live-cell-imaging-of-chromosome-segregation-during-mitosis

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

US Food and Drug Administration

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

Inhibition of hepatitis C virus by the cyanobacterial protein Microcystis viridis lectin: mechanistic differences between the high-mannose specific lectins MVL, CV-N, and GNA.

The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated.

TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection.

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

Article	Year
Inhibition of hepatitis C virus by the cyanobacterial protein Microcystis viridis lectin: mechanistic differences between the high-mannose specific lectins MVL, CV-N, and GNA. Molecular pharmaceutics\| PubMed ID: 24152340	2013
TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26170307	2015
PP2A-B56γ is required for an efficient spindle assembly checkpoint. Cell cycle (Georgetown, Tex.)\| PubMed ID: 28562161	2017

Kazuyo Takeda

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