Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis.

During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. Here, we explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, we observed that immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, we found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division.

Connections between sister and non-sister telomeres of segregating chromatids maintain euploidy.

The complete separation of sister chromatids during anaphase is a fundamental requirement for successful mitosis. Therefore, divisions with either persistent DNA-based connections or lagging chromosome fragments threaten aneuploidy if unresolved. Here, we demonstrate the existence of an anaphase mechanism in normally dividing cells in which pervasive connections between telomeres of segregating chromosomes aid in rescuing lagging chromosome fragments. We observe that in a large proportion of Drosophila melanogaster neuronal stem cell divisions, early anaphase sister and non-sister chromatids remain connected by thin telomeric DNA threads. Normally, these threads are resolved in mid-to-late anaphase via a spatial mechanism. However, we find that the presence of a nearby unrepaired DNA break recruits histones, BubR1 kinase, Polo kinase, Aurora B kinase, and BAF to the telomeric thread of the broken chromosome, stabilizing it. Stabilized connections then aid lagging chromosome rescue. These results suggest a model in which pervasive anaphase telomere-telomere connections that are normally resolved quickly can instead be stabilized to retain wayward chromosome fragments. Thus, the liability of persistent anaphase inter-chromosomal connections in normal divisions may be offset by their ability to maintain euploidy in the face of chromosome damage and genome loss.

Wolbachia action in the sperm produces developmentally deferred chromosome segregation defects during the Drosophila mid-blastula transition.

Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, we focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. We performed single embryo PCR and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, we find increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI.

The Cell Biology of Heterochromatin.

A conserved feature of virtually all higher eukaryotes is that the centromeres are embedded in heterochromatin. Here we provide evidence that this tight association between pericentric heterochromatin and the centromere is essential for proper metaphase exit and progression into telophase. Analysis of chromosome rearrangements that separate pericentric heterochromatin and centromeres indicates that they must remain associated in order to balance Cohesin/DNA catenation-based binding forces and centromere-based pulling forces during the metaphase-anaphase transition. In addition, a centromere embedded in heterochromatin facilitates nuclear envelope assembly around the entire complement of segregating chromosomes. Because the nuclear envelope initially forms on pericentric heterochromatin, nuclear envelope formation proceeds from the pole, thus providing time for incorporation of lagging and trailing chromosome arms into the newly formed nucleus. Additional analysis of noncanonical mitoses provides further insights into the functional significance of the tight association between heterochromatin and centromeres.

Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes.

The dramatic changes of subcellular structures during mitosis are best visualized by live imaging. In general, live imaging requires the expression of proteins of interest fused to fluorophores and a model system amenable to live microscopy. Drosophila melanogaster is an attractive model in which to perform live imaging because of the numerous transgenic stocks bearing fluorescently tagged transgenes as well as the ability to precisely manipulate gene expression. Traditionally, the early Drosophila embryo has been used for live fluorescent analysis of mitotic events such as spindle formation and chromosome segregation. More recent studies demonstrate that the Drosophila third instar neuroblasts have a number of properties that make them well suited for live analysis: (1) neuroblasts are distinct cells surrounded by plasma membranes; (2) neuroblasts undergo a complete cell cycle, consisting of G1, S, G2, and M phases; and (3) neuroblasts gene expression is not influenced by maternal load, and so the genetics are therefore relatively more simple. Finally, the Drosophila neuroblast is arguably the best system for live imaging asymmetric stem cell divisions. Here, we detail a method for live imaging Drosophila larval neuroblasts.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Mechanisms driving acentric chromosome transmission.

