Mitotic transmission of acentric chromosomes

University Of California Santa Cruz, Santa Cruz CA

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Abstract

The goal of this proposal is to elucidate the mechanisms that maintain genome integrity during anaphase and telophase. These are particularly perilous phases of the cell cycle, requiring separation and segregation of replicated sister chromosomes and their incorporation into newly formed daughter nuclei. Failure to completely and accurately separate the duplicated genome during these phases can lead to cancer development or cell death. However, while much work has been devoted toward elucidating the mechanisms that maintain genomic integrity during interphase and metaphase, less attention has been paid to the mechanisms that operate specifically during anaphase and telophase. One reason for this knowledge gap is that the prevailing view has been once checkpoints acting prior to anaphase are satisfied, anaphase and telophase proceed rapidly without error correction mechanisms. However, work over the past decade from our lab and others have upended this traditional view to reveal that the eukaryotic cell maintains a sophisticated and diverse set of mechanisms that function during anaphase-telophase to maintain genomic integrity. Our entry into this field originated from live observations of Drosophila neuroblasts. We discovered that chromosome fragments lacking a kinetochore (acentrics) successfully congress, undergo delayed but proper sister separation, efficiently segregate to opposing poles and incorporate into daughter nuclei well after nuclear envelope assembly. This was unexpected as all of these chromosome dynamics were thought to be largely driven by kinetochore-microtubule interactions. Our past studies have revealed a number of cellular adaptions such as protein-coated DNA tethers, cell and spindle elongation, and channels in the telophase nuclear envelope that ensure accurate acentric segregation and incorporation into daughter nuclei. During this last period of MIRA funding, we built upon this knowledge by determining that acentric sister separation is driven by plus-end-directed microtubule forces. We also acquired evidence that acentric poleward segregation relies on actin-based processes. Additionally, we characterized previously undescribed mechanisms such as ubiquitous DNA threads between separated sister and non-sister anaphase chromosomes that promote acentric rescue. Finally, we gathered preliminary evidence suggesting that these anaphase/telophase adaptations promoting acentric chromosome segregation exist and operate in human cells. The future Drosophila and mammalian cell line studies described here will determine the molecular underpinnings of these unexpected mechanisms operating during anaphase-telophase to preserve genome integrity through a combination of genetic, fluorescent and electron microscope studies as well as biochemical approaches. Additionally, we will apply our expertise in chromosome biology to elucidate the mechanisms by which the insect endosymbiont Wolbachia disrupts host chromosome segregation in order to promote its transmission through wild populations. All of these studies provide excellent opportunities for undergraduates, especially URM students to receive mentored hands-on lab experiences often leading to co-authorship on peer reviewed publications.