Ran in Mitosis - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 126

immobilised RCC1 assembles spindles in the Xenopus cell-free system adding
further evidence for this hypothesis (Halpin et al. 2011 ).
It is now widely accepted that RanGTP plays a key role for chromatin-driven
processes in spindle formation. These include MT nucleation as well as MT
organisation generating a bipolar structure. However, Ran stimulates additional
functions in M-phase MT assembly. Although inhibition of RanGTP production by
RanT24N had no influence on MT nucleation from isolated centrosomes (Carazo-
Salas et al. 1999 ), it greatly stimulated the nucleation of a radial MT array (“aster”)
formed around both isolated centrosomes (Gruss et al. 2002 ) and sperm centrioles
that stay in the vicinity of chromatin (Kalab et al. 1999 ; Wilde and Zheng 1999 ;
Zhang et al. 1999b ; Carazo-Salas et al. 2001 ): Moreover, RanGTP strongly
influenced MT dynamics in Xenopus cell-free extracts (Kalab et al. 1999 ;
Carazo-Salas et al. 2001 ; Wilde et al. 2001 ). All these data show that RanGTP
mediates a variety of different processes in spindle formation during M-phase. This
concept even expanded when Ran variants were coupled to beads and tested in
extracts at late stages of M-phase that would readily return to interphase. Remark-
ably, these beads assembled pseudo-nuclei with an intact NE and incorporated
NPCs allowing regular nucleocytoplasmic transport (Zhang and Clarke 2000 ).
Although these experiments strikingly demonstrated a second important function
of Ran in M-phase, they made it even more difficult to explain how Ran mecha-
nistically acts in these diverse and complicated processes. Moreover, a formal proof
to show if and how RanGTP was generated in M-phase in the absence of an intact
nucleus was still missing.
7.4 Production of RanGTP in Mitosis: A Diffusible
Gradient Around Chromatin
In interphase, the asymmetric distribution of RanGTP between the nucleoplasm and
the cytoplasm maintains the directionality of nucleocytoplasmic transport.
RanGTP, present at high concentrations in the nucleoplasm, dissociates import
complexes and assembles export complexes. In contrast, low cytoplasmic RanGTP
concentration allows import complex assembly and promotes disassembly of export
complexes after hydrolysis of GTP in Ran. An intact NE greatly increases the
efficiency of both processes. It dramatically limits diffusion of free RanGTP out of
the nucleus and maintains RanGTP concentration differences of almost 3 orders of
magnitude in between the nucleus and the cytoplasm (G¨rlich et al. 2003 ). More-
over, it enables strict separation of RCC1 and the combination of RanGAP and
RanBP1/BP2. This situation changes in open mitosis of metazoan cells after NE
breakdown. However, the accessory components of Ran stay active throughout the
cell cycle. In particular, RCC1 continues to work as a chromatin-bound guanine
nucleotide Ran exchange factor in M-phase (Fig. 7.1 ), which is well supported by
several detailed biochemical and structural studies on RCC1 binding to chromatin.