The kinetochore-microtubule association is a core, conserved event that drives chromosome transmission during mitosis. Failure to establish this association on even a single chromosome results in aneuploidy leading to cell death or the development of cancer. However, although many chromosomes lacking centromeres, termed acentrics, fail to segregate, studies in a number of systems reveal robust alternative mechanisms that can drive segregation and successful poleward transport of acentrics. In contrast to the canonical mechanism that relies on end-on microtubule attachments to kinetochores, mechanisms of acentric transmission largely fall into three categories: direct attachments to other chromosomes, kinetochore-independent lateral attachments to microtubules, and long-range tether-based attachments. Here, we review these "non-canonical" methods of acentric chromosome transmission. Just as the discovery and exploration of cell cycle checkpoints provided insight into both the origins of cancer and new therapies, identifying mechanisms and structures specifically involved in acentric segregation may have a significant impact on basic and applied cancer research.

ESCRT-III-mediated membrane fusion drives chromosome fragments through nuclear envelope channels.

Mitotic cells must form a single nucleus during telophase or exclude part of their genome as damage-prone micronuclei. While research has detailed how micronuclei arise from cells entering anaphase with lagging chromosomes, cellular mechanisms allowing late-segregating chromosomes to rejoin daughter nuclei remain underexplored. Here, we find that late-segregating acentric chromosome fragments that rejoin daughter nuclei are associated with nuclear membrane but devoid of lamin and nuclear pore complexes in Drosophila melanogaster. We show that acentrics pass through membrane-, lamin-, and nuclear pore-based channels in the nuclear envelope that extend and retract as acentrics enter nuclei. Membrane encompassing the acentrics fuses with the nuclear membrane, facilitating integration of the acentrics into newly formed nuclei. Fusion, mediated by the membrane fusion protein Comt/NSF and ESCRT-III components Shrub/CHMP4B and CHMP2B, facilitates reintegration of acentrics into nuclei. These results suggest a previously unsuspected role for membrane fusion, similar to nuclear repair, in the formation of a single nucleus during mitotic exit and the maintenance of genomic integrity.

Micronuclei Formation Is Prevented by Aurora B-Mediated Exclusion of HP1a from Late-Segregating Chromatin in Drosophila.

While it is known that micronuclei pose a serious risk to genomic integrity by undergoing chromothripsis, mechanisms preventing micronucleus formation remain poorly understood. Here, we investigate how late-segregating acentric chromosomes that would otherwise form micronuclei instead reintegrate into daughter nuclei by passing through Aurora B kinase-dependent channels in the nuclear envelope of Drosophila melanogaster neuroblasts. We find that localized concentrations of Aurora B preferentially phosphorylate H3(S10) on acentrics and their associated DNA tethers. This phosphorylation event prevents HP1a from associating with heterochromatin and results in localized inhibition of nuclear envelope reassembly on endonuclease- and X-irradiation-induced acentrics, promoting channel formation. Finally, we find that HP1a also specifies initiation sites of nuclear envelope reassembly on undamaged chromatin. Taken together, these results demonstrate that Aurora B-mediated regulation of HP1a-chromatin interaction plays a key role in maintaining genome integrity by locally preventing nuclear envelope assembly and facilitating the incorporation of late-segregating acentrics into daughter nuclei.

Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor.

Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. Here we demonstrate that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes.

Aurora B-mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments.

To determine how chromosome segregation is coordinated with nuclear envelope formation (NEF), we examined the dynamics of NEF in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late-segregating acentric fragments influence NEF to ensure their inclusion in daughter nuclei. Through live analysis, we show that acentric chromosomes induce highly localized delays in the reassembly of the nuclear envelope. These delays result in a gap in the nuclear envelope that facilitates the inclusion of lagging acentrics into telophase daughter nuclei. Localized delays of nuclear envelope reassembly require Aurora B kinase activity. In cells with reduced Aurora B activity, there is a decrease in the frequency of local nuclear envelope reassembly delays, resulting in an increase in the frequency of acentric-bearing, lamin-coated micronuclei. These studies reveal a novel role of Aurora B in maintaining genomic integrity by promoting the formation of a passageway in the nuclear envelope through which late-segregating acentric chromosomes enter the telophase daughter nucleus.