A spindle matrix has long been proposed to serve as a stationary or elastic molecular matrix substrate for the organization and activities of microtubules and motors, based on the consideration of a mechanical and functional support for the stabilization of microtubule spindle during force generation from mitotic motors. Recently, the identification of four Drosophila proteins, Skeletor, Megator, EAST and Chromator has provided molecular evidence for the existence of this macromolecular matrix structure during mitosis. All of these four proteins have been shown to interact with each other within a protein complex and redistribute from the nucleus at interphase to form a fusiform spindle-like structure that does not rely on polymerized microtubules from prophase until telophase. Especially, the discovery of the large coiled-coil domain in Megator suggests that Megator may provide the structural element of this matrix. Characterizations of these molecules all indicate their potential to constitute a bona fide spindle matrix. However, functional analysis is missing due to the limitation of existing mutant alleles and the multiple essential roles played by at least some of these proteins at different stages of the cell cycle.;Taking advantage of newly generated hypomorphic alleles of the Chro gene, different functional roles of Chromator were dissected and the results are presented in this dissertation. In transheterozygous Chro71/Chro612 mutants, interphase polytene chromosome structures are disrupted with misalignment of the band/interband pattern and numerous ectopic contacts between non-homologous regions. During mitosis a dramatically disorganized spindles and chromosome segregation defects are observed in dividing neuroblasts from mutant third instar larval brains. In addition, an interphase-specific interaction between Chromator and the H3 Serine 10 kinase JIL-1 as well as mitotic interactions of Chromator with the molecular motor Ncd and microtubules are described.;This dissertation provides the first reported detailed functional analysis of one spindle matrix candidate, Chromator, in animal system. Impaired Chromator function in the mutant allele causes improper spindle assembly and chromosome segregation during mitosis as well as defective interphase polytene chromosome structures. These findings, with the characteristic cell-cycle dependent distribution pattern of Chromator reveal that Chromator is a nuclear-derived multifunctional protein that performs its essential functions from interphase to mitosis.