(Support NIH/NCRR/P41 RR 01219 grant and NSF MCB 9420 772 to B.F. McEwen) The kinetochore in is a functionally complex organelle that attaches chromosomes to the spindle, generates poleward chromosome motion, and produces the anaphase cell cycle checkpoint. The central importance of these processes for mitotic and meiotic cell division has prompted several laboratories to identify molecular components of the kinetochore. Initial localization of kinetochore components is carried out via immunofluorescent light microscopy, but localization via immuno-electron microscopy is required for evaluating detailed hypotheses concerning the role of each kinetochore component. Thus far, immuno-electron microscopy of the kinetochore has been performed on extracted, chemically fixed, and solvent dehydrated specimens. The results are then interpreted in terms of the classical trilaminar structural model of the kinetochore. The goal of our study is to determine the accurate location of as many kinetochore proteins as possible. From this data we will construct hypotheses concerning the interactions between kinetochore components. Although we are using conventional pre-embedded labeling methods during our initial localization's, we will soon switch to post-embedded labeling of cells prepared via high-pressure freezing and freeze-substitution. Our initial investigations of kinetochore ultrastructure using these superior methods for structural preservation (see TRD subproject entitled """"""""Investigating the ultrastructure of the mammalian kinetochore using high-pressure freezing and freeze-substitution"""""""") reveal that the classical trilaminar model of kinetochore structure is highly inaccurate due to structural disruption from conventional methods for specimen preparation. Thus a truly accurate model of kinetochore function requires that immunolocalizations be based upon the updated view of kinetochore ultrastructure. Cells were fixed and stained for microtubules. Secondary staining was with fluoronanogold. This reagent is supposed to combine fluorescence and small-gold staining. The latter can be silver enhanced after viewing the fluorescence staining. The fluorescent signal was dimer than FITC, but visible by light microscopy. Silver enhancement did not work, as evidenced by no staining being visible either by light or electron microscopy. We traced the problem to the fluoronanogold reagent. We switched to using the straight nanogold as a tertiary antibody. This reagent worked for silver enhancement, as evidenced by clear microtubule staining both via LM and subsequent EM. PtK1 cells were fixed, stained with primary antibody, stained with a FITC-conjugated secondary antibody, and viewed via fluorescence LM. Well-stained cells were then stained with the nanogold tertiary, sliver-enhanced, processed for EM, and embedded in Epon. Sections were cut 80 nm thick and viewed on a conventional EM. We also made a preliminary attempt at staining for CENP-F, which did not work well. We went back to microtubule staining using the 1.0 nm gold reagent from Aurion. Staining was great for interphase cells but not as good for mitotic cells. Cells were viewed via LM (after FITC staining) and EM (after tertiary gold, silver enhancement, embedding, sectioning). MAD2-stained cells were sectioned 80 nm thick and viewed in the EM. Kinetochores were stained but the background was high. We requested more cells from the lab of Ted Salmon. We made another attempt at CENP-F staining, pulling back on the times of silver enhancement to lower the background. The Aurion reagent was used as a secondary antibody (i.e., FITC staining was omitted) and was silver enhanced. Cells were embedded and sectioned. We received another set of coverslips of PtK1 cells stained for MAD2 from the lab of Ted Salmon. This included taxol-treated cells and controls. We stained the chromosomes with Hoechst dye. Using phase-contrast and fluorescence LM, we photographed the chromosome and MAD2 staining independently. This gave us a clear map of where the MAD2-staining kinetochores were. Cells were then stained with a gold tertiary antibody, silver enhanced, embedded, and sectioned for EM. McEwen, B.F., and M. Marko 1999. 3-D transmission electron microscopy and its application to mitosis research. Methods in Cell Biology 61:82-113.
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