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First published online July 25, 2006
doi: 10.1242/10.1242/jcs.03048

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Contribution of microtubule growth polarity and flux to spindle assembly and functioning in plant cells

Pankaj Dhonukshe^*,, Norbert Vischer and Theodorus W. J. Gadella, Jr

Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands

View larger version (109K): [in a new window]   Fig. 1. Microtubule dynamics during NE breakdown. (A-H) Gradual appearance of GFP-MAP4-labelled microtubules around the NE before its disintegration. Note the GFP-MAP4-labelled microtubules penetrate inside the NE before spindle initiation (image C-F). (I-L) Dynamics of GFP-AtEB1-labelled microtubule plus ends during NE breakdown. Microtubule plus ends are in green and FM4-64-labelled endocytic vesicles are in red. White arrows indicate plus ends of microtubules penetrating inside NE from the polar region; white arrowheads indicate plus ends of microtubules penetrating inside NE in a direction perpendicular to the confocal section; the yellow arrow indicates integrity of the NE. Note the appearance of GFP-AtEB1 inside the NE by microtubule penetrations and the continuity of NE integrity as revealed by the persistence of endocytic vesicles outside the nuclear area until significant NE breakdown. Bars, 5 µm; time is indicated in seconds.

View larger version (131K): [in a new window]   Fig. 2. Microtubule dynamics during spindle initiation. (A-J) Microtubule dynamics revealed by GFP-AtEB1 microtubule labelling during spindle assembly. Red arrowheads (B) show microtubule plus ends and red arrows (D,E) show microtubule bundles. (K) Fluorescence intensity profile of GFP-AtEB1 along the spindle axis (longitudinal axis extending through poles), corresponding to A,D and H. (L-U) Combined dynamics of microtubule (green) and chromosome (red) during spindle assembly. White arrows and white arrowheads point to microtubule plus ends and the yellow arrow shows the nucleolus. Bar, 5 µm; time is indicated in seconds.

View larger version (122K): [in a new window]   Fig. 3. Growth polarity and growth speed of spindle microtubules during metaphase. (A) `xt' kymograph (horizontal line projection) of GFP-AtEB1-labelled spindle microtubules corresponding to the equatorial position in B (see corresponding arrows and arrowheads showing hardly any lateral movement of the areas containing chromosomes and those without chromosomes; yellow lines in A exactly match the areas shown by red lines in B, except that the yellow lines represent horizontal projection and the red lines vertical projection). (B) Metaphase spindle microtubules labelled with GFP-AtEB1. This is an initial image of a time-lapse series shown in Movie 2 from supplementary material. Panels C-H correspond to chromosomal areas and panels I-N correspond to overlapping microtubule areas without chromosomes near the equator. (C,D) `yt' kymograph obtained from the left line drawn in B. (I,J) `yt' kymograph obtained from the right line drawn in B. Rectangles in D and J indicate the areas taken for the Fourier analysis. The Fourier transformed images of the combined kymographs of all near chromosomal areas and of all overlapping microtubule areas without chromosomes in the entire spindle are given in E and K, respectively. All areas and individual kymographs are shown separately in supplementary material Fig. S2. The areas selected for obtaining the microtubule growth speeds from the Fourier images are shown in F and L. The specially developed image processing program Object-Image was used, which allows detector regions to be defined based on a local, polar co-ordinate system. A ring sector region of ±90 degrees was marked (F,L), with clockwise angles as positive and the x-axis corresponding to zero degrees for averaging the pixels with the same angle and yielding an angular intensity profile (G). (G,M) Angular intensity distribution of Fourier graph (F) and (L). (H,N) Graph with reciprocal x-axis (from graph G and M) showing microtubule growth speed distribution in spindle areas. Bar, 7 µm.

