-SLDRICH D9542 For nuclear staining
p-phenylenediamine SIGMA-SLDRICH P6001 For mounting medium
VWR Micro Slides, Frosted VWR International 48312-013 Micro slides 
Anti-CENP-A antibody Stressgen/Enzo Life Sciences KAM-CC006 Mouse monoclonal antibody; dilution ratio of 1:100 (IIF), 1:5000 (WB)
Anti-CENP-B antibody Novus Biologicals H00001059-B01P Mouse monoclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)
Anti-CENP-B antibody  abcam ab25734 Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)
Anti-centromere antibody (ACA) Fitzgerald Industries International, Inc. 90C-CS1058 Human centromere antiserum; dilution ratio of 1:2000 (IIF)
Anti-CENP-H antibody Bethyl Laboratories BL1112 (A400-007A) Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF)
Anti-CENP-H antibody BD 612142 Mouse monoclonal antibody; dilution ratio of 1:200 (IIF)
Anti-CENP-I antibody N/A, Dr. Katusmi Kitagawa N/A, Dr. Katusmi Kitagawa Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF); Niikura et al., Oncogene, 4133-4146 (2006)
Anti-KNL1 antibody Novus Biologicals NBP1-89223 Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Hec1 antibody Novus Biologicals / GeneTex NB 100-338 / GTX70268 Mouse monoclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Hec1 antibody GeneTex GTX110735 Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Ska1 antibody abcam ab46826 Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Flag antibody SIGMA-ALDRICH F3165 Mouse monoclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)
Anti-Flag antibody SIGMA-ALDRICH F7425 Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)
Anti-CUL4A antibody N/A, Dr. Pradip Raychaudhuri N/A, Dr. Pradip Raychaudhuri Rabbit polyclonal antibody; dilution ratio of 1:3000 (WB); Shiyanov et al., The Journal of biological chemistry, 35309-35312 (1999)
Anti-RBX1 antibody Cell Signaling 4397 Rabbit polyclonal antibody; dilution ratio of 1:2000 (WB)
Anti-GAPDH antibody Chemicon MAB374 Mouse monoclonal antibody; dilution ratio of 1:5000 (WB)
Alexa Fluor 488 Goat Anti-Mouse IgG Life Technologies/Invitrogen A11001 fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 594 Goat Anti-Mouse IgG Life Technologies/Invitrogen A11005 fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 488 Goat Anti-Rabbit IgG Life Technologies/Invitrogen A11008 fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 594 Goat Anti-Rabbit IgG Life Technologies/Invitrogen A11012 fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Non fat powdered milk (approved substitution for carnation powdered milk) Fisher Scientific NC9255871 (Reorder No. 190915; Lot# 90629) Skim milk
Leica DM IRE2 motorized fluorescence microscope  Leica motorized fluorescence microscope 
HCX PL APO 63x oil immersion lens Leica LEICA HCX PL APO NA 1.40 OIL PH 3 CS 63X oil immersion lens
HCX PL APO 100x oil immersion lens Leica LEICA HCX PL APO NA 1.40 OIL PHE 100X oil immersion lens
Leica EL6000 compact light source Leica External compact light source for fluorescent excitation
ORCA-R2 Digital CCD camera  Hamamatsu C10600-10B digital CCD camera 
Openlab version 5.5.2 Scientific Imaging Software  Perkin Elmer/Improvision For image observation, acquisition, quantification, and analysis
Velocity version 6.1.1 3D Image Analysis Software  Perkin Elmer/Improvision For image observation, acquisition, quantification, and analysis
Complete EDTA-free protease inhibitor cocktail Roche 11873580001/11836170001 Protease inhibitor cocktail tablets
PlusOne 2-D Quant Kit Amersham Biosciences 80-6483-56 Commercial protein assay reagent I for measurement of protein concentration (compatible with 2% SDS)
Bio-Rad Protein Assay Bio-Rad 500-0006 Commercial protein assay reagent II for measurement of protein concentration (compatible with 0.1% SDS)
Immobilon-FL EMD Millipore IPFL00010 PVDF membrane for transferring
IRDye 800CW Goat Anti-Mouse IgG LI-COR Biosciences 926-32210 IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
IRDye 680 Goat Anti-Rabbit IgG LI-COR Biosciences 926-32221 IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-Mouse IgG DyLight 549 Fisher Scientific PI35507 DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-Rabbit DyLight 649 Fisher Scientific PI35565 DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-mouse IgG-HRP Santa Cruz SC-2005 HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)
Goat anti-rabbit IgG-HRP Santa Cruz SC-2004 HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)
Openlab version 5.5.2. Scientific Imaging Software  Improvision/PerkinElmer Software A
Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition) PerkinElmer Software B1
Volocity version 6.3 3D Image Analysis Software (Volocity Quantification) PerkinElmer Software B2
Branson SONIFIER 450 Sonicator
Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm VWR Scientific Products Inc.  33995-325 Disruptor horn for sonication
Branson Ultrasonics sonicator Microtip Tapered 6.5 mm VWR Scientific Products Inc.  33996-185 Microtip for sonication
Odyssey CLx Infrared imaging System  LI-COR Biosciences Infrared imaging system for immunoblot detection
Image Studio Analysis Software Ver 4.0  LI-COR Biosciences Software C
Molecular Imager Versadoc MP4000 System  Bio-Rad Chemiluminescence imager for immunoblot detection
Quantity One 1-D analysis software  Bio-Rad Software D
SuperSignal West Femto Maximum Sensitivity Substrate Thermo 34095 Ultra-sensitive enhanced chemiluminescent (ECL) substrate
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ImmunofluorescenceEndogenousExogenousCentromere-kinetochore ProteinsCENP-ACell CultureTransfectionFixationPermeabilizationAntibody LabelingMicroscopy
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Niikura, Y., Kitagawa, K. Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins. J. Vis. Exp. (109), e53732, doi:10.3791/53732 (2016).
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