1 Department of Physiology, University of Connecticut Health Center, Farmington,
CT 06032, USA
2 Marine Biological Laboratory, Woods Hole, MA 02543, USA
3 Department of Cell Biology and Howard Hughes Medical Institute, Harvard
Medical School, Boston, MA 02115, USA
4 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany
* Author for correspondence (e-mail: terasaki{at}neuron.uchc.edu)
Accepted 3 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell division, Chromosomes, G-proteins, Meiosis, Mitosis, Nuclear proteins
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The M phase studies have made use of the cell-free extract system from
Xenopus eggs, where addition of sperm chromatin or DNA coated beads
induces the formation of a bipolar microtubule spindle centered on the added
DNA. The initial experiments indicated that generation of Ran-GTP was
necessary for assembly of the spindle (Wilde et al., 1999;
Ohba et al., 1999;
Kalab et al., 1999
;
Carazo-Salas et al., 1999
).
Importins
and ß have been identified as the target of Ran-GTP; it
appears that Ran-GTP displaces microtubule polymerization promoters such as
TPX2 or NuMA from importin (Gruss et al.,
2001
; Nachury et al.,
2001
; Wiese et al.,
2001
). A Ran-GTP gradient near the chromosomes in Xenopus
egg extracts has been visualized (Kalab et
al., 2002
). In addition to the work on spindle assembly, it has
also been shown that Ran has an essential role in nuclear envelope reformation
after mitosis (Zhang and Clarke,
2000
; Hetzer et al.,
2000
; Zhang and Clarke,
2001
). There remain unresolved issues about Ran's role in M phase;
for instance, verification in living cells of the roles for Ran identified in
extracts is required, and has begun to be addressed
(Bamba et al., 2002
), and to
what degree Ran is involved in all eucaryotic cell meioses and mitoses needs
to be resolved.
Light microscopic imaging seems likely to be useful for learning more about
Ran and its interactions. For many proteins, the amount of fluorescent
analogue required for imaging is in excess of the endogenous protein
concentration, which may perturb the natural pathways. Because Ran is one of
the most abundant proteins in the cell
(Bischoff and Ponstingl, 1991),
there should be less danger of disrupting the cell in this way. Another
advantage is that Ran's biochemical properties have been well characterized. A
major factor in this progress is that Ran is soluble and can be readily
produced as a functional recombinant protein (unlike all other small GTPases,
Ran has no lipid modifications). Lastly, there is only one isoform of Ran, one
known exchange factor and only a few GAPs (GTPase-activating proteins)
(Macara et al., 2000
), all of
which make it simpler to try to interpret observations in living cells. To
further investigate Ran during M phase, we have observed fluorescent Ran
localization and dynamics in several types of living cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For live cell imaging, Rh-Ran was injected into immature oocytes at a final
concentration of 1-2 µM. The endogenous concentration in Xenopus
oocytes has not been determined. In HeLa cells, Ran is 0.36% of total protein
(Bischoff and Ponstingl, 1991);
if the total protein concentration is 100 mg/ml in cells, this corresponds to
14 µM (Ran MW 25,000). After maturation, the egg was imaged using an MRC
600 confocal microscope (Bio-Rad; Cambridge, MA) with a krypton argon laser,
coupled to an upright microscope with a 40xPlan neofluar N.A. 1.3
objective lens (Zeiss; Thornwood, NY). For photobleaching, a macro was written
in the BioRad SOM software to control the scan and image acquisition while the
neutral density and laser power levels were switched manually. Quantification
was done using public domain NIH Image program (available at
http://rsb.info.nih.gov/nih-image/).
For imaging anaphase of meiosis II, frog eggs were activated by puncturing the egg surface with a microneedle. The eggs were in an open sided chamber made of silicon rubber (Ronsil, thickness 0.03 inches; North American Reiss, Corp, Blackstone, VA). The needle puncture was done on an upright microscope using a 10x objective lens, then the egg was transferred to a dissecting scope, where a #0 thickness cover slip was placed on top of the egg and maneuvered so that the white spot was positioned straight up. The white spot was located under a 40x oil immersion lens on the confocal microscope, and then scanning was begun to image the calcium wave. Sometimes the wave had already passed, and sometimes it failed to be initiated [later experiments suggested that Ins(1,4,5)P3 injection is more reliable]. With the confocal microscope set to continuous scanning, images were collected on an optical memory disk recorder (Panasonic TQ303F, Secaucus, NJ) with a custom-made trigger circuit.
