Stathmin-Tubulin Interaction Gradients in Motile and Mitotic Cells

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1 Science Supporting Online Material Niethammer, p. 1 Stathmin-Tubulin Interaction Gradients in Motile and Mitotic Cells Contents Materials and Methods SOM Text Fig. S1 References Movie S1 Philipp Niethammer, Philippe Bastiaens, Eric Karsenti doi: /science Materials and Methods Cloning and bacterial expression of proteins For bacterial expression, Xenopus stathmin cdnas encoding wild-type (wt) protein or Ser-to-Ala (aaa) and Ser-to-Glu (eee) mutants were subcloned into an ECFP-pHAT2- vector containing an N-terminal 6-His-ECFP sequence upstream to the introduced stathmin cdna. For high-level expression in Escherichia coli the second residue of the stathmin-cdnas had been mutated to Ala. Downstream of the 6-His-ECFP-OP18 sequence a citrine (S1) was introduced to yield a 6-His-ECFP-stathmin-citrine construct. The phat2-6-his-ecfp-stathmin-citrine-constructs were expressed in BL21 cells, grown at 37 C to an OD 600 of 0.5. Expression was induced with 0.5 mm IPTG for 5 hours at 20 C. Bacteria were pelleted at 4 C by centrifugation and resuspended within Hepes buffer (Hepes 25 mm, ph 7.5) containing protease inhibitors (leupeptin, pepstatin, and aprotinin, 100 µg/µl) and PMSF (1 mm). The cells were lysed using a French Press and the extract was clarified at 10,000 rpm for 30 min at 4 C. The supernatant was incubated with a metal affinity resin (Talon IMAC Resin; CLONTECH) for 1 hour at 4 C. After washing the resin with Hepes buffer (Hepes 25 mm, ph 7.5) the protein was eluted with 100 mm imidazole and buffer was changed to BRB80-buffer (80 mm potassium-pipes, 1 mm MgCl 2, and 1 mm EGTA, ph 6.8) using a PD10-column (Amersham Biosciences). Protein concentration was determined by absorption at A 280 using the theoretical absorption coefficient calculated by the Protean-software (DNASTAR). In vitro fluorescence assays Wavelength spectrum of COPY-wt. A 2 µm solution of recombinant COPY-wt (BRB80 ph6.8, RT) was excited with monochromatic light (430 nm) in a spectrofluorometer (QuantaMaster, Photon Technology, Inc.), and the emission spectrum ( nm) before and after addition of 30 µm tubulin (pig-brain) was recorded. The spectra were corrected for dilution of the sample. Ratiometric analysis of COPY-tubulin interaction. Recombinant COPY proteins were measured in BRB80 ph 6.8 at RT. CFP was excited with monochromatic light at a wavelength of 430 nm using a spectrofluorometer (QuantaMaster, Photon Technology, Inc.). CFP- and citrine-emissions were acquired simultaneously at 475 nm and 525 nm, respectively. Cycled tubulin was added stepwise to the COPY solution and the ratio between the citrine- and the CFP-emission was monitored for each step of tubulin addition.

