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First published online October 27, 2004
doi: 10.1242/10.1242/jcs.01475

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The p150-Glued Ssm4p regulates microtubular dynamics and nuclear movement in fission yeast

Teresa Niccoli^1,*, Akira Yamashita^2,*, Paul Nurse^4, and Masayuki Yamamoto^2,3,

¹ Cancer Research UK, London Research Institute, Cell Cycle Laboratory, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
² Molecular Genetics Research Laboratory, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
³ Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
⁴ The President's Office, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (20K): [in a new window]   Fig. 1. Domain arrangement of Ssm4p in Saccharomyces pombe and related proteins. Ssm4p domain structure and related proteins were identified in the Pfam database. The protein diagrams are not exactly to scale.

View larger version (69K): [in a new window]   Fig. 2. Ssm4p is essential for the horsetail movement in fission yeast. Nuclei of conjugated cells were monitored and the fluorescence of the artificial nuclear marker GST-NLS-GFP was followed. (A) Nuclear behaviour in living wild type, dhc1 and ssm4 strains. Serial images were obtained from a single cell at meiotic prophase. The numbers on the left indicate time span in minutes. (B) Time-lapse recording of a nucleus in a wild-type cell and a ssm4 cell undergoing meiosis. The numbers on the left indicate the time from the nuclear fusion in minutes. Bars, 10 µm. (C) Diploid wild-type and ssm4 cells were cultured to late log phase, synchronously induced to meiosis at 30°C by depleting nitrogen from the medium. The number of nuclei was counted at the times indicated. More than 200 cells were examined at each time point. Open circles, open squares, open triangles indicate percentage of cells with one nucleus, two nuclei and three or four nuclei, respectively.

View larger version (52K): [in a new window]   Fig. 3. Ssm4p and the dynein complex interact. (A) cyr1 sxa2 ssm4-HA and cyr1 sxa2 dhc1-myc fission yeast cells were grown in the absence (–P) and in the presence (+P) of pheromone for 6 hours. Boiled cell extracts were prepared as described and western blots were probed with anti-HA or anti-MYC antibodies. (B) cyr1 sxa2 dhc1-myc ssm4-HA cells were induced with pheromone for 6 hours. Native cell extracts were immunoprecipitated with anti-HA and anti-MYC antibodies. Western blots of immunoprecipitates (IPs), supernatants and total cell extracts were carried out as described and probed with HA and MYC antibodies (right panel). Control IPs with anti-HA of cyr1 sxa2 dhc1-myc did not precipitate any Dhc1p-myc (middle panel) and IPs with anti-MYC of cyr1 sxa2 ssm4-HA did not precipitate any Ssm4p-HA (left panel). (C) Two-hybrid interaction of Ssm4p with Dlc1p. ß-galactosidase activity was assayed in Saccharomyces cerevisiae cells expressing proteins as indicated. The combination of Ras and Raf was used as a positive control. (D) Localisation of Ssm4p-GFP in meiotic cells. Homothallic haploid cells that carried the ssm4-GFP fusion gene and the CFP-atb2 fusion gene encoding CFP-tubulin were subjected to nitrogen starvation to induce mating and subsequent meiosis in zygotes. They were monitored by fluorescence microscopy for localisation of Ssm4p and microtubules. Nuclear DNA was stained with Hoechst 33342. Merged images are shown in the right panels. Green, GFP; red, CFP; blue, DNA. The arrowheads and the asterisks indicate intense GFP fluorescence at the leading edge of the nucleus and at the microtubular tip contacting the cell cortex, respectively. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 4. Ssm4p is essential for the oscillatory nuclear movement in shmooing fission yeast cells. (A,B) cyr1 sxa2 cells (A) and cyr1 sxa2 ssm4 cells (B) carrying a nuclear GFP marker were treated with pheromone for 6 hours, placed on a slide, and filmed under a fluorescent microscope. Images were taken every minute. Bar, 3 µm.