Fully grown, immature mouse oocytes were collected from the ovaries of
PMSG-primed NSA (CF-1) mice (Harlan Sprague-Dawley) in a minimal essential
medium (MEM), then washed into MEM without dibutryl cAMP to induce maturation.
Matured eggs were microinjected as described previously
(Mehlmann and Kline, 1994) and
were examined within 30 minutes of injection while being maintained at
37°C on the confocal microscope stage. Rh-Ran was injected in a 3% volume
injection, with a resulting final concentration of 2.5 µM.
Starfish (Asterina miniata) were obtained from Bodega Bay, CA.
Methods for obtaining and handling the gametes were described previously
(Jaffe et al., 1993).
Mercury-loaded pipettes were used for quantitative microinjection; detailed
description of this method and equipment used is available at
http://egg.uchc.edu/injection/.
Embryos were kept in an 18°C incubator until imaged.
Mitotic NRK cells in an injection chamber on a heated microscope stage were pressure injected (<10% volume injection) with a 5:1 mix of Alexa 488 Ran (20 mg/ml) and 70 kDa Rh dextran (2 mg/ml). They were imaged with a Zeiss Planapo 1.4 numerical aperture objective lens using a Zeiss 510 confocal microscope.
Fluorescent Ran proteins
Tetramethylrhodamine or Alexa 488 was conjugated to the single exposed
cysteine (C112) of the three cysteine residues of human Ran
(Ribbeck et al., 1998). Both
Rh-Ran and Alexa 488-Ran (3 µM) stimulated import of NPC-M9 substrate into
digitonin-permeabilized cells as much as wild-type Ran. In this assay, many
GTPase cycles are required for import to occur, so if either hydrolysis or
exchange on RCC1 were impaired, there would be no import. GTP hydrolysis by
both fluorescent conjugates was stimulated by RanGAP with identical kinetics
to wild-type Ran. The labeling efficiency of both was greater than 92% in
comparison with absorption spectra with tetramethylrhodamine or Alexa 488, so
this activity cannot be due to the presence of an active unlabeled fraction.
We concluded that Rh-Ran and Alexa 488-Ran are as active as the wild-type
unlabeled protein.
GFP-Ran was isolated in a visual screen of a cDNA library concatenated with
GFP (VLP55) (Rolls et al.,
1999). It consists of an N-terminal GFP followed by a 22
amino-acid linker region (GGGLDPRVRSDGRGDASGRNAA) and the entire coding
sequence for human Ran. GFP-Ran was inserted into the pSP64-S expression
vector by standard PCR methods resulting in a consensus echinoderm kozak
sequence (aattcaaa). RNA was prepared in vitro using the SP6 mMESSAGE mMACHINE
kit (Ambion; Austin, TX). Owing to difficulties in producing GFP-Ran for
biochemical characterization, its activity was not tested in vitro.
Modeling
Photobleaching of chromosomal Ran was followed by exponential recovery.
This is consistent with a model based on Ran (or a binding unit containing
Ran) binding by mass action kinetics to a single type of binding site.
Assuming that the endogenous, fluorescent and bleached forms of Ran behave
similarly, the concentrations of bound fluorescent Ran, [R*B] and
bound endogenous Ran, [RB] are described by the equations:
![]() | (1.1) |
![]() | (1.2) |
![]() | (2) |
The experimentally observed exponential recovery does not necessarily mean that there is only one type of interaction of Ran with chromosomes. For instance, if there is a second interaction with much larger koff, there would be a fast recovery for this interaction, which would be complete by the time of the first experimental time point. Also, if the number of binding sites is very different, then the less-abundant site will make only a small contribution to the recovery curve.