2 Niethammer, p. 2 Calculation of K d for the COPY-tubulin interaction The concentration of free tubulin was calculated as [T free ] = [T total ] 2Y[S total ] with [T total ] and [S total ] being the total amount of tubulin and stathmin present in the experimental system and Y the fractional saturation of COPY. The plot of ν = 2Y versus [T free ] was fitted (SigmaPlot, SPSS Inc.) with a two-site binding model assuming two independent binding sites for COPY [ν = B max1 x/(k d1 + x) + B max2 x / (K d2 + x)]. Each fit yielded B max1 = B max2 1. In vitro phosphorylation of COPY-wt A reaction-mixture of 4 µm COPY-wt in BRB80 ph 6.8 containing 9 mm MgCl 2, 25 mm ATP, 2800 units activated p42 MAP kinase (New England Biolabs), 17,500 units camp-dependent protein kinase (catalytic subunit, New England Biolabs) was incubated for 4 hours at 27 C. Phosphorylation of COPY at Ser 16 was assayed with a phosphospecific antibody (sc-12948, 1:2000, Santa Cruz Biotechnology, Inc.). Nonphosphorylated COPY was detected by reprobing the membrane with an antibody against the N terminus of stathmin (sc-10899, 1:2000, Santa Cruz Biotechnology, Inc.). Western blot signals were quantified by band density analysis using the ImageJ analysis software (Version 1.30v; Wayne Rasband, NIH, USA). Cell culture and transfection For expression in Xenopus cells the ECFP-stathmin-citrine sequence was subcloned into a pcdna3.1(+) vector (Invitrogen). Xenopus A6 cells were cultured at 23 C in 70% L15 Leibowitz-Medium (Sigma-Aldrich) supplemented with 15% FCS and 2 mm l-glutamine. For transfection with Lipofectamine Plus (Invitrogen), cells were seeded in six-well dishes to reach a density of 80% and transfected in serum-free DMEM using 2 µg plasmid per well according to the Lipofectamine Plus-Protocol. Twenty-four hours after transfection, cells were replated on polylysine coated coverslips or, in case of live-cell experiments, on Matek-dishes (Matek Corporation). In order to determine the COPY expression level, untransfected and transfected cells were stained with a primary stathmin antibody and a Cy5-labeled secondary antibody and the average Cy5 fluorescence of both populations was compared. Xenopus XL177 cells were propagated like A6 cells, but transfected by electroporation as follows: Cells from one 70 80% confluent 90-mm Petri dish were collected, washed in 70% PBS, resuspended in 200 µl 70% PBS with 20 µg plasmid DNA and electroporated in 0.2 cm cuvettes (Gene Pulser, Biorad) at 220 V, 200 Ω, and 250 µf. Cells were subsequently plated on polylysine-coated coverslips. FRET was measured 48 hours after transfection. Drug treatment of cells Serum starved (12 16 hours) A6 cells were stimulated with serum-free L15-medium supplemented with 100 nm phorbol 12-myristate 13-acetate (TPA, Sigma) for 30 min at 23 C. Inhibition of phosphatases was performed by simultaneous incubation with 6 µm okadaic acid (sodium salt, Calbiochem). Fixation and immunostaining of cells Cells were fixed with 4% paraformaldehyde for 15 min at RT. Monoclonal α-tubulinspecific antibody (DM1α, Sigma) was applied 30 min at RT, 1:400 in TPBS (PBS + 0.1% Triton X-100). Fluorescence labeling of the primary antibody was performed by incubating the coverslips with a Cy5-labeled secondary antibody directed against mouse (Molecular Probes) for 30 min at RT. Coverslips were rinsed several times in PBS and mounted with Aqua Poly/Mount (Polysciences, Inc.).

