Probing Interactions Between Eb1, Microtubules and Actin

The cytoskeleton is a dynamic cellular 'scaffold' that is present in all eukaryotic cells. Interestingly, two major cytoskeletal elements Ì¢‰âÂ' microtubules (MTs) and actin Ì¢‰âÂ' are crucial for the cell survival: MTs drive essential cell activities ranging from separating chromosomes to transporting large particles, while actin filaments provide mechanical structure and motility. The dynamic behavior of MTs and actin are separately regulated by two groups of proteins: MT binding proteins (MTBPs) and actin binding proteins (ABPs). For cells to function properly, actin filaments and MT polymers must cooperate, but the mechanism of 'cross-talk' between actin and MT are as yet poorly understood. This thesis focuses on a unique MT binding protein called EB1, how it interacts with MTs, and our evidence demonstrates that it plays an important role in actin-MT cross-talk. EB1 is a highly conserved MT binding protein that dynamic tracks MT plus ends, and it plays a pivotal role in MT dynamics. However, its activity and mechanism remain unclear, in part because of variability in the reported behavior of EB1 in the literature. We hypothesized that some of the variability came from differences in how the EB1 proteins were prepared. In particular, we were concerned that purification tags might influence EB1 behavior. The first part of this thesis describes my work to determine EB1 activity by testing the interaction between native (untagged) EB1 and MT. Because EB1 contains a calponin homology (CH) domain (a domain that typically binds actin), we were suspicious EB1 may interact with the actin. I address the interaction between EB1 and actin in the second part of this study. To investigate the EB1-MT interaction, we initially used His-tagged EB1 proteins. However, we found that the activities of His-tagged proteins were significantly different from the proteins with the His-tags removed. This result suggested that the His-tag could influence EB1 activity. To resolve this issue and define the native EB1 activity, we developed a de novo purification strategy for EB1 proteins and successfully purified several native (untagged) EB1 constructs. We found that N-terminal His-tags does directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity. Moreover, we also noticed that the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. All these observations demonstrate that EB1 binding is not limited to the MT seam (as had been implied by previous studies), and they suggest that EB1 binds cooperatively to MTs. We believed that these binding relationships are important for helping to elucidate the complex of proteins at the MT tip. To address the interaction between EB1 and actin, similar strategies were used as in our previous EB1-MT studies. Remarkably, we discovered that EB1 is able to directly bind to actin filaments, and it also greatly promotes actin polymerization. This was surprising because up to this time, all studies of EB1 had focused it is MT-related functions. Moreover, we found that the interaction between EB1 and actin is mediated by the EB1 CH-domain, which is responsible for EB1-MT interactions. Interestingly, the binding of EB1 to actin, like MT-binding, is auto-inhibited by the EB1 C-terminal tail. These data provide evidence that EB1 can directly regulate actin-mediated processes. Since EB1 has such an important and conserved role in MT dynamics, these observations suggest that EB1 might have a role in actin-MT coordination.