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

Immunofluorescentie analyse van endogene en exogene Centromere-kinetochoor Eiwitten

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

"Centromeren" werden klassiek gedefinieerd als gebieden onderdrukt meiotische recombinatie in de genetica en later gezien als het belangrijkste vernauwing van mitotische chromosomen, die een essentiële rol speelt nauwkeurige chromosoomsegregatie in mitose. "Kinetochoren" werden beschreven als het meerlagige structuren die binden aan microtubuli aan het oppervlak centromeren, zoals aangetoond door elektronenmicroscopie; "Kinetochoren" werden later gedefinieerd als macromoleculaire complex localiseert in het centromeer van mitotische chromosomen. Ondanks een dramatisch verschil in centromeer DNA-sequenties bij vertebraten, kinetochoor structuur en samenstelling sterk geconserveerd. Een dynamische interactie tussen spindel microtubuli en het kinetochoor vereist voor trouwe segregatie van chromosomen tijdens de mitose en defecten in centromeer-kinetochoor functie leidt tot aneuploïdie en daardoor kanker.

Het centromeer in de meeste eukaryotengeen gedefinieerde DNA-sequentie, maar is samengesteld uit grote arrays (0,3-5 Mb) repeterende alphoid DNA bestaat uit 171 bp α-satelliet DNA. Behalve in gist, wordt centromeer identiteit bereikt niet de DNA-sequentie, maar door de aanwezigheid van een speciale nucleosomen die de histon H3 variant CenH3 bevat (centromeer eiwit A [CENP-A] bij de mens). 5 CENP-A nucleosomen lokaliseren de binnenste plaat van zoogdier kinetochoren 7 en binden aan de 171-bp α-satelliet DNA. Actieve centromeres vereisen CENP-A-bevattende nucleosomen voor de aanwerving van een constitutieve-centromeer geassocieerd netwerk (CKan) en de kinetochoor eiwitten te leiden, die samen reguleren van de aanhechting van de chromosomen van de mitotische spindel en de daarop volgende cyclus progressie door de spil checkpoint.

In het licht van het bovenstaande bewijs heeft CENP-A voorgesteld om de epigenetische merkteken van het centromeer 8 zijn; echter, het proces waardoor CENP-A opgenomen into centromeer DNA en de factoren die tot deze opname nog niet goed gekarakteriseerd. Een korte-centromeer doeldomein (CATD) ligt in het histon voudige regio CENP-A, en vervanging van het overeenkomstige gebied van H3 de CATD zorgdragen om H3 het centromeer. 9 Verschillende studies suggereerde functionele rol voor post-translationele modificatie (PTM) van CENP-A 12-16; De moleculaire mechanismen van deze PTMs van CENP-A in de rekrutering van centromeren nog niet opgehelderd. We hebben eerder gemeld dat CUL4A-RBX1-COPS8 E3 ligase activiteit is vereist voor CENP-A K124 ubiquitinering en lokalisatie van CENP-A naar centromeres. 17

De ontdekking en karakterisering van kinetochore eiwitten hebben geleid tot nieuwe inzichten over chromosoomscheiding. 18 meer dan 100 kinetochoor componenten zijn in gewervelde cellen die door verschillende benaderingen. 19,20 Een onderstaande hoe kinetochoren assembleren en functie komt ook uit het karakteriseren van de cellulaire functies van elke centromeer-kinetochoor eiwitten en eiwit-eiwit netwerk in cellen. 19 Directe visualisatie en geavanceerde imaging werkwijzen fluorescentiemicroscopie bieden opmerkelijke resolutie van centromeer-kinetochore componenten en laat directe observatie van de specifieke moleculaire componenten van de centromeren en kinetochoren. Bovendien, de methoden van indirecte immunofluorescentie (IIF) kleuring met behulp van specifieke antilichamen zijn cruciaal voor deze waarnemingen. Ondanks talrijke rapporten over IIF protocollen weinig besproken problemen specifieke centromeer-kinetochore eiwitten. 1-4 Zo ontwikkelen en rapporteringsmethoden van IIF kleuring en een kwantitatieve bepaling om IIF specifiek wat elke centromeer-kinetochoor eiwit is zeer belangrijk. In IIF kleuring, moet men overgaan tot de kleuringsprotocol verlies van het eiwit van belang voorkomen ofde rest van de cel. Echter, fixatie vernietigt antigene plaatsen af en verschillende antilichaam-antigeen combinaties werken slecht met een fixatief, maar zeer goed met elkaar, en 21 keuze fixeermiddel hangt grotendeels af van de eiwit (ten) van belang. Daarom verschillende fixerende methoden zijn van cruciaal belang in IIF kleuring van centromeer-kinetochoor eiwitten.

Hier geoptimaliseerde werkwijzen voor indirecte immunofluorescentie (IIF) kleuring en een test om lokalisatie van endogeen centromeer-kinetochore eiwitten, waaronder CENP-A en Flag-tagged exogene CENP-A eiwitten en kwantificering van deze eiwitten in menselijke cellen aanpakken ontwikkeld. Deze methoden kunnen worden toegepast op de analyse van centromeer-kinetochore eiwitten in andere species.

Protocol

1. Cel Cultuur en Transfectie Zet een deksel glas (22 mm x 22 mm) in een 6-well polystyreen plaat. Eventueel jas een cover glas met Poly-L-lysine, 0,1% w / v, in water (zie lijst van materialen / Equipment) tot mitotische cellen op de cover glas van de volgende stappen te houden: Let op: Optimale voorwaarden moeten worden bepaald voor elke cellijn en toepassing. Aseptisch laag kweekoppervlak met Poly-L-lysine, 0,1% w / v in water (0,4 ml / putje van een 6-well polystyreen platen). Rock zachtje…

Representative Results

Immunofluorescentie analyse van endogene CENP-A ondersteunt de hypothese dat CUL4A-E3 ligase nodig voor het lokaliseren van CENP-A centromeren Onze recente studies is gebleken dat CUL4A-RBX1-COPS8 E3 ligase activiteit is vereist voor ubiquitinering van lysine 124 (K124) op CENP-A en lokalisatie van CENP-A centromeren. 17 Aanvankelijk interfase-centromeer complex (Icen) werd geïsoleerd door anti-CENP-a: natieve chromatine immunoprecipitatie, 32-34<…

Discussion

De laatste jaren zijn veel studies verschillende kwantitatieve microscopie assays ontwikkeld voor gefixeerde cellen. 42 Progress in centromeer-kinetochoor biologie vereist vaak een begrip van de centromeer-specifieke of kinetochoor specifieke functie van eiwitten waarvan subcellulaire ruimtelijk-temporele regulering weerspiegelt de veranderende functies deze eiwitten tijdens celcyclus. Daarom hier ontwikkelden wij werkwijzen IIF kleuring en een kwantitatieve bepaling om IIF specifiek wat de relatieve niveaus …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dit werk werd ondersteund door NIH-subsidie ​​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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Tags

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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