Functional midbody assembly in the absence of a central spindle

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Caveolae promote successful abscission by controlling intercellular bridge tension during cytokinesis

During cytokinesis, the intercellular bridge (ICB) connecting the daughter cells experiences pulling forces, which delay abscission by preventing the assembly of the ESCRT scission machinery. Abscission is thus triggered by tension release, but how ICB tension is controlled is unknown. Here, we report that caveolae, which are known to control membrane tension upon mechanical stress in interphase cells, are located at the midbody, at the abscission site and at the ICB/cell interface in dividing cells. Functionally, the loss of caveolae delays ESCRT-III recruitment during cytokinesis and impairs abscission. This is the consequence of a 2-fold increase of ICB tension measured by laser ablation, associated with a local increase in myosin II activity at the ICB/cell interface. We thus propose that caveolae buffer membrane tension and limit contractibility at the ICB to promote ESCRT-III assembly and cytokinetic abscission. Altogether, this work reveals an unexpected connection between caveolae and the ESCRT machinery and the first role of caveolae in cell division.

The Abscission Checkpoint: A Guardian of Chromosomal Stability

The abscission checkpoint contributes to the fidelity of chromosome segregation by delaying completion of cytokinesis (abscission) when there is chromatin lagging in the intercellular bridge between dividing cells. Although additional triggers of an abscission checkpoint-delay have been described, including nuclear pore defects, replication stress or high intercellular bridge tension, this review will focus only on chromatin bridges. In the presence of such abnormal chromosomal tethers in mammalian cells, the abscission checkpoint requires proper localization and optimal kinase activity of the Chromosomal Passenger Complex (CPC)-catalytic subunit Aurora B at the midbody and culminates in the inhibition of Endosomal Sorting Complex Required for Transport-III (ESCRT-III) components at the abscission site to delay the final cut. Furthermore, cells with an active checkpoint stabilize the narrow cytoplasmic canal that connects the two daughter cells until the chromatin bridges are resolved. Unsuccessful resolution of chromatin bridges in checkpoint-deficient cells or in cells with unstable intercellular canals can lead to chromatin bridge breakage or tetraploidization by regression of the cleavage furrow. In turn, these outcomes can lead to accumulation of DNA damage, chromothripsis, generation of hypermutation clusters and chromosomal instability, which are associated with cancer formation or progression. Recently, many important questions regarding the mechanisms of the abscission checkpoint have been investigated, such as how the presence of chromatin bridges is signaled to the CPC, how Aurora B localization and kinase activity is regulated in late midbodies, the signaling pathways by which Aurora B implements the abscission delay, and how the actin cytoskeleton is remodeled to stabilize intercellular canals with DNA bridges. Here, we review recent progress toward understanding the mechanisms of the abscission checkpoint and its role in guarding genome integrity at the chromosome level, and consider its potential implications for cancer therapy.

Septin Remodeling During Mammalian Cytokinesis

Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.

Use of cell doublets for studying cytokinesis regulation reveals a new form of cytokinesis regression

Cytokinesis mediates separation of daughter cells at the end of cell division. We have developed a high-throughput approach for monitoring cell-autonomous cytokinesis in non-adherent cells. Focusing on cytokinesis termination, we show that chemical inhibition of protein phosphatase 1 (PP1) and PP2A specifically in late cytokinesis activates cytokinesis regression, which is distinct from any known cytokinesis failure, and is not a by-product of abnormal furrow ingression or chromatin bridges. This process is characterized by the formation of cortical blebs primarily at the intercellular bridge, reopening of the cleavage furrow and reassembly of an interphase-like microtubule network, but not by chromatin recondensation and mitotic spindle formation. Finally, cytokinesis regression is suppressed by chemical inhibition of aurora kinases but not Cdk1 or PLK1. Altogether, our results highlight a hitherto uncharacterized facet of the counter-activity of PP1/PP2A and aurora kinases in the final step of cell division, which ultimately secure the conclusion of cytokinesis, thereby preventing polyploidy and genomic instability.

Altered germline cyst formation and oogenesis in Tex14 mutant mice

ABSTRACT During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

First person – Nicholas Iannantuono

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nicholas Iannantuono is first author on ‘ Rab11FIP1 maintains Rab35 at the intercellular bridge to promote actin removal and abscission’, published in JCS. Nicholas is a PhD student in the lab of Dr Gregory Emery at the Institute for Research in Immunology and Cancer, Université de Montréal, Canada, investigating the mechanisms that govern how cells faithfully divide their contents and separate into two independent entities.

Rab11FIP1 maintains Rab35 at the intercellular bridge to promote actin removal and abscission

Cytokinesis occurs at the end of mitosis/meiosis wherein the cytoplasms of daughter cells are separated. Prior to abscission, an intercellular bridge containing the remaining furrowing machinery, mitotic spindle and actin cytoskeleton connects the two daughter cells. To remove this actin and allow for separation of daughter cells, Rab35 vesicles, loaded with the actin oxidizer MICAL1 and the inositol polyphosphate 5-phosphatase OCRL are recruited to the midbody in a fine-tuned spatiotemporal manner. Importantly however, the means by which these vesicles are recruited is currently unclear. Here, we demonstrate that Rab11FIP1 is recruited to the midbody after Rab35 to scaffold it at the bridge and maintain Rab35 in this region. In the absence of Rab11FIP1, Rab35 dramatically drops from the midbody, inducing defects such as cytokinetic delays and binucleation due to actin overaccumulation at the intercellular bridge, defects which can be rescued with Latrunculin A treatment. Importantly, we show that Rab11FIP1 is critical for Rab35 function in actin removal prior to cytokinesis.

Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge that connects the two newly formed sibling cells, which is ultimately resolved by abscission. While mitosis is completed within 15 min, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell–cell communication is fundamental for development, the role of intercellular bridges during embryogenesis has not been fully elucidated. In this work, we characterized the spatiotemporal characteristics of the intercellular bridge during early zebrafish development. We found that abscission is delayed during the rapid division cycles that occur in the early embryo, giving rise to the formation of interconnected cell clusters. Abscission was accelerated when the embryo entered the midblastula transition (MBT) phase. Components of the ESCRT machinery, which drives abscission, were enriched at intercellular bridges post-MBT and, interfering with ESCRT function, extended abscission beyond MBT. Hallmark features of MBT, including transcription onset and cell shape modulations, were more similar in interconnected sibling cells compared to other neighboring cells. Collectively, our findings suggest that delayed abscission in the early embryo allows clusters of cells to coordinate their behavior during embryonic development.

Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

During the final stages of cell division, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell division the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells. While much is known about intracellular MB-Rs, sMB-Rs are poorly understood. Here, we report the large-scale purification and biochemical characterisation of sMB-Rs released from colon cancer cells, including profiling of their proteome using mass spectrometry. We show sMB-Rs are an abundant class of membrane-encapsulated extracellular vesicle (200-600 nm) enriched in core cytokinetic proteins and molecularly distinct from exosomes and microparticles. Functional dissection of sMB-Rs demonstrated that they are engulfed by, and accumulate in, quiescent fibroblasts where they promote cellular transformation and an invasive phenotype.