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RESEARCH ARTICLE |
1 Departments of Biological Sciences, Anatomy & Cell Biology & Pathology, Columbia University, College of Physicians & Surgeons, 630 W. 168th St, Rm BB1213, New York, NY 10032-3702, USA
2 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA
3 Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
4 Cell Biology and Institute for Childhood & Neglected Diseases, Scripps Research Institute, La Jolla, CA 92037, USA
Author for correspondence (e-mail: jcb4{at}columbia.edu)
Accepted July 15, 2001
Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5xGFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5xGFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5xGFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5xGFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the tg of either 5xGFP-EMTB or full-length GFP-ensconsin was similarly rapid (
4 seconds), while in ATP-reduced and Taxol-treated cells, tg was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5xGFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is 40 µM in untreated living cells, compared with 1 µM in vitro. However, KD,app was not significantly changed in the presence of azide and was increased to 110 µM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5xGFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.
Key words: Fluorescent speckle microscopy, Photobleaching, Taxol, Microtubule-associated proteins, Phosphorylation, Epithelial cells
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