STAG2 Promotes Error Correction in Mitosis by Regulating Kinetochore–Microtubule Attachments

Mutations in the STAG2 gene are present in ∼20% of tumors from different tissues of origin. STAG2 encodes a subunit of the cohesin complex, and tumors with loss-of-function mutations are usually aneuploid and display elevated frequencies of lagging chromosomes during anaphase. Lagging chromosomes are a hallmark of chromosomal instability (CIN) arising from persistent errors in kinetochore-microtubule (kMT) attachment. To determine whether the loss of STAG2 increases the rate of formation of kMT attachment errors or decreases the rate of their correction, we examined mitosis in STAG2-deficient cells. STAG2 depletion does not impair bipolar spindle formation or delay mitotic progression. Instead, loss of STAG2 permits excessive centromere stretch along with hyperstabilization of kMT attachments. STAG2-deficient cells display mislocalization of Bub1 kinase, Bub3 and the chromosome passenger complex. Importantly, strategically destabilizing kMT attachments in tumor cells harboring STAG2 mutations by overexpression of the microtubule-destabilizing enzymes MCAK (also known as KIF2C) and Kif2B decreased the rate of lagging chromosomes and reduced the rate of chromosome missegregation. These data demonstrate that STAG2 promotes the correction of kMT attachment errors to ensure faithful chromosome segregation during mitosis

Eisosomes Provide Membrane Reservoirs for Rapid Expansion of the Yeast Plasma Membrane

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires \u27membrane reservoirs\u27 that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells

ETAA1 ensures proper chromosome segregation: A matter of S phase or mitosis?

ETAA1 activates the master checkpoint kinase ATR. Bass and Cortez (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201810058) recently reported an intra-mitotic function of ETAA1 that safeguards chromosome stability. In this issue, Achuthankutty et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905064) describe a mechanism controlling the ATR-activating potential of ETAA1 in S phase to preserve chromosome stability.Fil: González, Marina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Potent Single-Domain Antibodies that Arrest Respiratory Syncytial Virus Fusion Protein in its Prefusion State

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Numerical Chromosomal Instability Mediates Susceptibility to Radiation Treatment

The exquisite sensitivity of mitotic cancer cells to ionizing radiation (IR) underlies an important rationale for the widely used fractionated radiation therapy. However, the mechanism for this cell cycle-dependent vulnerability is unknown. Here we show that treatment with IR leads to mitotic chromosome segregation errors in vivo and long-lasting aneuploidy in tumour-derived cell lines. These mitotic errors generate an abundance of micronuclei that predispose chromosomes to subsequent catastrophic pulverization thereby independently amplifying radiation-induced genome damage. Experimentally suppressing whole-chromosome missegregation reduces downstream chromosomal defects and significantly increases the viability of irradiated mitotic cells. Further, orthotopically transplanted human glioblastoma tumours in which chromosome missegregation rates have been reduced are rendered markedly more resistant to IR, exhibiting diminished markers of cell death in response to treatment. This work identifies a novel mitotic pathway for radiation-induced genome damage, which occurs outside of the primary nucleus and augments chromosomal breaks. This relationship between radiation treatment and whole-chromosome missegregation can be exploited to modulate therapeutic response in a clinically relevant manner

Cdk1 and Plk1 mediate a CLASP2 Phospho-Switch that Stabilizes Kinetochore–Microtubule Attachments

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT-MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 phospho-switch that temporally regulates KT-MT attachment stability

Oxidant selection to treat an aged PAH contaminated soil by in situ chemical oxidation.

International audienceThis work is a part of the OXYSOL project aiming at the conception of a global treatment pathway including In Situ Chemical Oxidation to clean up soils of former metallurgical sites. It deals with the selection of the most adapted oxidants. Batch experiments were performed with aged contaminated soil samples of a former steel-making plant to degrade the 16 US EPA PAHs. In this research, hydrogen peroxide, modified Fenton's reaction, potassium permanganate, sodium percarbonate and sodium persulfate were compared at high and moderate doses. Hydrogen peroxide, modified Fenton's reagent, percarbonate and activated persulfate led to a maximum degradation ratio of 45%. A higher ratio (70%) was obtained with a high dose of permanganate. Except for permanganate, increasing oxidant dose did not improve degradation rates, especially with radical-based oxidative systems probably due to radical scavenging. Oxidant doses had an effect on pH that drastically increased or dropped in some cases, which was a drawback. Permanganate efficacy was mainly assigned to its persistence. In all cases, the low availability of PAHs, partly sequestrated in the aged soil, was identified as the most limiting factor for degradation performance. Oxidants were ranked according to their efficiency for PAH oxidation in soils. Efficiency was not correlated to the doses

Megadalton-Node Assembly by Binding of Skb1 to the Membrane Anchor Slf1

The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV