Sunday, April 28, 2024
4/28/2024
Project Summary The “brain-eating amoeba” Naegleria fowleri causes a disease with a 97% fatality rate. Current treatments are not reliable and risk significant side effects, including brain damage. Because cell proliferation is essential for disease progression, and the microtubule network in Naegleria has evolutionarily diverged from that in humans, targeting the mitotic spindle is a promising strategy to develop effective therapeutics with limited side effects. However, we lack key information about the basic cell biological mechanisms that organize the Naegleria mitotic spindle, hampering progress towards rational therapies. In particular, dynamic microtubule turnover is critical in other cells for assembling a bipolar spindle, but it is not known to what extent the Naegleria spindle relies on microtubule dynamics. A major obstacle is that inhibitors that block microtubule dynamics in other species are ineffective against Naegleria’s divergent tubulins. Further, while microtubule motor proteins play key roles in assembling spindles from diverse species, the function or even the identity of microtubule motors within the Naegleria spindle is completely unknown. Lack of knowledge about the mechanistic contributions of microtubule turnover and molecular motors to Naegleria spindle organization constrains identifying key proteins and processes to target for antimicrobial development. This proposal will address this knowledge gap by testing the hypothesis that microtubule dynamics and molecular motors both contribute to Naegleria spindle organization. To identify the role of microtubule dynamics, microtubules will be stabilized with a class of inhibitors that was recently shown to block Naegleria cell division, and the effect on the organization of the spindle will be measured with super-resolution microscopy. Comparing untreated and drug-treated spindles at different mitotic stages will reveal which stages require microtubule turnover. To determine the function of molecular motors in the spindle, motor genes upregulated during cell division will be identified with RNA sequencing of mitotically synchronized Naegleria cultures. These motors will be knocked down, and phenotypes scored by microscopy. The proposed project will provide comprehensive training to prepare the applicant for a career as an independent investigator. With support from a sponsor, who is a Naegleria expert, and a co-sponsor, who has decades of experience studying cell division, the applicant will learn: new experimental techniques, such as super-resolution microscopy and high-throughput sequencing; new conceptual approaches, including the biology of microtubules and mitosis; and career development skills. This proposal will determine the impact of microtubule stabilization on the Naegleria spindle and will identify the role of spindle associated molecular motors in spindle organization, providing new targets for future treatments for the devastating disease caused by Naegleria.
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