Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Kristen Johansen

Second Advisor

Jorgen Johansen

Abstract

ABSTRACT<

The nucleus undergoes a dramatic reorganization as the cell prepares to segregate its duplicated chromosomes during cell division. For many years s, the prevailing view was that in open mitosis the nucleus completely disassembled during early mitotic stages, thus enabling cytoplasmic microtubules emanating from the separated centrosomes to form a mitotic spindle. This cytocentric view largely discounted any nuclear contributions to regulation of mitotic progression (Johansen and Johansen, 2009; De Souza and Osmani, 2009; Simon and Wilson, 2011; Sandquist et al., 2011). A spindle matrix has long been proposed to serve as a relatively stable or elastic molecular matrix that interacts with the microtubule spindle apparatus, based on the consideration of a mechanical and functional support for the stabilization of the microtubule spindle during force generation; however, whether such a structure exists and its molecular and structural composition has remained controversial.

In Drosophila we have identified four nuclear proteins, Skeletor, Chromator, Megator, and EAST from two different nuclear compartments that interact with each other (reviewed in Johansen et al., 2012) and that redistribute during prophase to form a dynamic, gel-like spindle matrix that embeds the microtubule spindle apparatus, stretching from pole-to-pole (Yao et al., 2012a). In this dissertation, I present the dynamic distribution of the spindle matrix components using a live imaging approach by expressing GFP tagged spindle matrix complex components in Drosophila syncytial embryos. As shown in the dissertation, this matrix forms prior to nuclear envelope breakdown and specific interactions between spindle matrix molecules are necessary for complex formation and cohesion (Yao et al., 2012a). When microtubules are depolymerized with colchicine just prior to metaphase, the spindle matrix contracts and coalesces around the chromosomes suggesting that microtubules act as "struts" stretching the spindle matrix. Furthermore, in colchicine treated embryos free tubulin accumulates co-extensively with the spindle matrix proteins suggesting that this enrichment is dependent on one or more proteins within the spindle matrix with tubulin binding activity. Biochemical interaction assays show a potential direct interaction between Chromator and polymerized microtubules or free tubulin. This tubulin binding activity of Chromator provides support for the hypothesis that reorganization of nuclear proteins into a spindle matrix may play a wider functional role in spatially regulating cell cycle progression factors in conjunction with contributing to microtubule spindle assembly and dynamics. Moreover, we have demonstrated that the coiled-coil domain of Megator is responsible and required for Megator's spindle matrix localization and function.

During interphase Chromator localizes to the interband region of Drosophila polytene chromosomes and is required for the maintenance of chromosome morphology. Here I show that the N-terminus domain of Chromator is required for proper localization to chromatin during interphase and that chromosome morphology defects observed in Chromator hypomorphic mutant backgrounds can be largely rescued by expression of this domain. Furthermore, the Chromodomain can interact with histone H1 and this interaction is necessary for correct chromatin targeting (Yao et al., 2012b).

Copyright Owner

Changfu Yao

Language

en

File Format

application/pdf

File Size

155 pages

Share

COinS