Measuring kinetochore-microtubule interaction in vitro.
Methods in Enzymology(2014)
摘要
Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause effect relationships, for example, between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule-binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore microtubule attachments, which is believed to be critical for correcting erroneous kinetochore spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.
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关键词
chromosomal mis-segregation,kinase,laser trapping,mechanoregulation,mitosis,optical tweezers,microtubules,kinetochores
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