View larger version (58K): [in a new window]   Fig. 4. Effect of line width on spindle kymograph analysis. Spindle kymograph analysis was performed as described in Fig. 3 and supplementary Fig. S2. To create a kymograph, a vertical rectangular region of interest (roi) in the x-y scan (A) was used, from which a vertical single pixel column was generated via the brightest point horizontal (x-directed) projection. The width of the roi was varied to investigate the effect of horizontal sampling range on the Fourier analysis. Three line widths were used; 5, 10 and 20 pixels corresponding to 0.35, 0.7 and 1.4 µm, respectively, as shown in A. Panels B and C correspond to the analysis of the near chromosomal areas, also shown in Fig. 3G and H, respectively; panels D and E correspond to the analysis of overlapping microtubule areas, also shown in Fig. 3M and N, respectively.

View larger version (80K): [in a new window]   Fig. 5. Validation of Fourier-based kymograph analysis by simulation. (A-D) Left, Simulated kymograph; middle, power spectrum of Fourier transform and ring sector; right, angular distribution plots (corresponding to speeds) in the kymograph (blue, input; red, output as measured inside the ring sector). (A) Simulation of unidirectional growth with narrow distribution of growth speeds. (B) Simulation of bidirectional growth with symmetrical distribution of growth speeds. (C) Simulation of bidirectional growth with asymmetrical distribution of growth speeds; input frequencies differ by a factor of 10. (D) Simulation of unidirectional growth with broad distribution of growth speeds. (E) Measured versus theoretical minor peak position in simulation C, where the major peak remained fixed and the minor peak varied from 10 to 70 degrees. Vertical bars represent standard deviation of 10 independent simulations. (F) Theoretical peak width of measured velocities (FWHM) in simulation D; input peak width varied from 2 to 30 degrees. Vertical bars represent standard deviation of 10 independent simulations.

View larger version (106K): [in a new window]   Fig. 6. Microtubule polarity and growth speed along the spindle-axis. Kymograph obtained from a line drawn over a chromosomal area (A) and overlapping microtubule area without chromosome (B) displaying growth polarity of GFP-AtEB1-labelled spindle microtubules. Fourier graphs in C are obtained from four parts of the combined kymograph representing the entire spindle area, which is assembled from various kymographs obtained by drawing subsequent lines on whole spindle area. (D) Angular intensity distribution and growth speed distribution of the Fourier graphs from C.

View larger version (134K): [in a new window]   Fig. 7. Dynamics of chromosome movements during anaphase. Green: GFP-AtEB1, and Red: SYTO82-DNA staining (chromosomes inside and mitochondria outside the spindle area). (A-F) Combined microtubular and chromosome dynamics at anaphase (Movie 3 in supplementary material). (G) Kymograph obtained by drawing a line on one of the chromosomes from the anaphase spindle. (H) Phases of poleward motion of chromosome during anaphase. (I) Merged kymograph showing both microtubule growth and chromosome motion. (J) Fluorescence intensity profile of GFP-AtEB1 along the spindle axis (pole to pole axis) during anaphase obtained from lines drawn at three time points (shown in G). The x axis represents pole to pole distance in µm and y axis shows fluorescence intensity in an arbitrary scale. Note that all three panels are at the same fluorescence intensity scale. (K) Speed of individual chromosome motion (represented by each dot) along with its respective position in the spindle. 0 represents a pole-to-pole axis passing through the centre of the spindle equator and left and right sides depict the distance in relation to this spindle axis. The dots show the relative positions of various chromosomes from the spindle axis. Blue dots show the speed of chromosomes during the first (fast) phase of anaphase, whereas green dots show the speed of chromosomes during the second (slow) phase of anaphase. The y axis represents the speed (µm/minute) of individual chromosomes. Bar, 5 µm; time is indicated in seconds.

View larger version (89K): [in a new window]   Fig. 8. Occurrence of microtubule flux in plant spindles. (A-D) Movement of photobleached lines marked on spindle microtubules during metaphase (Movie 4 in supplementary material). (E-H) Movement of photobleached lines marked on spindle microtubules during anaphase (Movie 5 in supplementary material). Panels I and K represent fluorescence intensity profiles projected sequentially on time axes showing translocation of the photobleached area corresponding to images A-D and E-H, respectively. t represents time in seconds and d indicates distance in µm. J represents a difference kymograph of I, showing the gain of fluorescence along the spindle axis immediately after photobleaching, clearly demonstrating microtubule translocation towards the spindle poles. Bar, 5 µm; time is indicated in seconds

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