This work was supported by NIH grant GM68418.

1. Cell Culture and Transfection
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	<li>Put a cover glass (22 mm x 22 mm) in a 6-well polystyrene plate. Optionally coat a cover glass with Poly-L-Lysine, 0.1% w/v, in water (see List of Materials/Equipment) to keep mitotic cells on the cover glass following the steps below: 
	Note: Optimal conditions must be determined for each cell line and application.
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		<li>Aseptically coat culture surface with Poly-L-Lysine, 0.1% w/v, in water (0.4 ml/well of a 6-well polystyrene plate). Rock gently to ensure even co...

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.

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 ...

The overall goal of this indirect immunoflorescent assay is to optimize the localization and quantification of endogenous and exogenous centromere-kinetochore proteins, including centromere protein A and flag-tagged exogenous CENP-A proteins in human cells. This method can help answer key questions about how to identify and quantify centromere-kinetochore proteins. The main advantage of this technique is that it can be used to quanitfy different levels of centromere-kinetochore expression depending on the selected anaphase of interest.18 hours after seeding, wash the cultured cells one time with PBS and add 500 microliters of reduced serum medium to each well of the six well polystyrene culture plate. Next, transfect the cells in each well with freshly prepared A and B mixture solution and incubate the cultures at 37 degrees Celsius and 5%carbon dioxide. After four and a half hours, change the medium to high glucose DMEM supplemented with FBS and antibiotics and return the plate to the cell culture incubator.48 to 72 hours later, aspirate the cell culture medium and rinse the cells one time with PBS, adding the saline down the sides of the culture wells to avoid disturbing the cells. Then fix the cells with 4%paraformaldehyde for 30 minutes at 4 degrees Celsius. At the end of the fixation period, rinse the cells two times with Kb2 buffer and permeabilize the samples in Kb1 buffer for 30 minutes at room temperature.Then rinse the cells one time with Kb2 buffer and block any non-specific binding with the addition of fresh Kb2 buffer. After five minutes at room temperature, use forceps to remove the seeded cover glasses from each of the wells, and use a hydrophobic barrier pen to draw hydrophobic barriers around each cover glass sample. Transfer the cover glasses into a new six well polystyrene plate and immerse the samples in the appropriate primary antibody for one hour at 37 degrees Celsius.Then rinse the cells three times with Kb2 buffer and label them with the appropriate secondary antibody for another hour at 37 degrees Celsius. At the end of the incubation, rinse the samples with five 6 minute washes in Kb2 buffer. Then label the cell nuclei with Kb3 buffer containing DapE for five minutes at room temperature, followed by one to two washes in Kb2 buffer and refilling the wells with Kb2 buffer.Now place a drop of mounting medium in the center of one microscope slide for each cover glass of cells, and remove any liquid from the cell samples. Finally, carefully place each sample onto the center of one of the prepared microscope slides, and remove any excess mounting medium with a paper towel. As observed in this representative experiment, the levels of endogenous CENP-A protein expression in the total cell lysates of CUL4A siRNA transfected cells were similar to the levels of CENP-A expression in Luciferase siRNA transfected cells.Further, the protein levels of endogenous CENP-A in the total cell lysates of RBX1 siRNA transfected cells were similar to the endogenous CENP-A levels of the Luciferase siRNA transfected cells, suggesting the CUL4A RBX1 E3 ligase is required for the localization of CENP-A to the centromeres. CENP-A lysine mutants lose their ability to localize within the centromeres, suggesting that CENP-A K124 ubiquitylation is essential for the localization of CENP-A to the centromeres. Importantly, exogenous monoubiquitin fusion of CENP-A protein rescued the delocalization CENP-A K124R mutant, returning the protein to its centromeric location.Once mastered, the cell fixation and immunoflourescent staining procedures can be completed in five to six hours if they are performed properly. Remember to gently apply all of the solutions onto the sides of the wells so that the cells won't be dislodged from their cover glasses when rinsing or immersing the samples. Don't forget that working with heated paraformaldehyde outside a ventilated room or that flammable materials near fire can be extremely hazardous and that the appropriate precautions should always be taken while performing this procedure.

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15.1K visualizações.  Nationwide Children's Hospital. The overall goal of this indirect immunoflorescent assay is to optimize the localization and quantification of endogenous and exogenous centromere-kinetochore proteins, including centromere protein A and flag-tagged exogenous CENP-A proteins in human cells. This method can help answer key questions about how to identify and quantify centromere-kinetochore proteins. The main advantage of this technique is that it can be used to quanitfy different levels of centromere-kinetochore expression dep...