View larger version (80K): [in a new window]   Fig. 5. Ssm4p is expressed during fission yeast mating and localises to microtubules. (A) cyr1 sxa2 ssm4GFP cells were induced for 6 hours with pheromone, cells were then fixed in methanol and immunostained with anti-tubulin (MT) and anti-Sad1 antibodies. Arrows indicate the SPB and arrowheads the point where microtubule plus ends contact the cell surface. (B) cyr1 sxa2 ssm4GFP cells were induced for 6 hours with pheromone, treated with MBC and imaged on a fluorescent microscope before and after the treatment. (C) cyr1 sxa2 ssm4GFP cells were induced with pheromone for 6 hours, placed on a slide and filmed with a fluorescence microscope. Frames are 8-10 seconds apart. White arrows indicate the microtubule tips and yellow arrowheads the fluorescent dot at the SPB, which can be identified as a point where more than one microtubule originates. The microtubular tip is anchored in a fixed position in a, c and d and the SPB is moving towards it as the microtubule depolymerises. In b, the SPB is not moving and the microtubule is depolymerising back towards it. Bars, 3 µm. (D) Fluorescence at the tip of microtubules in C (marked by the white arrow) was measured with NIH image for each frame and plotted.

View larger version (48K): [in a new window]   Fig. 6. Tip1p and Ssm4p collaborate to localise Dhc1p. (A) Localisation of Ssm4p-GFP in the dhc1 and tip1 mutant zygotes. (B) Localisation of GFP-Dhc1p in the ssm4, tip1 and ssm4 tip1 mutant zygotes. (C) cyr1 sxa2 dhc1GFP, cyr1 sxa2 ssm4 dhc1GFP, cyr1 sxa2 tip1 dhc1GFP, cyr1 sxa2 tip1 ssm4 dhc1GFP cells were grown in pheromone for 6 hours and then imaged with a fluorescence microscope. The white arrows indicate the microtubule tips and the yellow arrowheads the SPB. (D) cyr1 sxa2 ssm4 dhc1GFP, cyr1 sxa2 tip1 ssm4 dhc1GFP cells were grown in pheromone for 6 hours and then fixed and stained for tubulin. (E) ssm4 dhc1GFP, tip1 ssm4 dhc1GFP zygotes were fixed and stained for tubulin. (F) Colocalisation of Tip1p and Dhc1p. cyr1 sxa2 tip1YFP dhc1GFP cells were treated with pheromone for 5 hours. Cells were visualised with a confocal microscope using a CFP-YFP filter set which allows to separate the YFP and GFP signals. A single plane through the centre of the cells was taken. Arrows indicate fluorescence at the SPB and arrowheads indicate fluorescence at the microtubular tip. Bars, 10 µm (A,B); 3 µm (in C for C and D; F); 5 µm (E).

View larger version (66K): [in a new window]   Fig. 7. Microtubular dynamics in wild-type fission yeast cells are coordinated. (A) cyr1 sxa2 nmt1atb2GFP cells were treated with pheromone for 5 hours. Cells were then imaged every 6 seconds on a confocal microscope. The panels show the first 3.3 minutes of a typical time course. Images shown are projections of sections through the whole cell. + and – indicate growing and shrinking microtubules respectively. (B) Microtubule lengths for single cells were measured distinguishing the side of the cell in which they lie. The diagram describes the dynamics of the microtubules of the cell shown in A over the total filming period of 4.5 minutes. The concordance of dynamics at each cell end and between opposite cell ends was then calculated as a percentage of total time points, where eight cells were scored for a total imaging time of 45 minutes. (C) cyr1 sxa2 nmt1atb2GFP were induced with pheromone for 5-6 hours, placed on a lectin-coated glass bottom dish, bleached with a localised laser beam and then filmed every 6 seconds to monitor the pattern of recovery. Images show a single plane through the middle of the cell. The yellow arrows indicate the position of the SPB, from which more than one microtubule originates. The grey lines mark the initial bleached area. The bleached area moves in the same direction as the SPB, with the distance between the SPB and the further boundary of the bleached area remaining the same, as indicated by a green bar in two panels, and the fluorescence recovers from the boundary closer to the SPB. (D) cyr1 sxa2 nmt1atb2GFP cells were induced with pheromone for 6 hours. Microtubules were bleached with a pulse of laser light; MBC was then added to depolymerise the microtubules, which were filmed with a confocal microscope (see Materials and Methods for details). Images shown are single planes through a cell. The red arrow marks the initial bleaching position. Bar, 3 µm.

