JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Biology

Immunofluorescensanalyse av Endogene og eksogene cent-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

"sentromerer" ble klassisk definert som områder av undertrykt meiotisk rekombinasjon i genetikk og senere anerkjent som den primære innsnevring av mitotiske kromosomer, som spiller en viktig rolle i nøyaktig kromosom segregering under mitose. "kinetochores" ble beskrevet som den multilagsstrukturer som binder seg til mikrotubuli ved overflaten av sentromerer, som vist ved elektronmikroskopi; "kinetochores" ble senere definert som macromolecular kompleks som lokaliserer på cent av mitotiske kromosomer. Til tross for en dramatisk divergens av centromeric DNA-sekvenser blant virveldyr, kinetochore struktur og sammensetning er sterkt konservert. En dynamisk samspill mellom spindel mikrotubuli og kinetochore er nødvendig for trofast segregering av kromosomer under mitose, og defekter i cent-kinetochore funksjon fører til Aneuploidy og dermed kreft.

Cent i de fleste eukaryoterhar ingen definert DNA-sekvens, men er sammensatt av store matriser (0.3-5 MB) med repeterende alphoid DNA bestående av 171-bp α-satellitt-DNA. Bortsett fra i spirende gjær, er cent identitet oppnås ikke av DNA-sekvensen, men ved tilstedeværelsen av en spesiell nucleosome som inneholder histon H3 varianten CenH3 (cent Protein A [CENP-A] i mennesker). 5 CENP-A nukleosomer lokalisere til indre plate av pattedyr kinetochores 7 og binde seg til 171-bp α-satellitt DNA. Aktive sentromerer krever CENP-A-holdig nukleosomer til direkte rekruttering av et konstituerende cent-forbundet nettverk (ccan) og kinetochore proteiner, som sammen regulere feste av kromosomene til mitotisk spindel og påfølgende syklus progresjon gjennom spindel sjekkpunkt.

I lys av det ovenstående bevis har CENP-A blitt foreslått å være den epigenetisk merket på cent 8; imidlertid, den prosessen som CENP-A er inkorporert into centromeric DNA og de faktorer som er ansvarlige for dette inkorporering ennå ikke er godt karakterisert. En kort cent-målsøkende domene (CATD) befinner seg i den histon fold region av CENP-A, og erstatning av det tilsvarende område av H3 med CATD er tilstrekkelig til direkte H3 til cent. 9. Flere studier antydet funksjonelle roller for post-translasjonell modifikasjon (PTM) av CENP-A 12-16; Men de molekylære mekanismene for disse PTMs av CENP-A i rekrutteringen til sentromerer ennå ikke er klarlagt. Vi har tidligere rapportert at CUL4A-RBX1-COPS8 E3 ligase aktivitet er nødvendig for CENP-A K124 ubiquitylation og lokalisering av CENP-A til sentromerer. 17

Oppdagelsen og karakterisering av kinetochore proteiner har ført til ny innsikt om kromosom segregering. 18 Mer enn 100 kinetochore komponentene har blitt identifisert i virveldyrceller ved ulike tilnærminger. 19,20 En understående av hvordan kinetochores montere og funksjon kommer også fra karakteriseringen av de cellulære funksjoner av hver cent-kinetochore proteiner og protein-protein-nettverket i cellene. 19 direkte visualisering og avansert avbildningsmetoder i fluorescens mikroskopi gir bemerkelsesverdig oppløsning av cent-kinetochore komponenter og tillate direkte observasjon av spesifikke molekylære komponenter av sentromerer og kinetochores. I tillegg er fremgangsmåter for indirekte immunfluorescens (IIF) farging ved hjelp av spesifikke antistoffer er avgjørende for disse observasjonene. Men til tross for mange rapporter om IIF protokoller, få diskutert i detalj problemer med spesifikke cent-kinetochore proteiner. 1-4 Dermed utvikle og rapportering metoder for IIF flekker og en kvantitativ IIF analyse for å spesifikt analysere hver cent-kinetochore protein er ekstremt viktig. I IIF farging, bør man fortsette med fargingen protokollen for å unngå tap av proteinet av interesse ellerresten av cellen. Imidlertid ødelegger fiksering antigene seter fra tid til annen, og forskjellige antistoff-antigen kombinasjoner fungerer dårlig med en fikseringsmiddel, men svært godt med hverandre, 21 og valg av bindemiddel avhenger i stor grad av protein (er) av interesse. Derfor ulike festing metoder er avgjørende i IIF farging av cent-kinetochore proteiner.

Her optimaliserte fremgangsmåter for indirekte immunfluorescens (IIF) farging, og en analyse for å adressere lokalisering av endogene cent-kinetochore proteiner, inkludert CENP-A og Flagg-merkede eksogene CENP-A-proteiner, og kvantifisering av disse proteiner i humane celler har blitt utviklet. Disse metodene kan brukes til analyse av cent-kinetochore proteiner i andre arter.

Protocol

1. Cell Culture og Transfeksjon Sette et dekkglass (22 mm x 22 mm) i en 6-brønns polystyren plate. Eventuelt frakk et dekkglass med Poly-L-lysin, 0,1% vekt / volum, i vann (se liste over materialer / utstyr) for å holde mitotiske celler på dekkglass å følge trinnene nedenfor: Merk: Optimale betingelser må bestemmes for hver cellelinje og anvendelse. Aseptisk belegge kulturoverflaten med poly-L-lysin, 0,1% vekt / volum, i vann (0,4 ml / brønn av en 6-brønns polystyren plate). Rist for ?…

Representative Results

Immunofluorescensanalyse av endogent CENP-A støtter den hypotese at CUL4A-E3-ligase er nødvendig for lokalisering av CENP-A til sentromerer Våre nyere studier viste at CUL4A-RBX1-COPS8 E3 ligase aktivitet er nødvendig for ubiquitylation av lysin 124 (K124) på CENP-A og lokalisering av CENP-A til sentromerer. 17 I utgangspunktet var det inter-cent kompleks (ICEN) isolert av anti-CENP-A: innfødt kromatin immunopresipitering, 32-34 og det …

Discussion

I de senere år har mange studier har utviklet ulike kvantitative mikroskopi-analyser for fikserte celler. 42 Progress in cent-kinetochore biologi krever ofte en forståelse av den centspesifikk eller kinetochore-spesifikk funksjon av proteiner som subcellulære romlig-temporale regulering reflekterer de endrede funksjoner av disse proteinene i løpet av cellesyklusen. Derfor, her vi utviklet metoder for IIF farging og en kvantitativ IIF-analyse for spesifikt å analysere de relative nivåer av endogene og ek…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet ble støttet av NIH stipend 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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