For the binding experiments, the experimentally determined values are
[R*B]/[R*] as a function of added [R*]. The
total amount of fluorescent Ran was known from the quantitative injection. At
steady state, there is a higher concentration of Ran in the nucleus than in
the cytoplasm, so the total nuclear Ran was calculated by taking into account
this difference and the relative volumes of cytoplasm and nucleus. As follows
from Equations 1 under steady-state conditions (adding equations 1.1 and 1.2
then substituting for [RB]+[R*B] from the Bt equation):
![]() | (3) |
The last part of Equation 3 can be used for estimating K+[R] from
experiments with different amounts of [R*] by multiplying the
experimental [R*B]/[R*] by [R*], which puts
the right hand side into the form of the Michaelis-Menten equation with
parameters Vm=Bt and Km=K+[R]. The analysis
is made somewhat more complex because the endogenous Ran concentration is not
known and it is not known whether free Ran or complexed Ran is binding to the
chromosomes. When is equal to the fraction of the total nuclear Ran
(either endogenous or fluorescent), which is available for binding, the
expression for Km is (K/
)+[R].
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
For imaging Ran localization and dynamics in living eggs, we used a
rhodamine-labeled Ran (Rh-Ran). The activity of Rh-Ran was tested in GTPase
assays in vitro and behaved identically to unmodified Ran (see Materials and
Methods). Microinjected Rh-Ran accumulated at the chromosomes
(Fig. 1B). A GFP chimera of Ran
was isolated in a visual screen of a cDNA library concatenated with GFP
[termed VLP55; (Rolls et al.,
1999)]. GFP-Ran, expressed by mRNA injection, also accumulated at
the chromosomes (data not shown).
Mature mouse oocytes are arrested in metaphase of meiosis II. As in
Xenopus eggs, microinjected Rh-Ran localized to the mouse egg
chromosomes (Fig. 1C)
(Verlhac et al., 2000).
The meiosis II metaphase arrest in Xenopus eggs was released by
raising intracellular calcium. The egg surface was punctured with a
microneedle (prick activation); a calcium wave begins at this point and takes
several minutes to cross the egg. Rh-Ran was co-injected with calcium green
dextran in order to determine exactly when the calcium wave rose in the
spindle region. The Rh-Ran labeled chromosomes began anaphase movements
between 12-16 minutes after the calcium rose in the spindle region
(n=3) (Fig. 2). The
timing corresponds well with anaphase onset in cell-free extracts stimulated
by addition of calcium (Murray,
1991; Desai et al.,
1998
). Experiments with Rh-tubulin and calcium green dextran gave
the same results (data not shown). Rh-Ran remained associated with the
chromosomes at approximately the same brightness. In later stages, Rh-Ran
gradually became incorporated into the newly forming nuclear envelope
(Fig. 2).
|
Chromosomal association of Ran in starfish oocytes and embryos
In order to image Ran throughout meiosis and mitosis, we used starfish
oocytes and embryos; they are smaller than frog eggs (180 µm versus
1.3 mm), are optically clear, and are easier to maintain on the
microscope stage than mouse eggs. Immature starfish oocytes are arrested in
late G2 of meiosis I and possess a large nucleus. Microinjected Rh-Ran, as
well as GFP-Ran expressed by mRNA injection, became localized to the nuclear
envelope. During maturation induced by 1-methyladenine, fluorescence at the
nuclear envelope disappeared with nuclear envelope breakdown, whereas the
fluorescent globules migrated to the animal pole
(Fig. 3A). In oocytes
co-injected with Rh-tubulin to label the microtubules, the fluorescent Ran
became part of the meiotic spindle and separated at the time of polar body
formation (Fig. 3B). This
corresponds exactly with the known distribution and behavior of chromosomes in
starfish oocytes (Shirai et al.,
1990
; Ookata et al.,
1992
).
|
Fluorescently labeled Ran was also observed during mitosis in developing
embryos. The fluorescent nucleotide Oregon Green dUTP is incorporated into
newly synthesized DNA (Carroll et al.,
1999) and was used in double labeling experiments in embryonic
blastomeres. There was a close correspondence of the Oregon Green dUTP-labeled
chromosomes and Rh-Ran throughout the entire mitotic phase
(Fig. 3C). In the mitotic cells
as well, microtubules and Ran localized as expected
(Fig. 3D).
