Discrete States of a Protein Interaction Network Govern Interphase and Mitotic Microtubule Dynamics

Abstract

The cytoplasm of eukaryotic cells is thought to adopt discrete “states” corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of “cytoplasmic states” regarding the microtubule system. When eukaryotic cells undergo cell division, a dramatic reorganization occurs during the transition from interphase to metaphase. The cell rounds up, chromosomes condense, the nuclear envelope breaks down, and microtubules (proteins that help maintain the cell's shape) become very short and dynamic before assembly of the mitotic spindle (the structure that pulls chromosomes apart). Although it is known that the CDK1 kinase induces this reorganization, the precise mechanisms that regulate such coordinated changes are not yet understood. To investigate the regulation of microtubule dynamics, we applied a new method, called visual immunoprecipitation (VIP), that enables simultaneous visualization of multiple protein interactions in cell extracts. There are two known major regulators of microtubule dynamics: a stabilizer (XMAP215) and a destabilizer (XKCM1); a series of other molecules (EB1, APC, and CLIP 170) are also involved, although their roles in the global regulation of microtubule dynamic instability are not as clear. We show here that microtubules are stable during interphase because the destabilizer is inhibited by the other molecules. During mitosis, however, the destabilizer is released, triggering the alteration of microtubule structure and dynamics. Thus, microtubule dynamics change in response to a dramatic switch in the interactions of a set of proteins.