Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.

ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes.

The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division.

ESCRT machinery and cytokinesis: the road to daughter cell separation.

The endosomal sorting complex required for transport (ESCRT) machinery is a set of cellular protein complexes required for at least three topologically equivalent membrane scission events, namely multivesicular body (MVB) formation, retroviral particle release and midbody abscission during cytokinesis. Recently, several studies have explored the mechanism by which the core ESCRT-III subunits mediate membrane scission and might be differentially required according to the functions of the pathway. In this review, we discuss the links between the ESCRT machinery and cytokinesis, with special focus on abscission initiation and regulation.

Building Blocks of Virtuous Science Communication: Grant Funding, Policy Making, and Public Engagement.

Grant writing, science policy and public engagement (PE) activities are forms of science communication, and all are essential for research to function and benefit our society. Scientists rely on competitive grant funding to finance their research; this is an opportunity for researchers to communicate their scientific vision and engage funders, major players in defining how public funds and government policies are prioritized to drive research and innovation. PE is often still seen as a box-ticking or persuasion exercise, yet establishing the right communication channels with the public is pivotal for a scientist's job to impact society. We believe that evidence-based communication is becoming essential in a world dominated by an excess of information, where verified sources are under pressure. Support in the form of dedicated training and allocated resources, particularly for early career researchers, can help establish what we describe as a long-lasting virtuous circle, in which public funds are spent effectively toward scientific advances that the public and policy makers can embrace.

CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope.

The coordinated reformation of the nuclear envelope (NE) after mitosis re-establishes the structural integrity and the functionality of the nuclear compartment. The endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodeling pathway that is highly conserved in eukaryotes, has been recently involved in NE resealing by mediating the annular fusion of the nuclear membrane (NM). We show here that CC2D1B, a regulator of ESCRT polymerization, is required to re-establish the nuclear compartmentalization by coordinating endoplasmic reticulum (ER) membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE. Accordingly, CC2D1B determines the spatiotemporal distribution of the CHMP7-ESCRT-III axis during NE reformation. Crucially, in CC2D1B-depleted cells, ESCRT activity is uncoupled from Spastin-mediated severing of spindle microtubules, resulting in persisting microtubules that compromise nuclear morphology. Therefore, we reveal CC2D1B as an essential regulatory factor that licenses the formation of ESCRT-III polymers to ensure the orderly reformation of the NE.

A homeostatic clock sets daughter centriole size in flies.

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing "mother" centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.

ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins.

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.

ESCRT-III governs the Aurora B-mediated abscission checkpoint through CHMP4C.

The endosomal sorting complex required for transport (ESCRT) machinery plays an evolutionarily conserved role in cytokinetic abscission, the final step of cell division where daughter cells are physically separated. Here, we show that charged multivesicular body (MVB) protein 4C (CHMP4C), a human ESCRT-III subunit, is involved in abscission timing. This function correlated with its differential spatiotemporal distribution during late stages of cytokinesis. Accordingly, CHMP4C functioned in the Aurora B-dependent abscission checkpoint to prevent both premature resolution of intercellular chromosome bridges and accumulation of DNA damage. CHMP4C engaged the chromosomal passenger complex (CPC) via interaction with Borealin, which suggested a model whereby CHMP4C inhibits abscission upon phosphorylation by Aurora B. Thus, the ESCRT machinery may protect against genetic damage by coordinating midbody resolution with the abscission checkpoint.

The UBAP1 subunit of ESCRT-I interacts with ubiquitin via a SOUBA domain.

The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.