804A Fairchild Center, M.C. 2450
New York, N.Y. 10027
The polymerization and depolymerization dynamics of microtubules (MTs) are
subject to exquisitely precise regulation in vivo, probably owing to the fact
that small perturbations in MT dynamics are sufficient to arrest cell division,
differentiation and motility processes. In fact, numerous chemotherapeutic drugs
target MT dynamics in order to halt tumor cell proliferation, metastasis or
viability; the slight alteration of MT dynamics produced by these drugs is often
sufficient to launch cells on an apoptotic cascade. Our laboratory studies
MT-associated proteins (MAPs) that play various roles in the regulation of MT
dynamics during the cell cycle and differentiation, as well as in response to
MT-antagonistic drugs. These proteins are also capable of modulating MT
functions, such as mitosis and vesicle transport.
One MAP we study, MAP4, is the most abundant MAP present in organisms ranging from the worm, Caenorhabditis elegans, to humans. MAP4 has been hypothesized to be an important regulator of MT dynamics. We and others had previously shown that increased expression of MAP4, including novel isoforms, contributes to increased MT stability in muscle cells. Interestingly, we found that MAP4 has a dual function in proliferating cells: it stabilizes MT dynamics and regulates MT polymer level (1). We recently used site-directed mutations in MAP4 phosphorylation sites to test the hypothesis that MAP4's phosphorylation state regulates its activity at the G2 : M transition of the cell cycle. Analysis of phosphorylation-defective mutants demonstrated that in vivo phosphorylation of MAP4 by cdc2 kinase reduces MAP4's capacity to stabilize MTs, and these phosphorylation events are necessary for efficient progression from interphase into mitosis, when the interphase MT array must be disassembled and the dynamics of the MTs increased (2).
Our laboratory had previously shown that another MAP, ensconsin, which is present in a variety of human cells, does not regulate microtubule dynamics. Recent experiments revealed that increased expression of ensconsin is deleterious to cells treated with MT-stabilizing chemotherapeutics, e.g., Taxol (3), while a variety of biochemical experiments suggested that ensconsin's binding to MTs might be altered by Taxol treatment. To investigate this hypothesis, we developed a method called fluorescent speckle microscopy (FSM) that allows one to image single molecules in vivo; it optimizes visibility of fluorescent molecules in living cells and reveals assembly dynamics, movement, and turnover of protein assemblies (4). We used FSM to follow the dynamics of ensconsin molecules' association/dissociation with MTs in vivo. We found that ensconsin normally existed in dynamic association with MTs, but in cells that were Taxol-treated, or had their MT dynamics halted by other means (i.e., depleting cellular ATP levels or extracting cells with detergent) ensconsin became more statically associated with the MT(5). Quantifying ensconsin's residence time along the length of a MT by another technique, fluorescence recovery after photobleaching (FRAP) we found that the t1/2 of ensconsin's MT is rapidly increased up to 50-fold by decreasing the dynamics of the MTs to which the ensconsin is bound (5). These results suggest that ensconsin may function as a 'sensor' of MT dynamics, signaling to the cell that deleterious MT stabilization has occurred. We are currently testing this hypothesis. As a corollary, we have started to identify ensconsin binding partners, that is, proteins targeted to MTs by ensconsin, since these would be likely participants in signaling the state of cellular MT dynamics.
(1) Nguyen, H.L., Gruber, D. and Bulinski, J.C. 1999. Microtubule-associated
protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis
of tubulin in cultured cells. J. Cell Sci. 112:1813-1824.
(2) Chang, W., Gruber, D., Chari, S., Kitazawa, H., Hamazumi, Y., Hisanaga, S.I., and Bulinski, J.C. 2001. Phosphorylation of MAP4 affects microtubule properties and cell cycle progression. J. Cell Sci. 114:2879-2887. Article (.pdf)
(3) Gruber, D., Chang, W., Faire, K., and Bulinski, J.C. 2001. Abundant expression of the microtubule-associated protein, ensconsin (E-MAP-115), alters the cellular response to Taxol. Cell Motil. Cytoskel. 49: 115-129. Article (.pdf)
(4) Waterman-Storer, Desai, A., Bulinski, J.C. and Salmon E.D. 1998. Fluorescent speckle imaging: Visualizing the movement, assembly, and turnover of macromolecular assemblies in living cells. Current Biology 8:227-230. Article (.pdf)
(5) Bulinski, J.C., Odde, D., Howell, B., Salmon, E. D., and Waterman-Storer,
C. M. 2001. Rapid dynamics of the binding of ensconsin to microtubules in vivo.
J. Cell Sci. 114 (21), Article (.pdf)