Ran distribution in mitotic mammalian cultured cells
When fluorescent Ran was microinjected into mitotic mammalian cells, it was
found to be distributed throughout the mitotic spindle region, with no
apparent accumulation on the chromosomes
(Fig. 4). However, fluorescent
dextran was excluded from the chromosomal regions. When a mixture of 70 kDa
Rh-dextran and fluorescent Ran was injected into mitotic NRK (normal rat
kidney) cells, the fluorescent dextran was excluded from the region of the
chromosomes while the fluorescent Ran was not excluded
(Fig. 4). If Ran were not
interacting with chromosomes, it should have the same distribution as the
dextran. Thus, in mitotic mammalian cultured cells, Ran appears to be
associated with chromosomes, although to a lesser degree than in
Xenopus, mouse or starfish eggs.
|
Quantitative analysis of Ran association with chromosomes
Rh-Ran in a small region of chromosomes was photobleached with intense
laser excitation, and the redistribution of the remaining fluorescence was
monitored. In meiosis-I-metaphase-arrested Xenopus eggs, Rh-Ran
fluorescence redistributed to photobleached chromosomal regions with a 50%
recovery time of 11.7±4.0 second (s.d.; n=8 eggs)
(Fig. 5A,B). In blastula-stage
starfish embryos, a bleached area of metaphase-chromosome-associated Rh-Ran
recovered to 50% by 4.7±1.7 seconds (s.d.; n=5 cells)
(Fig. 5C,D). Owing to the
changing shape and position of anaphase chromosomes, we could not determine
whether the exchange rates change after the metaphase-anaphase transition.
|
The chromosomal association of Ran was examined more quantitatively in immature starfish oocytes, where the nucleus is intact but the chromosomes are partially condensed.
The photobleached chromosomal Ran recovery was found to be described very
well with an exponential rate constant 0.06 second-1,
corresponding to a 50% recovery time of
11 seconds
(Fig. 6A). The experimentally
observed exponential recovery is consistent with chromosomal Ran undergoing
one major interaction, of the simple type described above, and also allows one
to equate the exponential rate constant to the koff. Had
the experimentally observed recovery been non-exponential, it would not be
possible to draw these conclusions.
|
For simple binding interactions, a standard procedure is to measure the bound ligand as a function of total ligand concentration; this analysis yields the dissociation constant Kd and the concentration of binding sites. Four different amounts of Rh-Ran were injected into starfish oocytes and allowed to come to equilibrium, then measurements of the brightest part of the chromosome (typically a 1-2 µm diameter region of interest) along with a neighboring nuclear region were made. In oocytes injected with 10 kDa rhodamine dextran and Alexa 488-Ran, the fluorescent dextran was just as bright in the nucleus as in regions of the chromosomal Rh-Ran labeling (Fig. 6B), leading to the conclusion that the soluble Ran can diffuse into the regions of the chromosomes. All of the chromosomes are present in a small space in mitotic cells (Fig. 4C), so it seems reasonable that the scattered individual and more extended chromosomes in immature oocytes do not exclude soluble molecules. We therefore subtracted nuclear from chromosomal fluorescence to get the value for bound Ran. As with the FRAP recovery, the relationship between bound Ran and added Ran appears to be characteristic of a single type of binding site (Fig. 6C). Fitting the curve yields a value of 30 µM for the concentration of Ran-binding sites within the space occupied by the chromosomes.
To determine the Kd, it is necessary to know the amount of free
ligand that is available for binding. However, the endogenous Ran
concentration in starfish oocytes is unknown. Furthermore, the fraction
of the nuclear Ran that is available for binding to chromosomes is
unknown. Much of nuclear Ran is probably bound to proteins such as
import/export factors while a smaller amount exists as free Ran-GTP and
Ran-GDP. It is not known which of these is the chromosome-binding unit. These
uncertainties do not affect the above conclusions about binding to a single
type of site, or the concentration of the sites, nor do they affect the FRAP
determination of koff. Also, even with these
uncertainties, it is possible to estimate the sum (Kd/
) +
[R], where [R] is the total endogenous nuclear Ran and
is the fraction
of the total nuclear Ran that is available for binding to chromosomes (see
Materials and Methods). The data suggested that the sum of
(Kd/
) + [R] was 9-10 µM. Estimates of nuclear Ran
concentration in other cell types are in the range of 8-10 µM. With these
values, Kd<1 µM and is smaller for smaller
.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An earlier report found no association of Ran with chromosomes in fixed or
digitonin-permeabilized mitotic mammalian cells in culture
(Ren et al., 1993). However,
formaldehyde was used to fix the cells for immunofluorescence in that study,
whereas we find that methanol fixation preserves the chromosomal distribution.
