Ran in Mitosis - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 128

Although indirect evidence and computational models (G¨rlich et al. 2003 )
strongly suggested a diffusible gradient of RanGTP around chromatin, direct
proof only came from the visualisation of RanGTP by specific biosensors using
F ¨ rster Resonance Energy Transfer (FRET). In 2002, Petr Kalab pioneered this
approach when he engineered a FRET sensor comprising EYFG and ECFP as donor
and acceptor for FRET, connected by a high-affinity RanGTP binding site derived
from yeast RanBP1 (Kalab et al. 2002 ). The conformational change in the RanBP1
peptide upon RanGTP binding diminished FRET and allowed imaging RanGTP
specifically. Strikingly, the sensor detected a spherical gradient of RanGTP around
mitotic sperm chromatin in Xenopus cell-free extracts. Importantly, measurable
size (17 m) and steepness of the gradient matched computational simulation using

the known biochemical parameters of the Ran system (Kalab et al. 2002 ;G¨rlich
et al. 2003 ). Spindle size (ca. 30

ʼ

ʼ m), however, seemed to exceed the size of the

gradient.

7.5 Targets of the Ran System in M-Phase

The considerable complexity of Ran functions in M-phase strongly suggested the
presence of a variety of different molecular targets in spindle formation and nuclear
reformation. However, it initially remained unclear how Ran would activate these
downstream activities.
After being produced and diffusing away from chromatin in M-phase, RanGTP
will meet its most abundant interaction partners as a function of time, among them
the Ran binding proteins RanBP1 and RanBP2. They, in concert with RanGAP,
promote GTP hydrolysis in Ran. These interactions seem to be non-productive.
When RanGTP, however, meets importin

-like transport receptors (importins and
exportins), it changes their binding properties in the same way as in the interphase
nucleus. For example, RanGTP binding to importin

ʲ

ʲ

generally releases importin-
bound protein. Several experiments in Xenopus cell-free extracts using importin
fragments and an excess of nuclear localisations signal (NLS)s to compete
importin-substrate interactions initially revealed that the release of spindle assem-
bly factors from importin is a key mechanism of Ran-dependent

spindle formation (Gruss et al. 2001 ; Nachury et al. 2001 ; Wiese et al. 2001 )
(Fig. 7.2 ). In support of this model, it could be shown recently that a small molecule
inhibitor of importin

ʱ / ʲ or importin ʲ

, importazole, not only blocks nuclear import into semi-
permeabilised human cells but abolishes spindle formation in intact cells and
Xenopus cell-free extracts (Soderholm et al. 2011 ). Interestingly, the idea of
Ran's mechanism in M-phase was also directly confirmed by FRET probes to
monitor the activity of RanGTP in M-phase. In these probes, a conformational
change in importin

ʲ

ʲ , leading to the dissociation of the N-terminus of importin ʱ

(importin binding domain, IBB), was turned into an increasing FRET signal. This

strongly indicated dissociation of endogenous NLS proteins from importins and
suggested the presence of free spindle assembly factors. The range of the gradient

ʲ