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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02433

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Dynamic interaction of NtMAP65-1a with microtubules in vivo

Hsin-Yu Chang^*, Andrei P. Smertenko^*, Hisako Igarashi, David P. Dixon and Patrick J. Hussey

The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, DH1 3LE, UK

View larger version (169K): [in a new window]   Fig. 1. NtMAP65-1a N-terminal GFP fusion protein binds to microtubules in the cells of various organs of 7-day-old Arabidopsis seedlings. (A-D) GFP signal detected with a scanning confocal microscope in root tip (A), hypocotyl (B), leaf epidermis (C) and trichome (D). Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 2. GFP:NtMAP65-1a signal recovery after photobleaching. (A) GFP:NtMAP65-1a signal in the hypocotyl of 7-day-old Arabidopsis seedlings. The white rectangle outlines the photobleached area. (B) Time series (top to bottom) of GFP:NtMAP65-1a signal recovery during a FRAP experiment within the area indicated by the rectangle in A. The numbers on the right-hand side indicate the time in seconds when each of the frames was collected with 0 corresponding to the image before the photobleaching onset and 4.766 just after the photobleaching. (C) The first FRAP measurement was taken just before the photobleaching and corresponds to point 0. The grey sector represents the duration of photobleaching, after which 14 images were collected and measured at approximately 4.14-second intervals. The fluorescence signal was measured in 24 cells and expressed as the mean percentage of the signal before photobleaching. The error bars indicate s.d. of the mean. Bar, 5 µm.

View larger version (10K): [in a new window]   Fig. 3. NtMAP65-1a does not interact with tubulin dimers. NtMAP65-1a was covalently attached to the matrix of the Biacore chip, then solutions containing tubulin, buffer alone, taxol-stabilised microtubules and again buffer alone (as indicated at the top of the chart) were sequentially passed over the chip and the binding response or surface plasmon resonance (SPR) was measured over time and plotted (black line). The empty chip surface was used as negative control (grey line). The concentration of the tubulin dimers and microtubules was 0.1 mg/ml.

View larger version (41K): [in a new window]   Fig. 4. Random recovery of GFP:NtMAP65-1a signal over the microtubule bundle length. (A) The first measurement was taken just before the photobleaching and corresponds to point 0 (the image of the microtubule at this time point is represented in part B point 0). The grey sector represents the duration of photobleaching, after which 35 images were collected and measured at approximately 2.5-second intervals. The fluorescence signal on the chart is expressed in arbitrary values. (B) Time series (left to right) of GFP:NtMAP65-1a signal recovery after photobleaching the microtubules shown in A. The numbers at the top of each image indicate time in seconds when each of the frames was collected with 0 corresponding to the image before the photobleaching onset and 12.0 just after the photobleaching. The images shown follow the microtubule to complete recovery and this occurred after 34.5 seconds. (C) Changes in time (x axes) of the distribution of the fluorescence signal (z axes) along the length of the microtubule (y axes) shown in A and B. The signal intensity is expressed in arbitrary values.

View larger version (109K): [in a new window]   Fig. 5. Interaction of NtMAP65-1a with microtubules during cell division. GFP:NtMAP65-1a signal in the preprophase band (A), mitotic spindle (B) and anaphase to telophase transition (C). The numbers in the images in C indicate time in seconds from the beginning of image acquisition. Bar, 10 µm.

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