Kinesin-8s and Microtubule Severing Proteins Regulate Microtubule Dynamics in Mitosis
The end goal of mitosis is equal partitioning of genetic material into respective daughter cells. When this is not achieved, genomic instability may result, the end result of which may be cancer. The mitotic spindle, a dynamic structure comprised of microtubules, is essential in this process. Though several different classes of proteins are known to regulate microtubule dynamics, the functions of many proteins in these classes remain to be illuminated. Here we aim at understanding the mitotic roles of human Kinesin-8 family members Kif18B and Kif19 and the microtubule severing protein Katanin-like 1 (KL1) in the formation of the mitotic spindle. Knockdown of either Kif18B or Kif19 increases the mitotic index by increasing the percentage of cells in prometaphase. However, the different phenotypes that arise from their knockdown imply that they function via different pathways. Knockdown of Kif19 by siRNA increased by about ten fold the percentage of multipolar cells and cells whose spindle were not parallel to the plane of view. In contrast Kif18B knockdown by siRNA resulted in 45 percent of mitotic cells developing abnormal prometaphase spindles. These abnormalities include misaligned chromosomes, curled spindle poles, and collapsed spindles.;We also have identified human KL1 as a novel microtubule severing enzyme that is involved in the regulation of mitotic spindle architecture. Overexpression of KL1 results in the complete disassembly of cellular microtubules. This strongly suggests that KL1 is an active microtubule severing protein. During mitosis, the localization of KL1 is restricted to spindle poles and is notably absent from centrosomes. This is in clear contrast to conventional Katanin whose mitotic localization extends from centrosomes onto poles. Consistent with KL1's localization, siRNA depletion results in a specific and significant reduction in the density of microtubules at spindle poles and a significant increase in spindle length. Depletion of KL1 also alters the distribution of gamma--tubulin at centrosomes and spindle poles. Despite its impact on spindle morphology, we could find no evidence that KL1 influences anaphase chromosome motility. Based on our findings, we propose that KL1-mediated microtubule severing is utilized to generate microtubule seeds within the poles, and that loss of this activity alters the normal balance of motor-generated forces that determine spindle length. Through these studies, we characterize the cellular functions of three novel microtubule regulators, which enhance our understanding of microtubule dynamic regulation in mitosis. Our work also provides the basis for future studies to elucidate the mechanisms of each individual regulator.
Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: 4000.;Advisors: David Sharp.