Accurate segregation of chromosomes during mitosis requires the proper assembly of a bipolar spindle. Errors in this process can result in aneuploidy, a harbinger of malignant transformation and a major cause of birth defects. Spindle assembly results from stochastic interactions between microtubules and transiently associated microtubule motor proteins. The broad goal of this work is to better understand how these interactions and the forces generated by them contribute to spindle assembly and function. This research is based upon observations of spindles in Xenopus egg extracts and is focused on a dynein-dependent mechanism that facilitates spindle fusion. The specific aims of this proposal are 1) to characterize the mechanism of dynein-dependent microtubule-bundling and force generation at microtubule plus ends, and 2) to identify additional molecular components required for antiparallel microtubule interactions and minus end-directed force generation. High resolution multimode digital microscopy will be used to analyze antiparallel interactions of fluorescently labeled microtubules and microtubule motor protein dynamics in the microtubule "bridge" observed to form between fusing spindles. Spindle fusion will be further characterized in Xenopus extracts depleted of known motor and non-motor microtubule proteins thought to be involved in antiparallel microtubule interaction and force generation. The contribution of dynein and newly identified proteins to intrinsic spindle tension and steady state length maintenance will be evaluated by measuring changes in interkinetochore stretch and spindle length following pharmacological inhibition, function blocking antibody addition, or immunodepletion in Xenopus egg extracts. Errors in mitotic spindle assembly and function have been linked to cancer and birth defects. This research will increase our collective understanding of the basic mechanisms that contribute to the formation of a functional spindle and may ultimately lead to novel therapeutic strategies to reduce the prevalence and occurrence of these afflictions. [unreadable] [unreadable] [unreadable]