Supplementary Information
Supplementary Figures
Supplementary Figure S1. Rango in HeLa extract. Emission spectrum of 0.5
M Rango in HeLa extract (green), plus 10 M RanQ69L-GTP (red) or 10 M
RanQ69LRan-GTP + 10 M importin 71-876 (blue). Arrows indicate emission
peaks at 474nm (ICFP) and at 525nm (IFRET).
1
Supplementary Figure S2.
Estimation of the apparent dissociation
constant for Rango-importin . Increasing amounts of importin  were added
to 6 nM Rango and the IFRET/ICFP ratio was determined by spectrofluorimetry.
Half-maximal binding was observed at about 2 nM importin .
2
Supplementary Figure S3. Disruption of the Ran-importin  interaction in
vivo is reported by the Rango fluorescence lifetime signal. The upper panel
shows the same cells displayed in Fig. 1c together with an image of the
fluorescent dextran tracer in the rhodamine channel to indicate the Rangoexpressing cell (left) that was coinjected with importin 71-876 (420 M in the
needle). The microinjection of importin 71-876 resulted in a strong increase of
Rango fluorescence lifetime =2.44 ± 0.03 ns N=4) indicative of nearly complete
binding of homologous endogenous cargo. In the lower panel, a mitotic HeLa cell
expressing Rango was injected with rhodamine-tubulin and RanQ69L (420 M),
abolishing the mitotic Rango gradient =2.21 ± 0.03 ns, N=5). Bar, 20 m.
3
Supplementary Figure S4. Comparison of Rango FRET signal in mitotic
cells and interphase nuclei by acceptor bleach. Rango FRET signal was
measured in interphase HeLa, in mitotic HeLa and BHK cells (averaged
cytoplasmic + chromatin signal), and in HeLa interphase cells microinjected with
Rango and importin 71-876 as indicated. The ratio of Cerulean CFP fluorescence
recovery after / before YFP photobleaching was used as a measure of the Rango
FRET signal. Boxes include the 25th to 75th percentile. In addition, the median is
represented by a horizontal line and minimal and maximal values indicated by
the error bars. The Rango FRET signal is similar in the cytoplasm of mitotic HeLa
and BHK cells (P> 0.05), but smaller than the Rango FRET signal in interphase
HeLa nuclei (P<0.01), and larger than in nuclei of HeLa cells coinjected with
Rango and importin 71-876 (P<0.01). The statistical analysis was performed with
one-way ANOVA and Dunnett’s multiple comparison tests.
4
Supplementary
Figure S5. Fractional occupancy detected by FLIM in
mitotic HeLa cells. The scatter plot displays the Rango fractional occupancy in
individual mitotic cells (N=36) in the chromatin region (red) and in the cytoplasm
(N=36) (blue). The difference between cytoplasmic and perichromatin fractional
occupancy in individual cells is displayed in orange (N=36, “gradient”). The
horizontal bars in plots represent means +/- standard deviation.
We excluded from the analysis of the Rango gradient additional 10 cells in
which the difference between chromatin and cytoplasmic donor was found to be
lower than the typical pixel-to-pixel noise in our conditions (0.04ns). If this data
were included, the average Rango occupancy in the chromatin changes from 18
+/- 9% (N=36) to 18 +/- 10% (N=46) and from 31 +/- 12% (N=36) to 29 +/- 13%
(N=46) in the cytoplasm.
5
IYFP
IFRET/ICFP
ICFP after/before YFP bleach
Supplementary Figure S6. The Rango gradient detected by confocal
microscopy in mitotic Hela cells. Rango is excluded from mitotic chromatin, as
shown in the YFP fluorescence image (left). Pseudocolor images of the I FRET/ICFP
ratio (center) and ratio of ICFP after and before YFP photobleaching (right)
demonstrate a significant increase in importin free Rango in the perichromatin
area. Linescans below the ratio images display the signal averaged over 5 m
wide area in linear section across cells in the ratio images, marked with a line.
Bar, 10 m.
