The nucleoskeleton
and the dynamics
P. Hozaks, J. Blafkovas, A. Philimonenko§,
Nowam, P. deLanerolleT[
of the cell nucleus
V. Philimonenko§,
L. Pestic-DragovichT[, G.
9 Department of Cell Ultrastructure & Molecular Biology, Institute of Experimental Medicine, Academy of
Sciences, Videnska 1083,142 20 Prague 4, Czech Republic
l/Department of Physiology, University of Illinois at Chicago, Chicago, IL.
Electron microscopy reveals that the cell nucleus is a complex structure with many elements.
However, it is frequently not clear what functions can be ascribed to these structures, or how nuclear
order is maintained. Initially, cells in early S-phase were encapsulated in agarose microbeads,
permeabilized and incubated with biotin-1 l-dUTP in a ‘physiological’ buffer; then sites of DNA
synthesis were immunolabelled. Electron microscopy of thick resinless sections from which -90%
chromatin had been removed revealed that these sites are attached to a nucleoskeleton. Replication
sites were then analyzed throughout the S-phase. The factories are assembled at the end of G,-phase
and quickly become active; as S-phase progresses, they increase in size and decrease in number like
sites of incorporation seen by light microscopy. A dynamic study on precursor incorporation into the
replication factories suggests that DNA polymerization occurs as the template moves through them.
However, pulse-chase experiments using labelled nucleotides suggest that the chromatin loops are
relatively fixed within these foci and do not spread over large distances during the replication process
WI.
Sites of transcription of ribosomal RNA in HeLa cells were also visualized by electron
microscopy. The dense fibrillar component is the site of rRNA transcription. After dispersing the
granular component and the dense fibrillar component by a hypotonic treatment, removal of most
cbromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton.
Only transcribed parts of ribosomal genes were remained attached to the nucleolar skeletal structures,
when most of the chromatin was removed by eelectroelution. The non-transcribed spacers of the
ribosomal genes were not attached. These features were incorporated into a dynamic model for rRNA
transcription [3,4].
In all above described experiments, a diffuse nucleoskeleton was found to be connected with
all synthesizing structures. We now show that the nucleoskeleton, besides the lamina, consists at least
from three structural components: the ‘core filaments’ (10 nm thick, probably intermediate filaments),
the ‘dit%.tseskeleton’ that covers the core filaments and ramifies throughout the interior of the nucleus
(contains eg hnRNPs, lamin A, RNA polymerase II), and an actin skeleton. A basic model of the
nucleoskeleton is presented [5].
A novel nuclear 120kDa protein has been characterized as myosin I isoform. The protein has
predominantly
nuclear localization showed by epifluorescence
and confocal microscopy. The
intranuclear location was confirmed by electron microscopy and by Western blot analysis of extracts
from 2x washed nuclei that are free of cytosolic contamination. This protein also binds n51-calmodulin
and is photoaffinity labeled with ATP. Column fractions containing the 120 kDa protein contain
K+-EDTA ATPase activity. The partially purified 120 kDa protein also binds actin in the absence,
but not the presence, of ATP. A mass analysis of tryptic peptides showed that 14 of the 16 peptides
generated from the 120 kDa protein match the masses of tryptic peptides of Dictyostelium myosin lE.
hnmunogold staining was then used to local&e myosin I and actin in cell nuclei at the ultrastructura
level in various mouse and human cell types, labelling mostly small nucleoplasmic areas, frequentl)
colocalizing with actin (FIG. 1). In nucleoli, a typical arrangement was found with actin locatec
predominantly in the ‘fibrillar centers’ that serve as skeletal structures in nucleoli. In contrast, myosir
I was found in the ‘dense fibrillar component’ that surrounds the the fibrillar centers and in which
rDNA transcription occurs. Other experiments addressing biological roles of the novel nuclear myosir
t will be discussed. We propose that the 120 kDa protein is a previously undescribed myosin I isofom
that is an intranuclear actin-based molecular motor, possibly involved in actin/based intranucleal
Events such as chromatin organization and transcription [6].
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FIG. 1 - Immunogold staining of a 3T3 cell section (anti-myosin antibodies, 5 mn gold, thick arrows;
mti-actin antibodies, 10 mn gold). Sites where both proteins co-localize are marked by long arrows.