Because microtubules are required for cells to divide, rapidly growing tissues, including tumors, are susceptible to treatments that disrupt microtubule function. One such class of anti-cancer agents is Taxol and the Taxol-like compounds that bind to a specific site on tubulin, the protein that forms microtubules. We are developing an experimental system and testing the hypothesis that localized interactions with this site serve to selectively stabilize individual microtubules within the cell. We have thoroughly established the in vitro approaches described in our experimental design and have purified highly active mammalian tubulin protein. We have also covalently labeled tubulin with either alexa-488 or alexa-560 fluorescent molecules. This dual labeled tubulin was then utilized to establish conditions for obtaining both stabilized and dynamic microtubules with clearly distinguishable ‘minus’ and ‘plus’ end polarity. A dual-channel total internal reflection fluorescence (TIRF) microscopy based assay was developed for the visualization of individual dynamic microtubules in vitro. This assay is adaptable for both direct imaging of polarity marked microtubules or simultaneous visualization of dynamic microtubules and fluorescence-associated Taxol molecules. Earlier in the year we were able to successfully complete the design for the synthesis of a biotin-conjugated Taxol compound with a sufficient intervening linker and anticipate completion of the synthesis in the coming month. Upon completion, we will be poised to proceed directly with experiments to probe the role of the Taxol-binding site in the stabilization of end-on microtubule attachments.

Because microtubules are required for cells to divide, rapidly growing tissues, including tumors, are susceptible to treatments that disrupt microtubule function. One such class of anti-cancer agents is Taxol and the Taxol-like compounds that bind to a specific site on tubulin, the protein that forms microtubules. Binding of Taxol stabilizes microtubules, prevents their function during cell division, and ultimately can lead to cell death. Understanding the molecular details of Taxol-mediated microtubule stabilization will directly impact our understanding of extremely active cancer therapies, provide rationales for combinatorial therapy, and potentially identify novel therapeutic targets to enhance treatment or avoid unwanted side effects.

We have purified highly active mammalian tubulin protein and covalently labeled tubulin with either alexa-488 or alexa-560 fluorescent molecules. This dual-labeled tubulin was then utilized to establish conditions for obtaining both stabilized and dynamic microtubules with clearly distinguishable ‘minus’ and ‘plus’ end polarity. A dual-channel total internal reflection fluorescence (TIRF) microscopy based assay was developed for the visualization of individual dynamic microtubules in vitro. This assay is adaptable for both direct imaging of polarity marked microtubules or simultaneous visualization of dynamic microtubules and fluorescence-associated Taxol molecules.

The development of these methods and assays now positions us to perform the proposed experiments to directly localize substoichiometric molecules of Taxol during microtubule stabilization both in general and, specifically, in the stabilization of end-on microtubule attachments. We have also been actively engaged in the synthesis of a biotinconjugated Taxol compound with a sufficient intervening linker to maintain maximal biological activity. A four-month production cycle was initially estimated for this synthesis; however, the time required for synthesis has exceeded this estimate. The primary experimental strategy has not been entirely abandoned and, in fact, methods have been obtained that achieve at least 75% pure biotin-Taxol conjugate. Pilot experiments have also proved promising for obtaining a high degree of further purification of this desired product. Furthermore, alternate experiments using an Oregon Green 488-conjugated Taxol molecule have been initiated. To date, conditions have been established for simultaneously stabilizing microtubules and visualizing this Taxol analogue. We are currently at the stage of optimizing visualization to achieve single-molecule detection and resolution.