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Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary

¹ Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA
² Laboratory of Cell Motility, A. N. Belozersky Institute, Moscow State University, Moscow, Russia

View larger version (90K): [in a new window]   Fig. 1. Path photobleaching reveals persistent MT growth in the interior of a CHO cell. (A) Upper panels: low magnification images of Cy3-labeled MTs before and after bleaching a zone approximately 20x3 µm passing through the centrosomal region. The path of reduced fluorescence allows visualization of nascent centrosomal MTs. Lower panels: three MTs oriented along the direction of the path (colored yellow, red and green) can be seen to elongate persistently. Numbers in top-left corners indicate time in seconds. Images were acquired every 4 seconds. Bar, 5 µm. (B) Life history plots (length vs time) of colored MTs. Zero length represents position of the centrosome; zero time is time of bleaching. MT plus ends arrived at the plasma membrane in 60 seconds. (C) Instantaneous velocities of nascent MTs were measured as the displacement of MT ends between successive frames. The histogram shows that episodes of shortening and pause were infrequent.

View larger version (110K): [in a new window]   Fig. 2. Life cycle of MTs visualized in centrosome-containing CHO cytoplasts. (A) Time-lapse sequence shows selected panels from life histories of four MTs. (B) Two MTs (colored blue and red) were nucleated at the centrosome (at 3 seconds and 39 seconds, respectively) and persistently grew towards the cell margin. These MTs underwent transition to shortening only after they arrived at the cell margin (arrowheads: red MT, at 102 seconds; blue MT at 120 seconds). The length of the blue MT is greater than the red MT because it grew in a curved path and along the cell margin before beginning to shorten. (C) Two MTs (colored yellow and green) were initially tracked while near the cell margin. These MTs showed repeated episodes of growth and shortening near the membrane and the green MT eventually underwent a shortening excursion back to the centrosome. Numbers in the top-left corners indicate time in seconds. Bar, 5 µm. Life history plots constructed as indicated in Fig. 1 legend.

View larger version (99K): [in a new window]   Fig. 3. Selective visualization of growing MT ends with CLIP-170. CHO cells were transfected by nuclear microinjection of GFP-CLIP-170 DNA, allowed to express GFP-CLIP protein, then microinjected with Cy3-tubulin and, finally, enucleated to generate cytoplasts. Time-lapse sequences were obtained to show dynamics of MTs and CLIP-170. (A) Low magnification of MTs, CLIP-170 and merged image (green, Cy3-MTs; red, CLIP-170). (B) Time-lapse sequence of region boxed in panel A. Two dynamic MTs are indicated by arrowheads. CLIP-170 is present at their plus ends during growth phases but disappears within 5 seconds after transition from growth to pause or shortening phase. Numbers in top-left corners indicate time in seconds. Interval between acquisitions of images in alternating channels was 2.5 seconds, which gave an interval between successive images in either the GFP or Cy3 channel of 5 seconds. Scale bar, 5 µm. (C) Life history plots of MTs (green) and CLIP-170 tracks (red) indicated by arrowheads in panel B. CLIP-170 data points were time-shifted by 2.5 seconds to compensate for the delay between acquisition of CLIP and MT images. Absence of red data points from segments of the plots indicates when CLIP-170 disappeared from the MT end. (D) Example plots of nascent MTs (green) growing off the centrosome and corresponding CLIP-170 tracks (red) after time-shifting of 2.5 s. Plots were arbitrarily staggered along the time axis for clarity. Scales of axes indicated in upper right corner of graph. For some MTs either near the centrosome or in areas of high MT density, the MT end could not be clearly visualized. Nevertheless, CLIP-170 movement could be seen. This is represented in the plots as red points without corresponding green ones. In all cases, clear growing MTs had CLIP-170 at their ends.

