Wimmer-Kleikamp Sabine H, Janes Peter W, Squire Anthony, Bastiaens Philippe I H, Lackmann Martin
Dept. of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, Clayton, Victoria 3800, Australia.
J Cell Biol. 2004 Mar 1;164(5):661-6. doi: 10.1083/jcb.200312001.
Eph receptors and their cell membrane-bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5-coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph-Eph interactions.
Eph受体及其细胞膜结合的ephrin配体调节细胞定位，从而建立或稳定细胞组织模式。尽管人们认识到ephrin聚集对于Eph功能至关重要，但将ephrin密度信息传递到细胞生物学反应中的机制却知之甚少。我们通过共聚焦延时显微镜和荧光共振能量转移显微镜证明，在与ephrin-A5包被的珠子结合的几分钟内，EphA3受体组装成大的簇。虽然Eph簇仍位于ephrin接触位点周围，但其大小超过相互作用的ephrin表面几倍。具有受损的ephrin结合能力的EphA3突变体，单独时无法形成簇或磷酸化，当与功能性受体共表达时，能被有效招募并发生磷酸化。我们的研究结果揭示了ephrin促进的Eph簇形成和簇传播的连续启动，后者独立于ephrin接触和胞质Eph信号功能，但涉及直接的Eph-Eph相互作用。