3 Niethammer, p. 3 Confocal microscopy Ratiometric measurement in mitotic Xenopus egg extracts. Recombinant COPY-wt (6 µm) was added to mitotic Xenopus egg extract (S2). The extracts were incubated 30 min at 20 C with and without addition of 4 µm okadaic acid (sodium salt, Calbiochem). With an SP2 confocal microscope (Leica), 12-bit confocal images of the extract were acquired with an HCX PL APO 63.0x1.32 objective. CFP was excited with a 405-nm laser diode (50% power). CFP emission was sampled between 470 and 500 nm, YFP emission was sampled between 525 and 650 nm (4 lineaverages, pinhole: 5.23 airy). Fluorescence images were smoothed (median filter) and the YFP:CFP images were calculated. Ratiometric measurement in Xenopus cells. With an SP2 confocal microscope (Leica), 12-bit confocal images of COPY in Xenopus A6-cells were acquired with an HCX PL APO 63.0x1.32 objective. CFP was excited with a nm argon laserline (68% power). CFP emission was sampled between 470 and 500 nm, YFP emission was sampled between 525 and 610 nm (time lapse: 16 line averages, pinhole 1 airy; others: 8 line-averages, pinhole 3 airy). Cy5 emission was excited at 633 nm and sampled between 650 nm and 800 nm. For mitotic XL177 cells, confocal z-stacks were acquired and z-projected (average method) to yield the CFP and YFP intensity images (16 line-averages, pinhole: 1 airy). All image processing was done with ImageJ. The average background was measured in a square near the cell and subtracted globally. Images were smoothed (median filter) and a threshold was applied. The ratio map of each cell was calculated by dividing the corrected fluorescence images (YFP:CFP). Acceptor photobleaching. With an SP2 confocal microscope (Leica), 12-bit confocal images of COPY in Xenopus A6-cells were acquired with an HCX PL APO objective. The YFP signal was bleached (514-nm laser line) in rectangular quarters of the cells containing central, as well as peripheral, regions. CFP was excited with a 405-nm laser diode (33% power). CFP emission was sampled between 450 and 490 nm before and after bleaching (8 line-averages, pinhole: 1 airy). After background-correction and smoothing (median-filter) the prebleach-image was subtracted from the postbleach-image. The subtraction-image was divided (for all pixel values >0) by the postbleach-image to yield the FRET-efficiency image. The mean FRET efficiency was measured in two arbitrary rectangular regions at the leading edge and in one region at the cell center. The average FRET difference was calculated for each cell and averaged for the whole population. Statistical analysis of ratio-images SIH assay. A square was drawn to fit the ratio image of the cell (Fig. 2C, upper panel). Squares of 10 by 10 pixels were placed at the sites, where the diagonals hit the edges of the cell (r 1 to r 4 ) or each other (c) in the center. The mean ratio values in these squares were acquired. Relative ratio changes ( (r n -c) /c) from the periphery to the inside were calculated and averaged for each cell. From these single-cell SIHvalues, the average for the whole population was calculated Mitotic gradient analysis. Mean ratios were measured within three circular areas around the center of the spindle (r 1 to r 3; Fig. 5B, upper panel). The mean ratio in the rest of the cell (c) was set as base value. The relative difference ( r) between the basal ratio value and the mean ratio of the three circular regions was calculated.

4 Niethammer, p. 4 Calculation of gradient size By FRAP experiments, we approximated the diffusion constant of COPY to be D COPY µm 2 /s. The concentration of PP2A in Xenopus egg extracts was reported to be 3 6 µm (S3). This gives a total phosphatase activity at subsaturating conditions of s 1 given the K m (10.8 µm) and k cat (1.2 s 1 ) values for protein phosphatase PP2A (S4). The stathmin phosphorylation gradient will decay to 1/e (S5) of its initial intensity at 4 to 8 µm given these values. Supporting Text In order to evaluate the homogeneity of COPY distribution in cells we cotransfected Xenopus A6 cells with CFP-stathmin and YFP. We analyzed the YFP/CFP ratio images of eight cells and found an average SIH of 0.05 ± 0.01 (±SEM), which does not exceed the basal SIH-levels for unstimulated COPY-wt or COPY-aaa, as well as TPA-stimulated COPY-aaa cells.

5 Niethammer, p. 5 Supporting figures Fig. S1. Formation of the T 2 S complex is detected by a FRET-based approach using a ratiometric stathmin-sensor (COPY). Xenopus stathmin was N-terminally tagged with a CFP and C-terminally with a YFP (citrine). Upon tubulin binding, the two fluorescent protein tags move apart from each other, which results in a decrease of FRET. References and Notes S1. O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, R. Y. Tsien, J. Biol. Chem. 276, (2001). S2. A. W. Murray, Methods Cell Biol. 36, 581 (1991). S3. X. H. Lin et al., Proc. Natl. Acad. Sci. U.S.A 95, (1998). S4. N. E. Price, M. C. Mumby, Biochemistry 39, (2000). S5. G. C. Brown and B. N. Kholodenko, FEBS Lett. 457, 452 (1999).