Tight regulation of the spindle microtubule (MT) dynamics is vital for the success and fidelity of mitosis, but the mechanism for regulating spindle MT dynamics remains unknown. Without this knowledge, a complete understanding of spindle regulation during mitosis is impossible. In recent years, a number of MT regulatory proteins have been identified, but little is known of how they interact with each other to collectively manipulate spindle MT dynamics. The first endeavor along this direction is the recent identification of a network of five regulatory proteins (KLP59C, KLP67A, Mast, EB1 and Msps) that governs kinetochore MT (kMT) plus-end dynamics during metaphase. This network utilizes a complex balance between MT polymerases and depolymerases (instead of polymerases alone) to induce net polymerization at kMT plus-ends, which counteracts constant depolymerization at minus-ends to maintain the metaphase kMTs in a steady state. The long-term goal is to elucidate the molecular events that drive the assembly and function of the mitotic spindle. The objective of this application is to determine how the actions of only a handful of MT regulatory proteins give rise to the broad range of dynamics at spindle MT plus-ends from prometaphase through anaphase. The central hypothesis is: the regulatory networks controlling spindle MT dynamics at other mitotic stages can be attained by shifting the balance among the components of the metaphase network. Guided by strong preliminary data, this hypothesis will be tested through the pursuit of three specific aims: (1) Determine the changes to the kMT regulatory network that transform the plus-end dynamics from net polymerization (metaphase) to net depolymerization (anaphase A). (2) Determine the kMT regulatory network that generates the plus-end dynamics driving chromosome congression during prometaphase. (3) Determine the regulatory networks governing non-kinetochore MT plus-end dynamics to establish/maintain a bipolar spindle during pre- anaphase and to promote spindle elongation during anaphase B.
These aims will be achieved using complementary computer simulation, a custom-developed automatic image tracking method, live cell imaging and RNAi-based protein knockdowns. By bridging hypothesized molecular interactions with cellular-scale experimental observables quantitatively and rigorously, simulations allow us to discriminate alternative molecular mechanisms that experiments alone cannot due to lack of necessary spatial and temporal resolution. The innovation of this plan stems from both the novelty of its hypotheses and the broad and unique array of tools it wields to test them. The proposed research is significant because it will provide a systems-level understanding of an essential module of the spindle machinery, which will fill a severe gap in the current knowledge of mitosis. Moreover, the knowledge thus gained will deepen the understanding of the mechanisms of aneuploidy--the underlying cause of many forms of cancers.
The proposed studies are of an important and under-investigated area of mitosis that has potential applicability to understanding the mechanisms of aneuploidy--the underlying cause of many forms of cancers. The proposed research has relevance to public health, because the fundamental mechanisms to be investigated are expected to be conserved across the phyla. Thus, the findings are ultimately expected to be applicable to the health of human beings.
