Spatiotemporal Dynamics And Regulatory Mechanisms Of Microtubule Plus-end-tracking Protein EB1

As a key member of cytoskeletons, microtubules participate in multiple cellular events such as morphology formation and maintenance, cell movement, transport of protein, RNA and vesicles. Microtubules are polarized and maintain polymerizing and depolymerizing activities. A series of proteins called "plus-end tracking proteins (+TIPs)" locate at the plus-ends of microtubules. There are22kinds of well-known plus-end tracking proteins, and most of them bind to microtubule plus-ends through a core family called "End-binding (EB)" family proteins. Despite the broad studies on these plus-end tracking proteins, we have little knowledge about the regulation of their network. In this issue, we will introduce the acety lation of EB1K220, the most popular homolog of EB family, inhibits its interaction with other plus-end tracking proteins. Utilizing techniques of biochemistry, biophotonics and structure biology, we will uncover the detail molecular mechanism of this inhibition, and its antagonism on the plus-end localization of other+TIPs. Acetylation level of EB1K220increases in mitosis, but artificially suppress deacetylation will activate spindle assembly checkpoint, and finally results in mitotic arrest. Our results indicate EB1K220acetylation will take part in error correction of kinetochore-microtubule attachment. We also identified another EB1acetylation site on its N-terminal CH domain, K66. Acetylation of this site does not affect kinetochore-microtubule attachment, but controled mitotic spindle orientation and positioning. Further work presented K66acetylation orchestrated the interaction between EB1CH domain and microtubule, affected the plus-tracking property of EB1, so that regulated astral microtubule dynamics and spindle microtubule flux, and finally controlled spindle orientation and positioning. These results enlightened our knowledge on accurate mitosis regulation by dynamic acetylation and deacetylation of EB1proteins.Because of heterogeneity of microtubule dynamics seen at cellular level, it is useful and necessary to observe the precise organization of microtubule plus-ends in various cell compartment. But the structural unstable and sensitivity to formaldehyde and glutaraldehyde fixation bring microtubule disadvantage on its application of electron microscopy. To this end, it is of great value to develop novel observation methods, especially in live cell at single molecular level. Here we use recently established photoactivated localization microscopy (PALM) to break the200nm conventional resolution burrier, and observe the behavior of microtubule plus-end tracking protein EB1in live cell at a resolution of almost30nm. We founded a photoactivatable complementary fluorescent system (PACF) to image dimerized EB1molecules on microtubule plus-ends and reduce the background noise. With PACF, we successfully recorded two distinct types of EB1localization in migrating MCF7cells, and identified their preference in leading edge and cell body. This discovery is de novo as the various EB1localization types can not be distinguished by the conventional microscopy. During the time of PACF system construction, we also occasionally noticed the importance of previous ignored flexible Linker region on EB1plus-end localization, which lead us to discover the function of positive charged amino acids on regulating the folding of Linker region. Our experiments indicated the folding of Linker region restricted the two N-terminal CH domain from dimerized EB1in a special distance or position, and also provided flexibility, resulted in the special capacity of tracking dynamical microtubule plus-ends.In conclusion, this issue will elaborate the influence of EB1acetylation on its interaction with other plus-end tracking proteins, the molecular mechanisms and its significance on mitosis. It will also report the observation of dimerized EB1behavior in live cell at single molecular level, and the identification of two different types of EB1localization in leading edge and cell body of migrating cells. We will also introduce the meaning of EB1Linker region on its plus-end tracking property. These findings will promote our understanding on microtubule plus-end dynamics and its regulation mechanisms. At last but not least, our newly established PACF system provides a unique approach to probe precise protein interactions and delineate spacial dynamics of homo-and hetero-dimerized proteins at nanoscale in live cells.