Also, there was no energy source in the digitonin-permeabilization medium.
Stably associated molecules may remain bound under these conditions, but our
data indicate a dynamic association of Ran with chromosomes, and it would not
be expected that Ran would remain bound to the chromosomes under these
conditions.
Our findings are more in agreement with recent in vivo and in vitro work.
Ran was localized by immunofluorescence at kinetochores of chromosomes in
C. elegans (Bamba et al.,
2002). Transient association of Ran with condensed chromatin added
to Xenopus extracts was seen
(Zhang et al., 1999
), and Ran
binding to nucleoplasmin-decondensed sperm chromatin has been demonstrated
(Bilbao-Cortes et al.,
2002
).
It is not known what constitutes the Ran chromosomal binding unit. Ran, in
its GTP or GDP form, may bind by itself, or may bind to another protein(s),
which then binds to chromosomes (see further discussion below). Even with
these uncertainties, photobleaching and binding experiments indicate that the
association of the Ran-binding unit follows mass action kinetics to a single
type of binding site of relatively high concentration within the space
occupied by the chromosomes (30 µM) and that the koff for
binding is
0.06 second-1. Because we are using a fluorescently
tagged human Ran in starfish oocytes, the values for endogenous Ran may be
different.
As for the chromosomal binding site for Ran, one possibility is that it is
located on the protein RCC1. This protein is thought to be continuously
associated with the chromosomes
(Carazo-Salas et al., 1999),
and since it is the only known GDP/GTP exchange factor for Ran, there must be
a steady-state concentration of Ran bound to RCC1 owing to the turnover
through RCC1. From in vitro studies of purified proteins, three kinetic steps
are involved in the GDP/GTP exchange that occurs on RCC1; the slowest
koff, and therefore the likely rate-determining step, is
20
second-1 (Klebe et al.,
1995b
). This is much greater than the koff value of
0.06 second-1 measured for Ran association with starfish
chromosomes. The discrepancy suggests that most of the chromosomal Ran is
involved in an interaction with another protein and that the steady-state
concentration of Ran bound to RCC1 is a small fraction of the chromosomal Ran.
In support of this, a significant amount of Ran can still bind to sperm
chromatin depleted of RCC1 (Bilbao-Cortes
et al., 2002
). Another possibility is that RCC1 is regulated
during mitosis by modification or association with other proteins and that its
kinetic properties measured in vitro are different from those in vivo.
Recent ideas regarding Ran's role in spindle assembly had predicted a
Ran-GTP gradient at the chromosomes; this gradient was recently demonstrated
(Kalab et al., 2002). However,
there have been no predictions of a high concentration of Ran at the
chromosomes. One possibility is that the chromosomal interactions that we
observe keep Ran-GTP near the chromosomes. An analogy may be made with the
sarcoplasmic reticulum in striated muscle. Calcium is stored in the
sarcoplasmic reticulum lumen, but the calcium-binding protein calsequestrin is
only located in the terminal cisternae, near the calcium release sites
(Franzini-Armstrong and Protasi,
1997
). In a similar way, Ran associations with chromosomes may
serve to concentrate Ran-GTP in the area of the cell where it will function.
