MECHANICAL CONTINUITY AND REVERSIBLE CHROMOSOME DISASSEMBLY WITHIN INTACT GENOMES REMOVED FROM LIVING CELLS

Chromatin is thought to be structurally discontinuous because it is pa ckaged into morphologically distinct chromosomes that appear physicall y isolated from one another in metaphase preparations used for cytogen etic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physi cally connected in interphase as well as mitosis. To address this para dox directly, we used a microsurgical technique to physically remove n ucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chro mosome out from interphase or mitotic cells resulted in sequential rem oval of the remaining nucleoli and chromosomes, interconnected by a co ntinuous elastic thread. Enzymatic treatments of interphase nucleoplas m and chromosome chains held under tension revealed that mechanical co ntinuity with in the chromatin was mediated by elements sensitive to D Nase or micrococcal nuclease, but not RNases, formamide at high temper ature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by g elsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensatio n. As a result of these dynamic structural alterations, the mitotic ch ains also became sensitive to disruption by restriction enzymes. Ion-i nduced chromosome decondensation could be blocked by treatment with DN A binding dyes, agents that reduce protein disulfide linkages within n uclear matrix, or an antibody directed against histones. Fully deconde nsed chromatin strands also could be induced to recondense into chromo somes with pre-existing size, shape, number, and position by adding an ti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surround ing cytoskeletal networks function as a structurally-unified system. M echanical coupling within the nucleoplasm may coordinate dynamic alter ations in chromatin structure, guide chromosome movement, and ensure f idelity of mitosis. (C) 1997 Wiley-Liss, Inc.