Chromosome structure: improved immunolabeling for electron microscopy
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To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIα (topoIIα) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIα and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIα and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIα tether chromatin to loops according to a scaffolding-type model.
KeywordsGold Particle Chromosome Structure Mitotic Chromosome Chromatin Fiber Emerin
We are very grateful to T. Durussel for her competent technical assistance, and Drs. E. Käs and C. Bauer for comments on the manuscript. We thank Drs. K. Wilson and T. de Lange for the anti-Emerin and TRF-1 sera, respectively. The Louis-Jeantet Medical Foundation, the Swiss National Fund, and the Canton of Geneva supported this work.