Chromosome structure: improved immunolabeling for electron microscopy

Abstract

To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIalpha (topoIIalpha) 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 topoIIalpha 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 topoIIalpha 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 topoIIalpha tether chromatin to loops according to a scaffolding-type model.

Micromechanical studies of mitotic chromosomes

“…EM visualization of DNA loops extending from a protein-rich chromosome body after histone depletion (Paulson & Laemmli 1977), plus visualization of structures consistent with a loop organization in serial-sectioned fixed cells (Marsden & Laemmli 1979, Adolph 1980, 1981, Adolph & Phelps 1982, Adolph et al 1986, Maeshima et al 2005, support a model for mitotic chromosome structure based on chromatin loops interconnected by a non-histone-protein-rich Fscaffold_ (Marsden & Laemmli 1979). In human cells, mitotic loops observed in EM experiments are 50Y100 kb in size.…”

“…Condensin units have been observed to be axially organized in mitotic chromatids (Figures 3 and 4) (Hirano & Mitchison 1994, Maeshima & Laemmli 2003, Ono et al 2003, Kireeva et al 2004, Maeshima et al 2005. Immunofluorescence studies indicate that in animal cells, condensin II may be localized nearer to the chromatid axis than condensin I (Ono et al 2003), reflecting the loading of condensin II before condensin I.…”

“…As mentioned above, topo II has been observed to be axially or helically organized in mitotic chromosomes (Boy de la Tour & Laemmli 1988, Sumner 1996, Tavormina et al 2002, Cuvier & Hirano 2003, Maeshima & Laemmli 2003, Kireeva et al 2004, Maeshima et al 2005, although the degree to which an axial distribution is observed appears sensitive to experimental details (Hirano & Mitchison 1993).…”

“…Electron-microscopic studies have indicated that topo II can bind a crossover of two DNAs (Zechiedrich & Osheroff 1990), and topo II has been observed to be able to recondense protease-decondensed chromosomes (Bojanowski et al 1998). Immunofluores-cence experiments have observed topo II localized in chromatid-axial patterns in mitotic chromosomes (Boy de la Tour & Laemmli 1988, Saitoh & Laemmli 1994, Maeshima & Laemmli 2003, Kireeva et al 2004, Maeshima et al 2005 (Figure 3).…”

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

“…5-7). These proteins are variously assumed to mediate the folding of chromatin fibers into radially oriented loops (8)(9)(10), a hierarchy of helical structures (11,12), or an irregular network (13). Despite their disparities, all of these hypotheses assume that before higher-order compaction, the nucleosome chain forms a 30-nm chromatin fiber as the second level of DNA folding.…”