This work was supported by NIH grant GM68418.

The authors declare that they have no competing financial interests.

In recent years many studies have developed different quantitative microscopy assays for fixed cells.42 Progress in centromere-kinetochore biology often requires an understanding of the centromere-specific or kinetochore-specific function of proteins of which subcellular spatial-temporal regulation reflects the changing functions of these proteins during cell cycle. Therefore, here we developed methods of IIF staining and a quantitative IIF assay to specifically analyze the relative levels of endogenous and ex...

&#34;Centromeres&#34; were classically defined as regions of suppressed meiotic recombination in genetics and later recognized as the primary constriction of mitotic chromosomes, which plays an essential role in accurate chromosome segregation during mitosis. &#34;Kinetochores&#34; were described as the multilayered structures that bind to microtubules at the surface of centromeres, as revealed by electron microscopy; &#34;kinetochores&#34; were later defined as the macromolecular complex that localizes at the centromere of mitotic chromosomes. Despite a dramatic divergence of centromeric DNA sequences among vertebrates, kinetochore structure and composition are highly conserved. A dynamic interaction between spindle microtubules and the kinetochore is required for faithful segregation of chromosomes during mitosis, and defects in centromere-kinetochore function lead to aneuploidy and thereby cancer.
The centromere in most eukaryotes has no defined DNA sequence, but is composed of large arrays (0.3-5 Mb) of repetitive alphoid DNA consisting of 171-bp &#945;-satellite DNA. Except in budding yeast, centromere identity is achieved not by the DNA sequence but by the presence of a special nucleosome that contains the histone H3 variant CenH3 (CENtromere Protein A [CENP-A] in humans).5 CENP-A nucleosomes localize to the inner plate of mammalian kinetochores7 and bind to the 171-bp &#945;-satellite DNA. Active centromeres require CENP-A-containing nucleosomes to direct the recruitment of a constitutive centromere-associated network (CCAN) and the kinetochore proteins, which together regulate the attachment of chromosomes to the mitotic spindle and subsequent cycle progression through the spindle checkpoint.
In light of the above evidence, CENP-A has been proposed to be the epigenetic mark of the centromere8; however, the process by which CENP-A is incorporated into centromeric DNA and the factors responsible for this incorporation have not yet been well characterized. A short centromere-targeting domain (CATD) resides in the histone fold region of CENP-A, and replacement of the corresponding region of H3 with the CATD is sufficient to direct H3 to the centromere.9 Several studies suggested functional roles for post-translational modification (PTM) of CENP-A12-16; however, the molecular mechanisms of these PTMs of CENP-A in the recruitment to centromeres have not yet been elucidated. We previously reported that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A K124 ubiquitylation and localization of CENP-A to centromeres.17
The discovery and characterization of kinetochore proteins have led to new insight regarding chromosome segregation.18 More than 100 kinetochore components have been identified in vertebrate cells by various approaches.19,20 An understanding of how kinetochores assemble and function also comes from the characterization of the cellular functions of each centromere-kinetochore proteins and protein-protein network within cells.19 Direct visualization and 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 Thus, developing and reporting methods of IIF staining and a quantitative IIF assay to specifically analyze each centromere-kinetochore protein is extremely important. In IIF staining, one should proceed with the staining protocol to avoid loss of the protein of interest or the rest of the cell. However, fixation destroys antigenic sites occasionally, and different antibody-antigen combinations work poorly with one fixative, but very well with another,21 and choice of fixative depends largely on the protein(s) of interest. Therefore, different fixative methods are crucial in IIF staining of centromere-kinetochore proteins.
Here optimized methods of indirect immunofluorescent (IIF) staining and an assay to address localization of endogenous centromere-kinetochore proteins, including CENP-A and Flag-tagged exogenous CENP-A proteins, and quantitation of these proteins in human cells have been developed. These methods can be applied to the analysis of centromere-kinetochore proteins in other species.

<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.

Immunofluorescence analysis of endogenous CENP-A supports the hypothesis that CUL4A-E3 ligase is required for localization of CENP-A to centromeres 
Our recent studies showed that CUL4A-RBX1-COPS8 E3 ligase activity is required for ubiquitylation of lysine 124 (K124) on CENP-A and localization of CENP-A to centromeres.17 Initially, the interphase-centromere complex (ICEN) was isolated by anti-CENP-A:native chromatin immunoprecipitation,32-34 and...

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).

"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 Views.  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).

Video: 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.

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

Genetics

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.

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; ...

Biochemistry

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.

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

Developmental Biology

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).

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

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.

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.

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.

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.

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.

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.

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

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Chapters in this video

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

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

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

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

Imaging Centrosomes in Fly Testes

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

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

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

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

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