Mounting evidence suggests that the nucleus has a well-differentiated functional organization and raises the question of whether the chromosomes or loci arrange themselves within this environment. We are studying the formation of centromere clusters in early G 1 as a tool to examine early events in nuclear assembly. Nucleoli contain at least one, usually several, associated centromeres. Using a cell line that expresses a centromere protein linked to green fluorescent protein, we are recording the 3-dimensional trajectory of each centromere during chromosome segregation and subsequent nuclear reassembly in G 1 and then are using fluorescent in situ hybridization to identify specific chromosomes. Our goal is to determine whether centromeres move in early G 1 to form clusters or whether cells emerge from mitosis with clustered centromeres already proximal to one another.
Initial results suggest the latter possibility.
This system provides a model for studying the establishment, maintenance, and transmission of structures at the suprachromosomal level of nuclear organization. According to current understanding, the nucleus is a compartmentalized organelle in which numerous biochemically specialized domains carry out specific tasks. It has long been known that viral replication occurs in viral compartments within infected cell nuclei. We use a cell line that expresses a fusion protein consisting of green fluorescent protein and histone H3 to quantitatively examine chromatin structure at high resolution. With this method, we can study the structural response of host cell nuclei to viral infection in living cells.