6
Supplementary Figure S7. Rango FRET gradient in mitotic cells. The Rango
FRET signal was measured by the acceptor bleach method in mitotic cells
transfected with Rango, and the Rango gradient was calculated as a difference
between the average signal over the chromatin area vs. cytoplasm in individual
cells (bar height = mean +/- standard deviation). Boxes include the 25th to 75th
percentile, with the median represented by a horizontal line and minimal and
maximal values indicated by shorter horizontal lines. Already at the permissive
temperature (tsBN2 33oC) the average Rango gradient in tsBN2 cells was
smaller than in BHK21 cells (P<0.05) and in HeLa cells (P<0.01) indicative of a
decreased activity of RCC1 carrying temperature sensitive mutation. However,
treatment with the non-permissive temperature (tsBN2 40oC) abolished the
gradient, as indicated by a significant difference between tsBN2 at permissive
and non-permissive temperature (P<0.05). Statistical analysis was performed
with one way ANOVA with Dunnett’s multiple comparison test.
7
Supplementary Figure S8. Computational model of mitotic cytoplasm
We built a computational model of a minimal Ran system 1 coupled to the
minimal set of nuclear transport receptors, including importin  and Rango, using
Virtual Cell2, 3. This model applies kinetic parameters as described by Görlich et
al. (2003) 1 and Riddick and Macara (2005) 4.
As expected, in initial simulations we found that the relative concentrations
of transport receptors and their cargoes, have a large effect on the resulting
Rango fractional occupancy at equilibrium. To compare the response of this
model system to the experimental perturbations in the extracts (i.e. the addition
of importin  and RanQ69L), we set the initial reactant conditions to reflect the
measured Rango fractional occupancy at equilibrium in mitotic Xenopus egg
extracts.
Concentrations of Ran-transport system components used in the model (in M)
RanGTP
3
RanBP1
2
RanGDP
3
NTF2
0.6
GDP
1.6
Importin 
3
GTP
470
Importin  cargos
8
RCC1
0.25
Rango
2
RanGAP
0.4
Importin -like
transport receptors
5
Under these conditions, the response of this minimal system to importin 
and RanQ69L is qualitatively similar to our in vitro observations (Fig. 3c). This
suggests that the response of a localized importin  cargo release in vivo can be
quantitatively described in a simple mathematical model.
The models can be accessed at the published models webpage of the Virtual
Cell project (http://www.nrcam.uchc.edu/ User: Pralle, password: Ran ).
8
a
RanQ69L, 420 M
40 min
b
importin , 80 M
40 min
c
importin 71-876, 25 M
105 min
Supplementary Fig S9. Examples of phenotypes induced by microinjection
of HeLa cells with Ran system components. Mitotic HeLa cells were
microinjected, allowed to recover and then fixed and stained for tubulin and DNA.
The concentration of individual proteins in the needle, and time of recovery after
injection are indicated below the figures. a, Ectopic
microtubule asters
connecting to chromatin observed in a prophase cell microinjected with
RanQ69L. b, Prophase reversal: example of a HeLa cell that was microinjected
in prophase-to- metaphase state was found to revert to an interphase-like
microtubule pattern with decondensing chromatin. c, HeLa cell arrested in a
prolonged prophase state following microinjection of
concentration of importin 71-876.). Scale bar, 10 m
9
a relatively low
Supplementary Table
Protein
BSA
RanQ69L
Imp. 66-529
Imp.71-876
Importin 

M
prophase
reversal
550
420
310
40
180
420
25
80
410
3
8
3
0
4
5
2
2
6
prophase
stall /
defect
0
14
18
3
9
19
2
8
9
ectopic
asters
0
10
0
0
0
0
0
0
0
spindle
pole
defect
1
9
4
0
0
0
5
1
7
% normal
N
70
29
52
77
30
58
54
46
22
37
62
56
13
23
63
33
26
31
Supplementary Table 1. Summary of phenotypes induced by HeLa cell
microinjections. Prophase and metaphase HeLa cells were injected with protein
solutions as indicated and incubated at 37oC, 5% CO2 before being fixed, stained
and analyzed by fluorescence microscopy. Coinjected fluorescent dextran was
used as a marker to locate the injected cells. All treatments contain data from
slides fixed at time intervals that varied between 20-105 min. While this allowed
us to qualitatively examine a wider spectrum of possible phenotypes during
mitosis, a quantitative comparison of defect penetrance is limited.
Highlighted in red are the most prominent mitotic phenotypes induced by
individual microinjected proteins. Some cells that were injected in prophase-tometaphase state were found with interphase-like microtubules and a single mass
of decondensing chromatin, indicative of aborted mitosis (Supplementary Fig.