This is supported by the finding that the chromatin-bound Ran increases
membrane vesicle association with chromatin in an in vitro assay for nuclear
envelope reformation (Bilbao-Cortes et al.,
2002
).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bamba, C., Bobinnec, Y., Fukuda, M. and Nishida, E. (2002). The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo. Curr. Biol. 12,503 -507.[CrossRef][Medline]
Bilbao-Cortes, D., Hetzer, M., Langst, G., Becker, P. B. and Mattaj, I. W. (2002). Ran binds to chromatin by two distinct mechanisms. Curr. Biol. 12,1151 -1156.[CrossRef][Medline]
Bischoff, F. R. and Ponstingl, H. (1991). Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. Proc. Natl. Acad. Sci. USA 88,10830 -10834.[Abstract]
Carazo-Salas, R. E., Guarguaglini, G., Gruss, O. J., Segref, A., Karsenti, E. and Mattaj, I. W. (1999). Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400,178 -181.[CrossRef][Medline]
Carazo-Salas, R. E., Gruss, O. J., Mattaj, I. W. and Karsenti, E. (2001). Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly. Nat. Cell Biol. 3,228 -234.[CrossRef][Medline]
Carroll, D. J., Albay, D. T., Terasaki, M., Jaffe, L. A. and Foltz, K. R. (1999). Identification of PLC gamma-dependent and -independent events during fertilization of sea urchin eggs. Dev. Biol. 206,232 -247.[CrossRef][Medline]
Dasso, M. (2001). Running on Ran: nuclear transport and the mitotic spindle. Cell 104,321 -324.[Medline]
Dasso, M., Seki, T., Azuma, Y., Ohba, T. and Nishimoto, T. (1994). A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation. EMBO J. 13,5732 -5744.[Abstract]
Desai, A. and Hyman, A. (1999). Microtubule cytoskeleton: No longer an also Ran. Curr. Biol. 9,R704 -R707.[CrossRef][Medline]
Desai, A., Maddox, P. S., Mitchison, T. J. and Salmon, E. D.
(1998). Anaphase A chromosome movement and poleward spindle
microtubule flux occur at similar rates in Xenopus extract spindles.
J. Cell Biol. 141,703
-713.
Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L.,
Presley, J. F., Worman, H. J. and Lippincott-Schwartz, J.
(1997). Nuclear membrane dynamics and reassembly in living cells:
targeting of an inner nuclear membrane protein in interphase and mitosis.
J. Cell Biol. 138,1193
-1206.
Franzini-Armstrong, C. and Protasi, F. (1997).
Ryanodine receptors of striated muscles: a complex channel capable of multiple
interactions. Physiol Rev.
77,699
-729.
Gallo, C. J., Hand, A. R., Jones, T. L. Z. and Jaffe, L. A.
(1995). Stimulation of Xenopus oocyte maturation by
inhibition of the G-protein s subunit, a component of the
plasma membrane and yolk platelet membranes. J. Cell
Biol. 130,275
-284.[Abstract]
Gorlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15,607 -660.[CrossRef][Medline]
Gruss, O. J., Carazo-Salas, R. E., Schatz, C. A., Guarguaglini, G., Kast, J., Wilm, M., le Bot, N., Vernos, I., Karsenti, E. and Mattaj, I. W. (2001). Ran induces spindle assembly by reversing the inhibitory effect of Importin alpha on TPX2 activity. Cell 104,83 -93.[Medline]
Heald, R. and Weis, K. (2000). Spindles get the ran around. Trends Cell Biol. 10, 1-4.[CrossRef][Medline]
Hetzer, M., Bilbao-Cortes, D., Walther, T. C., Gruss, O. J. and Mattaj, I. W. (2000). GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5,1013 -1024.[Medline]
Jaffe, L. A., Gallo, C. J., Lee, R. H., Ho, Y. K. and Jones, T. L. (1993). Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein. J. Cell Biol. 121,775 -783.[Abstract]
Kahana, J. A. and Cleveland, D. W. (1999).
Beyond nuclear transport. Ran-GTP as a determinant of spindle assembly.
J. Cell Biol. 146,1205
-1210.
Kahana, J. A. and Cleveland, D. W. (2001). Cell
cycle. Some importin news about spindle assembly.
Science 291,1718
-1719.
Kalab, P., Pu, R. T. and Dasso, M. (1999). The ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9,481 -484.[CrossRef][Medline]
Kalab, P., Weis, K. and Heald, R. (2002).
Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus
egg extracts. Science
295,2452
-2456.
Klebe, C., Bischoff, F. R., Ponstingl, H. and Wittinghofer, A. (1995a). Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochem. 34,639 -647.[Medline]
Klebe, C., Prinz, H., Wittinghofer, A. and Goody, R. S. (1995b). The kinetic mechanisms of Ran-nucleotide exchange catalyzed by RCC1. Biochemistry 34,12543 -12552.[Medline]
Lemaitre, J.-M., Géraud, G. and Méchali, M.
(1998). Dynamics of the genome during early Xenopus
laevis development: karyomeres as independent units of replication.
