Abstract
The mammalian kinetochore is a multi-layered protein complex that forms on the centromeric chromatin. The kinetochore serves as the attachment hub for the plus ends of microtubules emanating from the centrosomes during mitosis. For karyokinesis, bipolar kinetochore-microtubule attachment and subsequent microtubule depolymerization lead to the development of inter-kinetochore tension between the sister chromatids. These events are instrumental in initiating a signaling cascade culminating in the segregation of the sister chromatids equally between the new daughter cells. Of the hundreds of conserved proteins that constitute the mammalian kinetochore, many that reside in the outermost layer are loaded during early mitosis and removed around metaphase-anaphase. Dynamically localized kinetochore proteins include those required for kinetochore-microtubule attachment, spindle assembly checkpoint proteins, various kinases, and molecular motors. The abundance of these kinetochore-localized proteins varies at prometaphase, metaphase, and anaphase, and is thus considered diagnostic of the fidelity of progression through these stages of mitosis. Here, we document detailed, state-of-the-art methodologies based on high-resolution fluorescence confocal microscopy followed by quantification of the levels of kinetochore-localized proteins during mitosis. We also document methods to accurately measure distances between sister kinetochores in mammalian cells, a surrogate readout for inter-kinetochore tension, which is essential for chromosome segregation. Key features • Immunostaining of cultured and suitably fixed adherent mammalian cells growing as monolayers. • Confocal fluorescence imaging for the purpose of fluorescence quantification. • 2D and 3D image reconstruction and analysis of the acquired images using appropriate background correction and normalization. • Quantification of the inter-sister kinetochore distances using 3D image reconstruction. Graphical overview Key steps involved in fluorescence quantification of spindle assembly checkpoint (SAC) protein loading at the kinetochores and in the calculation of inter-sister kinetochore distances. High-resolution confocal image datasets of cells subjected to appropriate treatment as per the experimental need (drugs, inhibitors, gene-specific siRNA, etc.) are used for quantifying the levels of SAC proteins at the kinetochores, measuring inter-sister kinetochore distances, or both. Quantification and analysis of both parameters are performed using image analysis software such as Fiji (open source) or the IMARIS software suite. Figure created with BioRender.com.