S9b). While such a “prophase reversal” phenotype was induced by all
microinjections, it was particularly frequent in cell injected with large
concentration of wt importin  (19%). This indicates the presence of a
checkpoint-like mechanism that is capable to revert progression of mitosis in
cells exposed to a variety of destabilizing insults, including disruptions of Ranimportin  regulation. Prophase cells with large monoastral MT arrays (Figure
4, cell injected with Importin 66-529) and all cells that were found in a prophase
state after more than 50 min of recovery (Supplementary Fig. S9c) are included
in “prophase stall / defect“ category. Cells with “ectopic asters” were those with
10
relatively small cytoplasmic microtubule asters that in some cases partially
interacted with chromatin (Fig. 4, cell injected with RanQ69L, Supplementary Fig.
S9a).
The category “spindle pole defect” includes cells with more robust,
supernumerary spindle pole–like microtubule structures associating with the
chromatin (Fig 4, cell injected with full length importin ), and cells with
apparently split, duplicated or malformed spindle poles.
Supplementary methods
Cloning and protein expression
To create a His6-tagged Rango E.coli expression plasmid (pKW1648), the
importin -binding domain of Snurportin15 (1-65) was inserted in frame between
EYFP (Clontech) and Cerulean6 cloned into pRSET-A (Qiagen). For expression
in tissue culture cells, the pKW1648 open reading frame was transferred into
pSG8, removing the N-terminal His6 tag (pKW1608). The pSG8 plasmid for kRango expression in cells (pKW1844) was produced by inserting the satellite
DNA binding domain of human Cenp-B7, at the 3’ of pKW1608 via BamH1 site.
The double protein A (ZZ) tagged-RanQ69L E.coli expression clone was
obtained by transferring ZZ-RanQ69L8 from pQE32 into pRSET-A (pKW1234).
The RBD-Cerulean clone for expression of Cerulean control protein in cells
(pKW1705) contains the Ran-binding-domain (RBD) of Yrb19 followed by
Cerulean.
Wild type Ran, zzRanQ69L, importin  and importin 71-976 were
expressed as described9. High yield of Rango proteins was obtained from BL21DE3 cells induced overnight at room temperature. The proteins were isolated
from French press lysates on NiNTA beads (Qiagen), dialyzed in XB (50 mM
sucrose, 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.7),
purified using a Superose 6 column (Pharmacia) in XB, snap frozen in liquid
nitrogen and stored at -80oC. Wheat germ agglutinin was purchased from Sigma,
rhodamine-tubulin from Cytoskeleton, and Lysine-fixable 75 kD Dextran-FITC
and Dextran-Rhodamine from Molecular Probes.
11
Spectrophotometry
Emission spectra were analyzed with a Fluorolog 2 spectrofluorimeter
controlled by Datamax 2.2 (Jobin Yvon Spex) and the Grams 3.04 II software
package (Galactic Industries, Salem, NH). Excitation wavelength, and excitation
(1.2-2.0 nm) and emission slits (2 or 5 nm) were set individually to minimize
sample-specific background and prevent saturation. Rango in Xenopus extracts
(Fig. 3c) was excited at 410 nm; excitation at 435 nm was used in Fig. 1b,c. A
blank was subtracted from all emission spectra except Fig. 1b. Rango in HeLa
extract (Supplementary Fig. 1) and in buffer (Supplementary Fig 2) was excited
at 410 nm and a corresponding blank was subtracted from the emission spectra.
Estimate of the importin free Rango fraction in X. laevis egg extract
We assumed that the IFRET/ICFP ratio measured at saturating concentrations of
RanQ69L (25 M, data not shown) corresponds to a concentration of free Rango
approaching 100%, and the IFRET/ICFP measured at saturating concentration of
importin  (20 M) corresponds to near complete Rango binding. Using this
assumption, we normalized IFRET/ICFP values measured in extract (Fig. 3c), and
IFRET/ICFP values measured in vitro (Fig. 1c) to a 0-1 scale. Next, we used the
IFRET/ICFP values measured in the extract to determine the closest corresponding
Rango fractional occupancy determined in in vitro.