J. Cell Biol. 142,1159
-1166.
Macara, I. G., Brownawell, A. and Welch, K. (2000). Ran. In GTPases (ed. A. Hall), pp. 198-221. Oxford: Oxford University Press.
Mattaj, I. W. and Englmeier, L. (1998). Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem. 67,265 -306.[CrossRef][Medline]
Mehlmann, L. M. and Kline, D. (1994). Regulation of intracellular calcium in the mouse egg: Calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation. Biol. Reprod. 51,1088 -1098.[Abstract]
Moore, J. D. (2001). The Ran-GTPase and cell-cycle control. Bioessays 23, 77-85.[CrossRef][Medline]
Murray, A. W. (1991). Cell cycle extracts. Methods Cell Biology 36,581 -605.[Medline]
Nachury, M. V., Maresca, T. J., Salmon, W. C., Waterman-Storer, C. M., Heald, R. and Weis, K. (2001). Importin beta is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104,95 -106[Medline]
Ohba, T., Nakamura, M., Nishitani, H. and Nishimoto, T.
(1999). Self-organization of microtubule asters induced in
Xenopus egg extracts by GTP-bound Ran.
Science 284,1356
-1358.
Ohtsubo, M., Okazaki, H. and Nishimoto, T. (1989). The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. J. Cell Biol. 109,1389 -1397.[Abstract]
Ookata, K., Hisanaga, S., Okano, T., Tachibana, K. and Kishimoto, T. (1992). Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes. EMBO J. 11,1763 -1772.[Abstract]
Ren, M., Drivas, G., D'Eustachio, P. and Rush, M. G. (1993). Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis. J. Cell Biol. 120,313 -323.[Abstract]
Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M. and Gorlich,
D. (1998). NTF2 mediates nuclear import of Ran.
EMBO J. 17,6587
-6598.
Rolls, M. M., Stein, P. A., Taylor, S. S., Ha, E., McKeon, F.
and Rapoport, T. A. (1999). A visual screen of a GFP-fusion
library identifies a new type of nuclear envelope membrane protein.
J. Cell Biol. 146,29
-44.
Sazer, S. (1996). The search for the primary function of the Ran GTPase continues. Trends Cell Biol. 6,81 -85.[CrossRef]
Sazer, S. and Dasso, M. (2000). The Ran
decathlon: multiple roles of Ran. J. Cell Sci.
113,1111
-1118.
Shirai, H., Hosoya, N., Sawada, T., Nagahama, Y. and Mohri, H. (1990). Dynamics of mitotic apparatus formation and tubulin content during oocyte maturation in starfish. Dev. Growth Diff. 32,521 -529.
Terasaki, M., Runft, L. and Hand, A. (2001).
Organization of the endoplasmic reticulum during Xenopus oocyte
maturation and egg activation. Mol. Biol. Cell
12,1103
-1116.
Verlhac, M. H., Lefebvre, C., Guillaud, P., Rassinier, P. and Maro, B. (2000). Asymmetric division in mouse oocytes: with or without Mos. Curr. Biol. 10,1303 -1306.[CrossRef][Medline]
Wiese, C., Wilde, A., Moore, M. S., Adam, S. A., Merdes, A. and
Zheng, Y. (2001). Role of importin-ß in coupling Ran to
downstream targets in microtubule assembly. Science
291,653
-656.
Wilde, A. and Zheng, Y. (1999). Stimulation of
microtubule aster formation and spindle assembly by the small GTPase Ran.
Science 284,1359
-1362.
Wilde, A., Lizarraga, S. B., Zhang, L., Wiese, C., Glicksman, N. R., Walczak, C. E. and Zheng, Y. (2001). Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. Nat. Cell Biol. 3, 221-227.[CrossRef][Medline]
Zhang, C. and Clarke, P. R. (2000).
Chromatin-independent nuclear envelope assembly induced by Ran GTPase in
Xenopus egg extracts. Science
288,1429
-1432.
Zhang, C. and Clarke, P. R. (2001). Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system. Curr. Biol. 11,208 -212.[CrossRef][Medline]
Zhang, C., Hughes, M. and Clarke, P. R. (1999).
Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in
Xenopus egg extracts. J. Cell Sci.
112,2453
-2461.