Biomedical Engineering Reference
In-Depth Information
allele using an isolated DNA fragment that efficiently restored wild-type behaviour
in
tsBN2
cells at restrictive temperature (Kai et al.
1986
). The cDNA
complementing the ts allele encoded a 421 amino acids open reading frame termed
regulator of chromosome condensation
, RCC1. RCC1 turned out to be highly
conserved from yeast to man consistent with a function in a basic cellular process
such as cell cycle control. The way that RCC1 was identified strongly suggested it
had an important role as a cell cycle regulator or, at least, as an effector of mitotic
entry and chromosome condensation. Consistent with being a key regulatory
component of the cell cycle, RCC1 function seemed to act upstream of Cdc25
phosphatase and Cyclin-dependent kinase 1 (Cdk1) (Seki et al.
1992
), two master
regulators of mitosis. Only later was the molecular function of RCC1 identified as
being the guanine nucleotide exchange factor for the small GTPase Ran (Bischoff
and Ponstingl
1991
), and the identification of Ran-regulated nuclear transport
receptors provided a consistent explanation for the initially observed phenotype
of
tsBN2
cells. The balance of nuclear import and export of key regulators for
mitotic entry, such as Cdc25, probably changed following the reduction of RCC1
activity in
tsBN2
cells at the restrictive temperature. Nuclear accumulation of
Cdc25 in
tsBN2
nuclei was proposed to account for premature entry into mitosis
as it dephosphorylated, and therefore activated, Cdk1 ahead of time. The exact
nature of nuclear transport defects in
tsBN2
cells stays unclear until now, but ever
since the characterisation of the primary functions of RCC1 and Ran, the connec-
tion of Ran components, nucleocytoplasmic transport and the cell cycle fertilised
and stimulated research in area of cell division. Important principles of mitosis and
many molecular details were worked after the initial observations had been made.
Our current knowledge is summarised in a number of excellent reviews, which I
recommend for further reading (Dasso
2001
; Moore
2001
; Weis
2003
; Clarke and
Zhang
2004
; Di Fiore et al.
2004
; Gruss and Vernos
2004
; Zheng
2004
; Ciciarello
and Lavia
2005
; Ciciarello et al.
2007
; O'Connell and Khodjakov
2007
; Clarke and
Zhang
2008
; Kalab and Heald
2008
; Rensen et al.
2008
; Sato and Toda
2010
; Kalab
et al.
2011
).
7.2 Uncoupling Interphase and Mitosis
Conceptually and experimentally, the intimate interdependence between cell cycle
progression and nucleocytoplasmic transport was very difficult to analyse in intact
mammalian cells. Chronic, incomplete loss of RCC1 activity in
tsBN2
cells would
certainly cause accumulating defects in nucleocytoplasmic transport over time.
Whether these primary defects were responsible for all cell division defects
observed or if the loss of RCC1 would also affect mitosis independently of
nucleocytoplasmic transport could not be dissected in intact cells. An idea to
solve this problem was to use cell-free extracts from amphibian eggs that were
naturally arrested in the second metaphase of meiosis and could proceed through
the cell cycle in a strictly defined manner. This biochemically accessible system
Search WWH ::
Custom Search