Mitosis, the process of cell division, involves unique interactions between spindle microtubules and chromosomes, which are regulated by protein structures located on chromosomes known as kinetochores. CENPF is a kinetochore-associated protein that is localized to chromatin during the G2 and M phases of mitosis. The main role of CENPF is to secure kinetochore proteins to the correct location surrounding the chromatid and centromere for proper function. Specifically, microtubule-dependent motor proteins work alongside the kinetochores to generate tension and ultimately power chromosomal movement. Outside of its role in cell division, CENPF also has a role in the regulation of the plasma membrane through its association with SNAP25 (a vesicle associated membrane protein). Research has implicated a wide variety of proteins as regulators of kinetochore and chromatin dynamics; however, this article will review how a CENPF antibody was used to investigate the role of PinX1, PHB2 and Sgt1 in chromosome segregation and kinetochore function.
CENPF Antibody [NB500-101] - Confocal immunofluorescent analysis of HeLa cells using CENPF antibody (NB500-101, 1:5). An Alexa Fluor 488-conjugated Goat to rabbit IgG was used as secondary antibody (green, A). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red, B). DAPI was used to stain the cell nuclei (blue, C).
Yuan et al used a CENPF antibody in their investigation of PinX1 as a novel microtubule binding protein required for chromosomal segregation. The protein of interest, PinX1 can be used to inhibit telomerase during interphase; however, its role in mitosis is unclear. After establishing that PinX1 localizes to chromosome periphery in control HeLa cell populations, and then witnessing chromosomal malfunction and kinetochore destabilization in its absence through siRNA, Yuan’s group set out to determine the exact process of these observations on a molecular level. Using a CENPF antibody, they were able to conclude that the suppression of CENPF did not in fact change levels of PinX1 expression. However, a close relative of CENPF, CENPE, did show a reduction in levels of PinX1 following suppression. PinX1 was also found to localize to spindle poles, and ultimately demonstrated that it is required for accurate chromosome segregation during mitosis.
Next, Lee et al took a closer look at PHB2, a member of the prohibitin family that has a role in cell cycle progression and was recently found to be involved in mitotic spindle formation and localization of key kinetochore components. Similar to Yuan’s study, first Lee’s group did a partial depletion experiment in order to study the affects of cell division in PHB2’s absence. As predicted, partial depletion of PHB2 yielded mitotic and chromosomal dysfunction. They also saw that PHB2 was not localized at the kinetochore, as previously predicted. However, using a CENPF antibody, they discovered that in PHB2 depleted cells, CENPF localization was entirely abolished at the kinetochore. This data suggests that the reduction of kinetochore proteins after PHB2 depletion acts through a Hec1-CENP-F pathway.
Lastly, Steensgaard et al examined the role of Sgt1 in kinetochore assembly; a protein that they had previously confirmed was required for kinetochore assembly in budding yeast. Again, this research began by depleting Sgt1 via RNA interference in HeLa cells to witness the affects on cell division. Steensgaard et al saw a dramatic alteration of the mitotic spindle, as well as chromosomal alignment. In addition, protein levels for the kinetochore proteins Mad1, Mad2, and BubR1 were greatly reduced. To confirm their suspicion of this effect being a result of mislocalization of other key kinetochore proteins, they used a CENPF antibody to examine localization of CENPF at the kinetochore. Their findings showed that CENPF, alongside CENPE and CENPI were all mislocalized. Overall, it is clear that the mechanisms of kinetochore assembly and subsequent chromosomal segregation involve an intricate pathway of diverse proteins.
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