View larger version (55K): [in a new window]   Fig. 8. Microtubules in ssm4 fission yeast mutants are less dynamic and do not oscillate. (A) cyr1 sxa2 ssm4 nmt1atb2GFP cells were treated with pheromone for 5 hours. Cells were then imaged every 12 seconds on a confocal microscope. Images shown are projections of sections through the whole cell. (B) The lengths of microtubules for single cells were measured distinguishing on which side of the cell they lie. The diagram describes the dynamics of the microtubules of the cell in A. The coordination of dynamics between opposite cell ends was then calculated as a percentage of total time points. Four cells were scored for a total imaging time of 20 minutes. At least 10 cells were observed for a total imaging time of 50 minutes and they all exhibited a similar behaviour. (C) cyr1 sxa2 ssm4 nmt1atb2GFP cells were induced with pheromone for 6 hours. Cells were imaged every 7 seconds on a confocal microscope. Images shown are projections of sections through the cell. The yellow arrow indicates where there is a change of fluorescence along a bundle. (D) cyr1 sxa2 ssm4 nmt1atb2GFP cells were induced with pheromone for 5-6 hours, placed on a lectin-coated glass bottom dish, bleached with a localised laser beam and then filmed every 6 seconds to monitor the pattern of recovery. Images show a single plane through the middle of the cell. The yellow arrows indicate the position of the SPB. The grey lines mark the initial bleached area. The bleached area recovers from the SPB. The time taken by fluorescence to recover over a 2.3 µm bleached area was 43.3±11.1 seconds (n=16) for the wild type (from Fig. 7C) and 40.3±11.3 seconds (n=17) for ssm4 cells. (E) cyr1 sxa2 ssm4 cells were induced with pheromone for 6 hours. Microtubules were bleached with a pulse of laser light, MBC was then added to depolymerise the microtubules and microtubules were filmed with a confocal microscope. Images shown are single planes through a cell. The yellow arrowheads mark the stud remaining after microtubules had depolymerised. Bar in A,C, 3.5 µm; D,E, 3 µm.

View larger version (86K): [in a new window]   Fig. 9. Microtubular dynamics have lost coordination in ssm4 tip1 cells. (A,B,C) cyr1 sxa2 tip1 nmt1atb2GFP and (D, E) cyr1 sxa2 tip1 ssm4 nmt1atb2GFP cells were treated with pheromone for 5 hours. (A,D) Cells were then imaged every 6 seconds on a confocal microscope. Images shown are projections of sections through the whole cell. Yellow arrows mark the position of the SPB. + and – indicate growing and shrinking microtubules respectively. (B,C,E) Microtubule lengths for single cells were measured distinguishing the side of the cell in which they lie. B describes the dynamics of the microtubules of the cell in A, diagram E corresponds to D, and diagram in C describes a different tip1 cell. The coordination of dynamics between opposite cell ends was then calculated as a percentage of total time points; eight cells were scored for a total imaging time of 45 minutes. Bar, 2 µm.

View larger version (26K): [in a new window]   Fig. 10. Model of microtubular oscillations. (1) The microtubules ahead of the nucleus depolymerise as they are pulled by Dhc1p, as proposed by Yamamoto et al. (Yamamoto et al., 2001). Ssm4p activates Dhc1p motor activity and ensures that the microtubules can interact with the cortical anchor, whose presence was proposed by Yamamoto et al. The microtubules behind the nucleus polymerise more slowly than the ones ahead are depolymerising and so they cannot reach the cell end and interact with the cortical anchor. (2) When the overlap region reaches the end of the cell, microtubules can no longer depolymerise and they dissociate from their cortical anchor, the putative dynein-dynactin complex, which is moving in the minus direction, is no longer anchored at the cortex and so could move towards the SPB. (3) When microtubules lose their cortical anchor they will no longer be pulled, the microtubules behind will be able to grow until they reach the cortical anchors in the opposite end. The anchors, or another factor at the cell ends, might induce microtubules to pause. Ssm4p will then accumulate at the microtubule tips, generating a stable interaction with the cortex and triggering microtubule depolymerisation. Dhc1p will also accumulate at the tips of microtubules, stabilised by Ssm4, which will also activate the motor activity of Dhc1p, which will start pulling the nucleus in the opposite direction. Alternatively, when the SPB reaches the cell end, it sends a signal to the opposite microtubule tip, activating it for an interaction with the cortical anchor.