Ran in Mitosis - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 124

enabled the RCC1 function in guanine nucleotide exchange to be interfered with by
using recombinant Ran variants [T24N, (Klebe et al. 1995 )], which efficiently
blocked exchange function at defined time points during cell cycle progression
(Clarke et al. 1995 ). Addition of recombinant RanT24N inhibited Cdk1 activity
independently of nucleocytoplasmic transport, suggesting that the Ran system may
function directly in mitosis independently of its role in nucleocytoplasmic trans-
port. The idea to use cell-free extracts from unfertilized amphibian eggs in combi-
nation with recombinant Ran variants further stimulated key observations that
elucidated direct functions of Ran in mitotic microtubule (MT) assembly,
M-phase spindle formation as well as nuclear re-formation after mitosis.
7.3 A Direct Function of Ran in Mitosis Uncoupled from Its
Role in Nucleocytoplasmic Transport
Cell-free extracts of Xenopus eggs preserve the natural arrest in metaphase II of
meiosis, which features unfertilised vertebrate eggs in general (Masui and Markert
1971 ). However, these extracts can recapitulate minimal embryonic cell cycles
ex vivo when released from their arrest. Released extracts show a short interphase
with low cyclin B and Cdk1 activity levels, after which constantly accumulating
cyclin B accelerates Cdk1 activity up to maximum in proceeding M-phase. Cdk1-
stimulated rapid degradation of cyclin B then leads to a sharp drop in Cdk1 activity.
Cyclin B levels oscillate in that way in cell-free egg extracts independently of
nuclear structures. However, nuclei added from the beginning of the reaction will
follow the cell cycle, replicate their DNA at low Cdk1 activity and build up bipolar
spindles when the Cdk1 activity rises (Murray 1991 ; Sawin and Mitchison 1991 ).
Inducing the cycling reaction in the presence of male sperm, for instance, results in
sperm chromatin decondensing, duplicating, re-condensing and finally aligning on
the metaphase plate of the bipolar spindle. The female cytoplasm also turns back
the basal body of the sperm into a functional centrosome, which was lost from the
female cytoplasm during the long growth phase of the oocyte. This first centrosome
duplicates concomitant with DNA replication to ensure that one centrosome can
localise to either of the two poles of the spindle. The formally meiotic egg extracts
recapitulate general principles of MT assembly seen during both meiotic spindle
formation in oocytes and mitotic spindle assembly in embryos and somatic cells.
Very dynamic M-phase (meiotic and mitotic) MTs in egg extracts exclusively but
strongly nucleate form the spindle poles and the chromatin to finally build up a
bipolar spindle.
When several groups addressed a possible role of Ran in M-phase MT assembly
using egg extracts of the African clawed frog Xenopus laevis , they consistently
observed that the sole addition of any stable form of RanGTP, e.g. the hydrolysis-
deficient Ran variants RanQ69L (Klebe et al. 1995 ), Ran G19V or Ran L43E
(Carey et al. 1996 ) loaded with GTP, or Ran (wild-type or G19V) preloaded with