Biochemistry and Pharmacology - Research Publications

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    Tracking protein aggregation and mislocalization in cells with flow cytometry
    Ramdzan, YM ; Polling, S ; Chia, CPZ ; Ng, IHW ; Ormsby, AR ; Croft, NP ; Purcell, AW ; Bogoyevitch, MA ; Ng, DCH ; Gleeson, PA ; Hatters, DM (NATURE PUBLISHING GROUP, 2012-05)
    We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.
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    Selective STAT3-α or -β expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes
    Ng, IHW ; Ng, DCH ; Jans, DA ; Bogoyevitch, MA (PORTLAND PRESS LTD, 2012-10-01)
    Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3β spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3β at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3β following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3β enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3β R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3β, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3β-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3β as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation.
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    WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression
    Bogoyevitch, MA ; Yeap, YYC ; Qu, Z ; Ngoei, KR ; Yip, YY ; Zhao, TT ; Heng, JI ; Ng, DCH (COMPANY BIOLOGISTS LTD, 2012-11-01)
    The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.
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    Selective STAT3-α or -β expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes
    Ng, Ivan H. W. ; Ng, Dominic C. H. ; Jans, David A. ; Bogoyevitch, Marie A. (Portland Press, 2012)