Functional Delineation Of Microtubule Ends Associated Protein CSPP1 And EB1

Adherens junctions play important roles during development. They keep the same cells together and generate boundaries between different cell groups, making sure of the tissue homeostasis. Microtubule is of great importance during the formation of adherens junctions, as adherens junctions are enormously impaired when epithelial cells are treated with nocodazole, a microtubule poison. However, the linkage between adherens junctions and microtubules is still unknown. Further, besides some centrosomal microtubules, there are lots of non-centrosomal microtubules in epithelial cells. And 90 percent of microtubules are quiescent in interphase cells, indicating that both ends of non-centrosomal microtubules are protected. Although the proteins specifically bound to the minus end of microtubule have been characterized, there is no microtubule plus end capping protein reported till now. Microtubule plus end tracking proteins (+TIPs) associate with the plus ends, however, they specifically resides on dynamic growing microtubule tips and they have no obvious effect on stabilizing microtubule ends. Here we report that CSPP1 is a new member of adherens junctions. Via binding to p120, CSPP1 helps to maintain the integrity of adherens junctions. In mechanism, CSPP1 caps both ends of non-centrosomal microtubule in vitro and in vivo. Binding of CSPP1 to microtubule ends completely inhibits catastrophe and restrains the velocity of polymerization, demonstrating that CSPP1 stabilizes microtubule ends. Thus, CSPP1 is the only protein identified that caps both ends of still microtubules. Furthermore, the microtubules bound CSPP1 are resistant to MCAK mediated depolymerization. So, we propose that CSPP1 links non-centrosomal microtubules to adherens junctions, thus facilitating assembly and maintenance of adherens junctions.EB1 is a microtubule plus end tracking protein, mediating the load of a series of +TIP proteins onto growing microtubule plus end. In mitosis, EB1 is regarded as a kinetochore protein, which specifically localizes at trailing kinetochore where attached microtubules are growing. However, not all+TIP proteins reside on kinetochore with EB1, demonstrating a regulation between EB1 and other +TIPs in mitosis. P300/CBP associated factor (PCAF), an acetyltransferase, was reported to resides on kinetochore during mitosis. Our research showed that PCAF interacts with and acetylates EB1. The acetylation occurs at Lys220 of EBH domain within the C terminus of EB1. With the antibody specific to acetyl-K220 (AcK220), we found that the acetylation in mitotic cells is higher than that in interphase cells by four fold. Biochemically, EB1-K220 acetylation down-regulates its interaction with SxIP motif containing +TIPs, which is essential to proper mitotic progression. Furthermore, we show that AcK220 antibody stains kinetochore, which is independent of microtubules, arguing against the previous result saying that EB1’s kinetochore residence reqires growing microtubule. But, AcK220 indeed preferentially localizes on trailing kinetochores, demonstrating a regulatory mechanism. Besides EB1, we found that EB2 and/or EB3 also contribute to AcK220 signal, implying the acetylation of EB2 and/or EB3, However, more experiments should be done to confirm. The N terminus of EB1 mediates its direct interaction with microtubule. Our results also suggest that PCAF could acetylate EB1-K66 and this acetylation regulates binding of EB1 to microtubules.