View larger version (88K): [in a new window]   Fig. 4. Persistent MT growth confirmed by long CLIP-170 tracks. GFP-CLIP-170 movement was analyzed by time-lapse microscopy. Images were acquired every 3 seconds. (A) Three next-nearest frames are shown, displaying radial organization and movement of CLIP-170 patches. Tips of four CLIP-170 patches are indicated over time by arrowheads. Numbers in the upper left corner indicate time in seconds; bar, 5 µm. Diagram shows 25 CLIP-170 tracks over a 90 second period of which four tracks (1-4) correspond to CLIP patches indicated by arrowheads on the images. Many tracks are long indicating continuous MT growth; radial pattern of tracks permits identification of the centrosome region (dashed circle) as the point from which the tracks originate. The centrosome is indicated as two dots in the diagram. (B) Histogram of the length distribution of CLIP-170 tracks. Mean length, 17.3±4.8 µm (n=78). (C) Histogram of length distribution of CLIP tracks normalized against the distance between the centrosome and plasma membrane. The histogram shows that the majority of MTs born at the centrosome grow persistently to 85% of the cell radius.

View larger version (95K): [in a new window]   Fig. 5. MT treadmilling and GFP-CLIP-170. MT dynamics and GFP-CLIP-170 movement were analysed in cytoplasts lacking the centrosome. (A) MTs and CLIP-170 images (two left pictures) and time lapse sequence of merged images with GFP-CLIP-170 set to the red channel and MTs to the green channel, respectively. Plus and minus ends of treadmilling MTs are indicated by arrowheads. CLIP-170 labels the plus ends of treadmilling MTs while minus ends are always CLIP-170-negative. Numbers in the top-left corner indicate time in seconds; bar, 5 µm. (B) Example histories of CLIP-170 tracks in cytoplasts lacking a centrosome. Individual plots are arbitrarily staggered along the time axis for clarity. Scale of axes is indicated in the top-right corner of the graph. (C) Life history plot of a treadmilling MT (plus and minus ends are green) and CLIP-170 (red). Time shift between plus end history and CLIP-170 history is 2.5 seconds. CLIP-170 persists at the growing plus end of the treadmilling MT.

View larger version (77K): [in a new window]   Fig. 6. Analysis of MT shortening. (A) Time-lapse sequence shows a rare event of plus end shortening (arrowheads) from the plasma membrane back to the centrosome within 12 seconds. (B) Histogram of instantaneous rates of MT shortening from the plasma membrane. (C) Length distribution of MT shortening episodes. MT ends adjacent to the cell margin were followed and the length of a continuous shortening episode was determined. The histogram shows small shortening episodes were frequent and long ones rare (n=319). (D) Time-lapse sequence shows release of a MT from the centrosome followed by shortening from the minus end (arrowheads). Time in seconds; bars, 5 µm.

View larger version (59K): [in a new window]   Fig. 7. Population analysis of MTs. (A) Sequential subtraction analysis for identification of growth and shortening. Image of Cy3-labeled MTs from a time-lapse series and corresponding differential image (In — In+1) obtained by subtracting from an image (In) the next image (In+1) in the series. Black and white segments represent MT growth and shortening, respectively, during the time interval. Five trapezoidal zones, each one-fifth of the cell radius, indicate the regions where growth and shortening events were scored. In the two right-hand panels, the fifth trapezoid is enlarged and growth and shortening excursions are shown in colors, — green and red, respectively. Parallel black and white segments arise from lateral shifts of MTs and were not scored. (B) Frequency distribution of growth and shortening velocities in region of 0.6-0.9 of cell radius fraction obtained by subtraction analysis. (C) Drift coefficient as calculated from histogram of growth and shortening velocities. Drift is positive and fairly uniform within the cell interior, dropping to negative values only near the cell boundary. (D) Distribution of MT ends. The position of active MT ends (growing or shortening) was scored by subtraction analysis and assigned to one of five trapezoidal zones shown in panel A (n=7421 episodes; 4244 growing; 3177 shortening; 4 cells). Data were fit to a single exponential function. The exponential functions taken separately for growth or shortening episodes were essentially identical.

View larger version (29K): [in a new window]   Fig. 8. MT life cycle and steady state length distribution (A). Life history of a typical MT reconstructed from data from two individual MTs. The first part (up to time axis break) traces persistent growth towards the cell boundary followed by catastrophes induced by the boundary that are rescued at high frequency. The second part (after time axis break) shows the rare event of shortening back to the centrosome. (B) Diagram of radial array of MTs showing distribution of lengths in accordance with exponential function fitted to data from subtraction analysis. Long MTs dominate over short ones.

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