Transposable element landscapes in aging Drosophila.

Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.

Nuclear envelope assembly defects link mitotic errors to chromothripsis.

Defects in the architecture or integrity of the nuclear envelope are associated with a variety of human diseases1. Micronuclei, one common nuclear aberration, are an origin for chromothripsis2, a catastrophic mutational process that is commonly observed in cancer3-5. Chromothripsis occurs after micronuclei spontaneously lose nuclear envelope integrity, which generates chromosome fragmentation6. Disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signalling7. Despite its importance, the basis of the fragility of the micronucleus nuclear envelope  is not known. Here we show that micronuclei undergo defective nuclear envelope assembly. Only 'core' nuclear envelope proteins8,9 assemble efficiently on lagging chromosomes, whereas 'non-core' nuclear envelope proteins8,9, including nuclear pore complexes (NPCs), do not. Consequently, micronuclei fail to properly import key proteins that are necessary for the integrity of the nuclear envelope and genome. We show that spindle microtubules block assembly of NPCs and other non-core nuclear envelope proteins on lagging chromosomes, causing an irreversible defect in nuclear envelope assembly. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective nuclear envelope assembly, prevent spontaneous nuclear envelope disruption, and suppress DNA damage in micronuclei. Thus, during mitotic exit in metazoan cells, chromosome segregation and nuclear envelope assembly are only loosely coordinated by the timing of mitotic spindle disassembly. The absence of precise checkpoint controls may explain why errors during mitotic exit are frequent and often trigger catastrophic genome rearrangements4,5.

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing.

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells.

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

Adaptive changes in the kinetochore architecture facilitate proper spindle assembly.

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

Microtubules and Lis-1/NudE/dynein regulate invasive cell-on-cell migration in Drosophila.

The environment through which cells migrate in vivo differs considerably from the in vitro environment where cell migration is often studied. In vivo many cells migrate in crowded and complex 3-dimensional tissues and may use other cells as the substratum on which they move. This includes neurons, glia and their progenitors in the brain. Here we use a Drosophila model of invasive, collective migration in a cellular environment to investigate the roles of microtubules and microtubule regulators in this type of cell movement. Border cells are of epithelial origin and have no visible microtubule organizing center (MTOC). Interestingly, microtubule plus-end growth was biased away from the leading edge. General perturbation of the microtubule cytoskeleton and analysis by live imaging showed that microtubules in both the migrating cells and the substrate cells affect movement. Also, whole-tissue and cell autonomous deletion of the microtubule regulator Stathmin had distinct effects. A screen of 67 genes encoding microtubule interacting proteins uncovered cell autonomous requirements for Lis-1, NudE and Dynein in border cell migration. Net cluster migration was decreased, with initiation of migration and formation of dominant front cell protrusion being most dramatically affected. Organization of cells within the cluster and localization of cell-cell adhesion molecules were also abnormal. Given the established role of Lis-1 in migrating neurons, this could indicate a general role of Lis-1/NudE, Dynein and microtubules, in cell-on-cell migration. Spatial regulation of cell-cell adhesion may be a common theme, consistent with observing both cell autonomous and non-autonomous requirements in both systems.