Regulating the dynamic behavior of microtubules is essential for many cellular functions. Several factors have been identified that play central roles in modulating microtubule dynamics in vivo. In Xenopus egg extracts, the protein XKCM1 provides the dominant microtubule destabilizing activity. XMAP215 stabilizes microtubules by opposing the function of XKCM1. However, the mechanism by which XMAP215 antagonizes XKCM1 activity remains unclear. I will establish in vitro assays, using purified, recombinant proteins, to examine the opposing relationship between XKCM1 and XMAP215. I have developed a system- using TIRF microscopy to investigate whether XMAP215 inhibits the binding of XKCM1 to ends of GMPCPP-stabilized microtubules, or its ability to promote microtubule depolymerization. I will expand this assay to investigate the behavior of XKCM1, and its interactions with XMAP215, on dynamic microtubules. And I will undertake a functional analysis of individual domains of XMAP215 to determine which regions of the protein are important for inhibiting XKCM1 function. Using this combination of techniques I will characterize the antagonistic relationship between XMAP215 and XKCM1, and begin to elucidate how the interplay between these proteins affects microtubule behavior. ? ?
| Brouhard, Gary J; Stear, Jeffrey H; Noetzel, Tim L et al. (2008) XMAP215 is a processive microtubule polymerase. Cell 132:79-88 |
