Este trabalho foi financiado pelo NIH conceder GM68418.

Os autores declaram que não têm interesses financeiros concorrentes.

Nos últimos anos, muitos estudos têm desenvolvido diferentes ensaios de microscopia quantitativa em células fixas. 42 Progress em biologia centrômero-kinetochore muitas vezes requer uma compreensão da função específica do centrômero ou específicos de kinetochore de proteínas das quais regulação espacial-temporal subcelular reflete a evolução das funções do estas proteínas durante o ciclo celular. Portanto, aqui desenvolvemos métodos de coloração IIF e um ensaio quantitativo para o IFI ana...

&quot;centrômeros&quot; foram classicamente definidos como regiões de recombinação meiótica suprimida em genética e, mais tarde reconhecida como a constrição primária de cromossomos mitóticos, que desempenha um papel essencial na segregação cromossômica precisas durante a mitose. &quot;cinetócoros&quot; foram descritos como as estruturas em camadas múltiplas que se ligam aos microtúbulos na superfície de centrómeros, conforme revelado por microscopia electrónica; &quot;cinetócoros&quot; foram definidos mais tarde, como o complexo macromolecular que localiza no centrômero dos cromossomos mitóticos. Apesar da divergência dramática de sequências de ADN centromérico entre os vertebrados, cinetócoro estrutura e composição são altamente conservadas. A interacção dinâmica entre os microtúbulos do fuso e do kinetochore é necessário para a segregação fiel de cromossomos durante a mitose e defeitos na função centrômero-kinetochore levam a aneuploidia e, assim, o câncer. O centrómero na maioria dos eucariotasnão tem nenhuma sequência de ADN definida, mas é composto por matrizes grandes (0,3-5) Mb de ADN repetitivo consistindo alphoid de ADN satélite α-171-pb. Salvo em brotamento de levedura, identidade centrómero é conseguido não por a sequência de ADN mas pela presença de um nucleossoma especial que contém a variante histona H3 CenH3 (centrómero Proteína A [CENP-A] em seres humanos). 5 nucleossomas CENP-A localizar a placa interna de cinetócoros mamíferos 7 e se ligam ao DNA α-satélite 171 pb. centrômeros ativos requerem CENP-A contendo nucleossomos para dirigir o recrutamento de uma rede constitutiva associada ao centrômero (CCAN) e as proteínas cinetócoro, que em conjunto regulam a fixação dos cromossomas ao fuso mitótico e subsequente progressão do ciclo através do ponto de verificação do fuso. À luz da evidência acima, CENP-A tem sido proposta para ser a marca epigenética do centrômero 8; No entanto, o processo pelo qual CENP-A é incorporada into de ADN centromérico e os fatores responsáveis ​​por essa incorporação ainda não foram bem caracterizados. Um domínio de direccionamento centrómero curta (CATD) reside na histona dobrar região de CENP-A, e a substituição da região correspondente de H3 com o CATD é suficiente para H3 directa ao centrómero. 9 Vários estudos sugeriram papéis funcionais para pós-translacional modificação (PTM) de CENP-A 12-16; No entanto, os mecanismos moleculares destes PTMs de CENP-A no recrutamento para centrómeros ainda não foram elucidados. Nós relatado anteriormente que a atividade ligase CUL4A-RBX1-COPS8 E3 é necessário para CENP-A K124 ubiquitinação e localização de CENP-A para centrômeros 17. A descoberta e caracterização de proteínas cinetócoro levaram a nova visão sobre a segregação de cromossomas. 18 mais de 100 componentes cinetócoro foram identificados em células de vertebrados por várias abordagens. 19,20 Um sobpe de como cinetócoros montar e função também vem da caracterização das funções celulares de cada rede centrômero-kinetochore proteínas e proteína-proteína dentro das células. 19 visualização direta e de imagem avançados métodos de microscopia de fluorescência fornecer notável resolução de componentes centrômero-kinetochore e permitir observação direta de componentes moleculares específicos dos centrômeros e cinetócoros. Além disso, métodos de imunofluorescência indirecta (IIF) A coloração por utilização de anticorpos específicos são cruciais para estas observações. No entanto, apesar dos numerosos relatórios sobre protocolos de IFI, alguns problemas discutidos em de proteínas específicas de centrómero-cinetócoro detalhe. 1-4 Assim, o desenvolvimento de métodos e relatórios de coloração IIF e um ensaio quantitativo para o IFI analisar especificamente cada proteína centrómero-cinetócoro é extremamente importante. Na coloração IIF, deve-se proceder com o protocolo de coloração para evitar a perda da proteína de interesse ouo resto da célula. Contudo, a fixação destrói os locais antigénicos ocasionalmente, e diferentes combinações de anticorpos para o antigénio funcionar mal com um fixador, mas muito bem com a outra, 21 e escolha de fixador depende em grande parte da proteína (s) de interesse. Portanto, diferentes métodos fixadoras são cruciais na coloração IIF de proteínas centrômero-kinetochore. Aqui métodos optimizados de imunofluorescência indirecta (IIF) e um ensaio de coloração para dirigir a localização de proteínas de centrómero-cinetócoro endógenos, incluindo CENP-A e CENP-A proteínas exógenas Bandeira-marcadas, e a quantificação destas proteínas em células humanas têm sido desenvolvidos. Estes métodos podem ser aplicados para a análise de proteínas de centrómero-cinetócoro em outras espécies.

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

immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins

"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.

Análise por imunofluorescência da endógena CENP-A suporta a hipótese de que CUL4A-ligase E3 é necessário para a localização de CENP-A a centrómeros Os nossos estudos recentes mostraram que a actividade de ligase CUL4A-RBX1-COPS8 E3 é necessária para a ubiquitinação de lisina 124 (K124) em CENP-A e localização de CENP-A a centrómeros. 17 Inicialmente, o complexo interfase-centrómero (ICEN) foi isolado pela anti-CENP-a: imunoprecipitaçã...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins at JoVE.com

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

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).

Proteínas Análise de imunofluorescência de endógenos e exógenos Centromere-cinetócoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

immunofluorescence-analysis-endogenous-exogenous-centromere

Research

JoVE Journal

Biology

15.1K visualizações.  Nationwide Children's Hospital. 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).

Vídeo: Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

Immunostaining para modificações do DNA: análise computacional de imagens Confocal

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genética

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Identificação da quinase Cyclin-dependente 1 sítios de fosforilação específica por uma quinase In Vitro do ensaio

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Bioquímica

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Avaliação da Integridade do Ponto de Verificação da Montagem do Fuso em Oócitos de Rato

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Biologia do Desenvolvimento

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Biologia

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrossomas de imagem na mosca Testículos

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Reação de Imunofluorescência Indireta para medir a variação nos níveis de proteína no centrossomas

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Avançadas técnicas de microscopia Confocal para estudar interações da proteína-proteína e cinética de lesões do ADN

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Imagem de fluorescência para a reconstrução da único-partícula de Chlamydomonas centríolos e isolamento

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examinação da dinâmica nuclear do fermento mitotic e Meiotic da fissão pela microscopia viva-pilha da fluorescência

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.