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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. Using light- and electron microscopy, Khodjakov, Mogilner and colleagues find that the shape of the kinetochore undergoes dramatic changes during mitosis. Computational analysis suggests that this facilitates correct chromosome attachment.

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.

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.

Defects in the architecture or integrity of the nuclear envelope are associated with a variety of human diseases 1 . Micronuclei, one common nuclear aberration, are an origin for chromothripsis 2 , a catastrophic mutational process that is commonly observed in cancer 3 – 5 . Chromothripsis occurs after micronuclei spontaneously lose nuclear envelope integrity, which generates chromosome fragmentation 6 . Disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signalling 7 . 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 proteins 8 , 9 assemble efficiently on lagging chromosomes, whereas ‘non-core’ nuclear envelope proteins 8 , 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 rearrangements 4 , 5 . The mitotic spindle prevents normal nuclear envelope assembly on missegregated chromosomes, leading to spontaneous envelope disruption of micronuclei and subsequent genome instability.

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. Author summary Transposable elements, also called transposons, are genetic parasites found in all animal genomes. Normally, transposons are compacted away in silent chromatin in young animals. But, as animals age and transposon-silencing defense mechanisms break down, transposon RNAs accumulate to significant levels in old animals like fruit flies. An open question is whether the increased levels of transposon RNAs in older animals also correspond to increased genomic copies of transposons. This study approached this question by sequencing the whole genomes of young and old wild-type and mutant flies lacking a functional RNA interference (RNAi) pathway, which naturally silences transposon RNAs. Although the wild-type flies with intact RNAi activity had little new accumulation of transposon copies, the sequencing approach was able to detect several transposon accumulation occurrences in some RNAi mutants. In addition, we found that some fly transposon families can also accumulate as extra-chromosomal circular DNA copies. Lastly, we showed that genetically augmenting the expression of RNAi factors can counteract the rising transposon RNA levels in aging and promote longevity. This study improves our understanding of the animal host genome relationship with transposons during natural aging processes.

Defects in the architecture or integrity of the nuclear envelope (NE) are associated with a variety of human diseases. Micronuclei, one common nuclear aberration, are an origin for chromothripsis, a catastrophic mutational process commonly observed in cancer genomes and other contexts. Micronuclei have a defective NE, with the extensive chromosome fragmentation that generates chromothripsis occurring after abrupt, spontaneous loss of NE integrity. After NE disruption, the exposed cytoplasmic DNA can additionally initiate proinflammatory signaling linked to senescence, metastasis, and the immune clearance of tumor cells. Despite its broad physiological impact, the basis for the nuclear envelope fragility of micronuclei is unknown. Here we demonstrate that micronuclei undergo markedly defective NE assembly: Only core NE proteins assemble efficiently on lagging chromosomes whereas non-core NE proteins, including nuclear pore complexes (NPCs), fail to properly assemble. Consequently, micronuclei have impaired nuclear import, and key nuclear proteins required to maintain the integrity of the NE and the genome fail to accumulate normally. We show that densely bundled spindle microtubules inhibit non-core NE assembly, leading to an irreversible NE assembly defect. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective NE assembly, prevent spontaneous NE disruption, and suppress DNA damage in micronuclei. Our findings indicate that chromosome segregation and NE assembly are only loosely coordinated through the timing of mitotic spindle disassembly. The absence of precise regulatory controls can explain why errors during mitotic exit are frequent, and a major trigger for catastrophic genome rearrangements.

ABSTRACT 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 answer 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 natural defenses like RNA interference (RNAi) pathways are compromised. By also incorporating droplet digital PCR to validate genomic TE loads, we confirm TL changes can occur in a single lifespan of Drosophila when TEs are not suppressed. 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 RNAi mechanisms generally mitigate genomic TL expansion despite the increase in TE transcripts during aging.