Three-Dimensional Structure of Eukaryotic 
Chromosomes 
John W. Sedat, Ph.D. — Investigator 
Dr. Sedat is also Professor of Biochemistry and Biophysics at the University of California, San Francisco. 
He received his Ph.D. degree in biology from the California Institute of Technology. His postdoctoral work 
with Fred Sanger was done at the Medical Research Council in Cambridge, England. Before joining the 
faculty at UCSF, Dr. Sedat was a research associate at Yale University. 
THE three-dimensional structure of chromo- 
somes, both in the nucleus and during cell 
division, remains a major unsolved problem in 
biology. Our laboratory, in collaboration with 
that of David Agard (HHMI, University of Califor- 
nia, San Francisco) , is investigating chromosome 
structure from the perspective of several inter- 
locking questions: 1) What is the architecture of 
the chromosome in the intact diploid nucleus? 
How does the three-dimensional structure 
change as a function of development, or progres- 
sion through the cell cycle? 2) What is the archi- 
tecture of a given gene in the nucleus? Do the 
structural attributes reflect the detailed molecu- 
lar information? 3) How do interphase chromo- 
somes condense to form the intricate mitotic 
structure at cell division? 
The fruit fly Drosophila melanogaster, well- 
known for its genetics, development, and bio- 
chemistry, was chosen as a model biological sys- 
tem. Although the initial emphasis is structural, 
molecular genetics and biochemistry provide 
functional correlations. 
The UCSF three-dimensional optical micro- 
scope has been developed to the point that data at 
several wavelengths can be routinely collected, 
even as a function of time (four-dimensional mi- 
croscopy) , and can be used without computer ex- 
perience. Still, we continue to perfect and en- 
hance the instrumentation. We increased the 
time resolution for data collection and greatly 
improved the image quality of the four- 
dimensional data, permitting analysis of much in- 
formation on biological structures. We continue 
to write software, with extensive mathematical 
analysis, to correct systematic image acquisition 
problems, to display results in a variety of for- 
mats, and to model and analyze, often quantita- 
tively, the complex three-dimensional data. We 
have started to develop a computer-based meth- 
odology to extract and analyze quantitatively the 
large- and small-scale motion of chromosomes or 
structure within the nucleus. 
Four-Dimensional Optical Microscopy 
We have continued to study the structure of the 
cellular nucleus in living Drosophila embryos. 
Nuclei were labeled by microinjection of fluores- 
cent histones, or other chromosomal proteins. 
Nuclear and chromosomal structures were fol- 
lowed throughout the cell cycle during embry- 
onic development. In addition to discerning 
structural changes, we can now infer function. 
Topoisomerase II — A Key Nuclear Protein 
Our studies include an effort to understand the 
role of various proteins in the organization and 
dynamics of chromosomes. We have therefore 
studied the distribution and dynamics of the DNA 
strand-passing (unknotting) enzyme topoisomer- 
ase II. High-resolution three-dimensional imag- 
ing of Drosophila embryonic chromosomes 
shows a heterogeneous distribution of topoiso- 
merase II along the chromosome. At metaphase 
and anaphase, the enzyme can be clearly seen to 
be situated adjacent to the chromosome. This 
suggests that its localization may be linked to its 
activity: the enzyme may concentrate at sites of 
chromosome condensation and/or segregation. 
These data argue against a purely structural role 
for the enzyme. 
We have studied the dynamics of localization 
by injecting fluorescently labeled antibodies 
against the enzyme, or the labeled enzyme itself, 
into live embryos and then imaging them by our 
three-dimensional microscopy as a function of 
time. The resulting time-lapse movies have 
shown that the concentration of nuclear topoiso- 
merase II changes dramatically throughout the 
cell cycle. The highest levels occur in late inter- 
phase, the lowest levels in telophase. A very dy- 
namic fibrillar complex is evident during inter- 
phase. Experiments that will disclose the 
functional relevance of this structure are in 
progress. 
A Molecular Dissection 
of the Nuclear Periphery 
Recently we showed that the lamin proteins of 
the nuclear envelope (NE) form a highly discon- 
tinuous network in somatic interphase nuclei. 
Several obvious questions arise. First, where are 
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