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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00796

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Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast

Department of Molecular and Cell Biology and Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720-3200, USA

View larger version (38K): [in a new window]   Fig. 1. Microtubule arrays in the fission yeast cell cycle are depicted by cartoon, still images and kymograph. (A) Description of how to construct a kymograph from a series of time-lapse images using DeltaVision software: 1. A series of time-lapse images were taken of a cell of interest. 2. The images were rotated to orient the microtubule of interest along the horizontal axis. 3. The images were then cropped around the cell boundary and the time-lapse images were converted into a 3D stack. 4. The stack of images were rotated 90° around the horizontal axis to align the time points in a vertical stack; this allows for the changes in microtubule length overtime to be seen as a line trace or kymograph. 5. The kymograph was then rotated so that time is on the horizontal axis. (B) The top panel shows a schematic demonstrating the different microtubule arrays as they appear in various stages of the cell cycle. The middle panel shows still images of a cell expressing GFP:-tubulin (YY105) taken from a time-lapse movie of a cell progressing from G2 through telophase. Images correspond to the cartoon drawings and to the areas marked on the kymograph (lower panel): (a) G2-interphase, (b) G2-M transition with stage 1 spindle, (c) phase 2 spindle, (d) early anaphase B, (e) late anaphase B, and (f) telophase. Bar, 2 µm. The lower panel shows a kymograph made from the same time-lapse movie as the images shown in the middle panel. The images a-f in the middle panel correspond to the indicated regions of the kymograph. The kymograph is presented with time in minutes progressing along the horizontal axis and the length of the cell on the vertical axis. The three spindle phases are labeled below the kymograph. The transition from G2-M is marked with an arrow. The white outline marks triangular area of fluorescence that marks the life history of an interphase half bundle. Bar, 2 µm.

View larger version (51K): [in a new window]   Fig. 2. Interphase microtubule arrays are composed of overlapping half bundles of 2-3 autonomously behaving microtubules. (A) Shown here are sequential images from a time-lapse movie of a cell expressing GFP:-tubulin (YY105). Images were taken at approximately 2-second intervals. Arrows and arrowheads mark the plus ends of individual microtubules, highlighting the presence of multiple microtubules in each bundle. The arrow points to a microtubule that is shrinking and the arrowheads to microtubules that are growing. The asterisk marks the site where antiparallel microtubules overlap. Bar, 2 µm. (B) The same time-lapse movie presented as a kymograph with time in seconds along the X-axis and length of cell along the Y-axis. The time points shown in A are delineated with a rectangle on the time line of the kymograph. The region of the microtubule overlap that corresponds to the still images is marked with an asterisk; this also marks the region where the SPB is found. The lines descending from the minus-end overlap zone towards the cell tip reflect growth of a single microtubule (white arrow). Lines ascending from the cell tip toward the minus-end overlap zone reflect depolymerization (black arrow). The triangular regions of increased fluorescence intensity where growth and shrinkage lines intersect is due to the presence of one growing and one shrinking microtubule. Bar, 2 µm. (C) Comparison of pixel intensity along the microtubule bundle at single time points. The graph on the left compares the pixel intensity plots of two consecutive time points (seen in Fig. 2A), 154 and 156. The plots are almost identical, showing that there is little change in the composition of the microtubule bundle and that the two intensity plots show similar but not identical patterns between the two time points. The plots show five major changes in fluorescence intensity across the bundle. The half bundle on the left (Fig. 2A, time 154) shows three intensity peaks on the graph, which are marked by arrows and arrow heads in Fig. 2A (time 154). The overlap zone shows the area of greatest intensity. The half bundle on the right in Fig. 2A (time 154) shows a more dramatic decrease in intensity from the overlap zone and a pixel intensity of two microtubules. This is also evident in the graph shown here. The graph on the right compares time 154 with time 179, showing there were major changes in the intensity distribution and therefore the number of microtubules along the length of the bundle over time (see Movie 1, http://jcs.biologists.org/supplemental/).