Microinjection
Cells were seeded at 25% confluency on glass coverslips 2-3 days prior to
microinjection. Proteins were diluted in XB or IB buffer (100 mM potassium
glutamate, 40 mM citric acid, pH 7.4), and centrifuged 5 min at 100 000xG prior
to microinjection, using an Olympus IX71 microscope equipped with a FemtoJet
microinjector (Eppendorf).
Immunofluorescence
Microinjected
HeLa
cells
were
fixed
in
0.05%
glutaraldehyde,
4%
paraformaldehyde (freshly prepared) in PHEM (60 mM Pipes, 25 mM Hepes, pH
12
6.9, 10 mM EGTA, 4 mM MgSO4) for 15 min at 37oC, followed by 5 min treatment
with fresh 0.5% (w/v) NaBH4 in PHEM and 20 min block in 100 mM Glycine made
in 6% heat-inactivated fetal bovine serum, 0.02% saponin, PHEM, pH 7.0. The
cover slips were incubated with anti -tubulin mouse monoclonal antibody E7
(1:350; Developmental Studies Hybridoma Bank at the University of Iowa)
followed by Donkey anti mouse IgG- Rhodamine or Donkey anti-mouse IgG-FITC
(Jackson Laboratories). DNA was stained with Hoechst 33342 dye (Sigma).
Samples were mounted with Anti-Fade Gold (Molecular Probes, Invitrogen).
Live cell imaging
During all live cell imaging experiments, cells were maintained at 30 oC in
a
temperature
controlled
imaging
chamber
(Warner
instruments).
For
epifluorescence and FLIM measurement, cells were kept in Dulbecco’s PBS
(Gibco, Invitrogen) supplemented with 1 mM sodium pyruvate (Gibco,
Invitrogen). During acceptor bleach experiments, cells were in Opti-MEM, 4%
FBS (Gibco, Invitrogen).
Epifluorescence
Live epifluorescence ratio imaging in Fig. 3a was performed with a Nikon
E600 microscope equipped with CFP-YFP FRET filter sets (Chroma) and
Hamamatsu C4742-98 CCD camera as described previously9, using Nikon Fluor
60x/1.00W water immersion lens (Nikon) for cells, and 40x/0.75 Nikon PlanFluor
lens for egg extract samples. Exposure time was 0.5-4 s and always identical for
YFP, CFP an FRET channels within a given sample. Image processing was
performed with Metamorph (Molecular Devices). The pixel alignment of the I YFP,
ICFP, and IFRET images was verified and adjusted, the background taken outside
the cells subtracted and IFRET/ICFP and ICFP/IYFP ratio computed. The 60x oil
immersion lens on Nikon E600 microscope was used to count rhodamine labeled
microtubule asters in Fig. 3c.
Live samples of rhodamine labeled microtubules in Xenopus egg extracts
(Fig. 3c) were examined and photographed using 60x oil immersion lens and all
fixed immunofluorescence samples using 100/1.30x oil immersion lens, both on
Olympus BX51 microscope equipped with Hamamatsu CA 742-98 CCD camera.
13
Confocal microscopy
Confocal microscopy was performed with a Zeiss LSM 510 META laser
scanning confocal microscope. Cerulean was excited by the 458 nm Argon laser
line and emission was simultaneously acquired at 462-505 nm (ICFP) and 537-569
nm (IFRET) by the META detector. YFP was excited by the 514 nm Argon laser
line, acquiring IYFP by 526-601 nm META detector. Four 12-bit image sets of
256x256 pixels were acquired (7.58 s per image) in each channel (I YFP, ICFP +
IFRET) prior to bleaching YFP by scanning with the 514 nm laser at full power for
100-200 iterations (20-30 s), followed by acquisition of an additional three sets of
images in all channels. As Rango is mostly excluded from condensed mitotic
chromatin, we used an 8 M optical slice in all experiments to obtain enough
signal. FRET efficiency was calculated as ratio of ICFP signal in the first image
following bleaching, and the ICFP signal in the image preceding the bleach. In
mitotic cells, the FRET efficiency was determined from an area covering the
entire cell, an area within the chromatin region, and two cytoplasmic regions
located approximately 70% distance between chromatin and the plasma
membrane. As a measure of the mitotic Rango gradient, we calculated the
difference between FRET efficiency within chromatin area and cytoplasmic
average.