View larger version (66K): [in a new window]   Fig. 3. At the G2-M transition, cytoplasmic microtubules disappear as the spindle forms inside the nucleus. (A) Shown here are still images from a time-lapse movie of a cell entering mitosis. The cell shown here is strain 2694 expressing both GFP:-tubulin and GFP:Swi6, a heterochromatin binding protein (Pidoux et al., 2000). The early spindle is seen as a small bar of fluorescence near the SPB on the inside of the nucleus (arrow, time 144). As mitosis progresses, the cytoplasmic bundles at the SPB persist whereas other nuclear-associated microtubules disappear (time 265). When the spindle reaches a length of approximately 1.0 µm, the cytoplasmic bundles at the SPB have also disappeared (time 342). These images are representative of the time points marked by the rectangle on the time line of the kymograph in B. Bar, 2 µm. (B) The kymograph is made from the same time-lapse movie shown in A. Cytoplasmic microtubules are seen as triangular projections extending from the SPB (as in Fig. 2). The pattern becomes much less complex around time point 110 as additional cytoplasmic bundles disappear. At time 230, microtubule growth is restricted to one side of the SPB. Once the phase 1 spindle has formed, the cytoplasmic microtubules become progressively shorter as seen by the decreasing length of the growth lines shown by the arrow and arrowhead. Cytoplasmic microtubules are gone by the time the spindle reaches a length of 1.0 µm (time 265). Bar, 2 µm.

View larger version (64K): [in a new window]   Fig. 4. Short dynamic microtubules are part of the early spindle. (A) INA microtubules appear at the earliest stages of spindle assembly. These still images are projections of three 0.4µm sections taken at 7-second intervals (strain, YY105). The fluorescent bar is the short spindle. The arrow at time point 35 shows the earliest detected INA microtubule before any visible spindle pole separation has occurred. The arrow at time 56 shows the same INA microtubule that has elongated towards the nuclear center. The arrow at time 119 shows two INA microtubules after spindle pole separation, one extending from each spindle pole. Bar, 1 µm (see Movie 2, http://jcs.biologists.org/supplemental/). (B) Sequential images of a single nucleus with an early phase 1 spindle and INA microtubules taken at approximately 2-second intervals. The arrows follow a single INA microtubule bundle as it sweeps across the nucleus. The same microtubule stops sweeping and appears to grow in length over the next several time frames. The cells in both A and B progressed normally through anaphase. Bar, 1 µm (see Movie 3, http://jcs.biologists.org/supplemental/).

View larger version (84K): [in a new window]   Fig. 5. During anaphase B, microtubule plus ends are dynamic but do not show depolymerization at minus ends. (A) Images of anaphase B spindle elongation. The region of overlap between the two half spindles gradually decreases as the spindle elongates. The strain used here is 2694. The GFP:Swi6-stained centromeres mark the location of the spindle poles in each daughter nucleus. Bar, 2 µm. (B) Kymograph of the anaphase B time-lapse movie seen in A. The increased fluorescence in the center of the kymograph, delineated by arrows, reflects the overlap of oppositely oriented polar microtubule plus ends. The triangular patterns reflect the growth and shrinkage of individual microtubules. The asterisk marks the distance between unique fluorescence patterns in the spindle microtubules due to uneven incorporation of GFP:-tubulin and the bright spindle pole labeled by GFP:Swi6 at the centromeres. This distance remains constant as the spindle elongates. SP, spindle pole; midzone, zone of overlap of antiparallel microtubules. Bar, 2 µm.

View larger version (114K): [in a new window]   Fig. 6. Spindle collapse occurs by sequential depolymerization of oppositely oriented microtubules. (A) A single microtubule is shown depolymerizing toward the lower spindle pole. The still images are sequential images taken at approximately 2-second intervals over 26 seconds. The arrowhead points to the plus end of the depolymerizing microtubule. Bar, 2 µm. (B) The same time-lapse movie is shown as a kymograph. The kymograph shows anaphase B spindle elongation and spindle collapse. After the spindle stops elongating (time 7 minutes), the microtubules begin to depolymerize one by one. This appears as a series of parallel lines sloping toward the spindle poles in an alternating pattern. Successive depolymerization lines are parallel, showing that rates of depolymerization are the same for each microtubule. SP, spindle pole; midzone, zone of antiparallel microtubule overlap Bar, 2 µm (see Movie 4, http://jcs.biologists.org/supplemental/).

View larger version (109K): [in a new window]   Fig. 7. Mitotic cytoplasmic astral microtubules behave like cytoplasmic interphase bundles. This kymograph is from a time-lapse movie of astral microtubule growth and shrinkage during anaphase B. It shows there are multiple microtubules in a bundle (arrow). Each individual microtubule only undergoes one growth and catastrophe cycle. SPB, spindle pole body. Bar, 2 µm.

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