Fluorescence lifetime microscopy
Datasets of spatially resolved, time-correlated single photon counting
(TCSPC) were acquired on an inverted Zeiss LSM510 Axiovert 200M microscope
with a NeoFLUAR 40
/1.3 NA oil-immersion immersion objective lens, equipped
with a TCSPC controller (Becker & Hickl SPC-730 card). Samples were excited
by 6 W, 435 nm generated by frequency-doubling 870nm pulses from a modelocked Ti:Sapphire laser (Tsunami, SpectraPhysics, 120-150 fs pulse width, 80
MHz repetition rate). One photon excitation of Cerulean at 435 nm was preferred
over two-photon excitation because it provided less direct excitation of YFP. The
emission light was filtered with a 480 nm bandpass filter (480BP40, Chroma) and
detected by a PMC-100 photomultiplier (Becker & Hickl) mounted to the fiber-out
port of the confocal scanhead. Recording conditions were chosen to achieve
14
approximately 105 counts per second, and images of 64
64 pixels (1024 time
bins/pixel) were averaged over 2-4 minutes.
For the calibration curve (Fig. 1), the fluorescence lifetime of a 1 M
solution of Rango in PBS, 0.03% Tween 20 was measured over 1 minute while
scanning the laser in an area of 20
20 m 30 m above the coverslip surface
at 23oC and 30oC. Off-line data analysis was done using pixel-based fitting
software (SPCImage, Becker & Hickl), assuming single exponential incomplete
decay during the 12.5 ns interval between laser pulses.
Xenopus laevis egg extracts
Assays for the detection of the Rango IFRET/ICFP signal during mitotic
spindle assembly in X. laevis egg extracts were performed as described
previously9 with rhodamine-tubulin and 2 M Rango in the extract instead of YIC.
For the experiment in Fig. 3c, mitotic egg extract containing 1 M Rango and 15
g/ml rhodamine-tubulin was divided into 57 l aliquots. Samples were then
supplemented with 3 l zzRanQ69L or importin , respectively, to achieve
concentrations indicated in Fig. 3c. Control samples received 3 l buffer only.
Samples were then transferred from ice to a 21 oC water bath and Rango
emission at 435 nm excitation was continuously monitored until a stable I FRET/ICFP
ratio was reached (60-90 min). At the end of the scans (90-120 min), rhodaminelabeled microtubule structures were examined and photographed live in 2-3
aliquots of samples.
To quantify the aster promoting activity in the samples, after the last scan,
30 l extract samples were fixed in freshly prepared 10% glycerol, 4%
formaldehyde, 0.1% glutaraldehyde, PHEM. Fixed samples were then spun on
12 mm round coverslips as described8, fixed in -20oC methanol (10 min), rinsed
with PHEM and mounted on microscope slides. The aster promoting activity,
defined as an average number of microtubule asters highlighted by incorporated
rhodamine-tubulin, was counted in 30 randomly selected visual fields (600x total
magnification).
HeLa cell extracts
15
HeLa S3 cells grown in suspension culture were washed with PBS by
centrifugation, pelleted and frozen in liquid nitrogen. The frozen pellet (1 ml) was
supplemented with 700 l M/PBS buffer (120 mM Na -glycerophosphate, 60
mM EGTA, 22.5 mM MgCl2, 72 mM NaCl, 1.3 mM KCl , 5 mM Na2HPO4. 8.5 mM
KH2PO4, pH 7.4.) containing 20 mg/ml each leupeptin, pepstatin and
chymostatin, and 1 mM PMSF. The tube was placed on ice and the liquid above
the pellet sonicated at 0.5 W using a microtip (Virsonic sonicator, The Virtis
Company Inc., Gardiner, NY) until the pellet became homogenized (about 5 min).
The sonicated lysate was clarified by centrifugation at 15,000 g for 10 min and
used immediately.
Rango – importin  dissociation constant
Aliquots of 40 nM Rango in PBS were diluted with increasing
concentrations of importin  in PBS to achieve a final concentration of 6 nM
Rango and 0-40 nM importin . IFRET/ICFP ratios in samples were measured at
25oC against a PBS blank in two samples (2-3 scans each) from each dilution
aliquot. The IFRET/ICFP values were plotted against the importin  concentration
and data were fitted with a down to bottom curve, standard slope using
GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego
California USA). The Rango-importin  dissociation constant was determined
using the importin  concentration at 50% amplitude of Rango IFRET/ICFP curve.
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3.
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17