Elucidating novel regulators of cytokinesis

Cytokinesis is the final event of cell division in which the mother cell splits into two daughter cells. During cytokinesis, the contractile ring is carefully positioned between the separating chromosomes by anaphase spindle. While the spindle midzone, located between segregating chromosomes, promotes the accumulation of contractile ring components at the equator, the centrosomal microtubule asters prevent the accumulation of contractile ring proteins at the cell poles. Despite rigorous research, the identity of aster derived inhibitory molecules(s) remains elusive and how cytokinesis regulators like Ect2 and RhoA are activated in a narrow equatorial zone is not properly understood. To identify novel regulators of this signalling pathway, high-throughput RNAi screen was performed in HeLa cells and cortical localization of contractile ring component GFP-tagged anillin was analyzed manually. In total, 7553 genes comprising druggable human genome were screened and 18 new genes were identified to play a role in regulating anillin localization at cell poles or equator. Among the 18 new candidate genes, Protein Kinase N2 (PKN2) and Septin 7 were the two most exciting candidates as they directly interact with RhoA and anillin, respectively and were further characterized. PKN2, a known RhoA effector, inhibited anillin localization on the cell poles whereas contractile ring component Septin 7 promoted anillin localization at the cell equator. Remarkably, the role of PKN2 and Septin7 in regulating anillin location during anaphase was found to be conserved in C. elegans one-cell embryos. It was previously shown that TPXL-1 mediated activation of Aurora A during anaphase is required for clearing anillin from the anterior pole in C. elegans one-cell embryos. To investigate whether Aurora A plays a similar role in clearing other contractile ring proteins from the anterior pole, localization of f-actin was analysed in TPXL-1 depleted embryos using a f-actin binding probe LifeAct fused to mKate2. Similar to anillin, TPXL-1 was found to be involved in clearing f-actin from the anterior pole during anaphase and the clearing defect was confirmed not to be a consequence of altered microtubule dynamics. Moreover, ectopic localization of Aurora A at the cell cortex induced by inhibiting PP6, a phosphatase that negatively regulates Aurora A activation, led to a significant reduction in anillin localization at cell equator and poles. Consistent with the observations in C. elegans, inhibition of Aurora A in HeLa cells by small molecular inhibitor MK-5108 resulted in increased accumulation of anillin on the polar cortex and a wider anillin zone at cell equator. Based on these findings, it is proposed that TPXL-1 activates Aurora A on the microtubule asters which diffuses to the adjacent cell poles and inhibits localization of contractile ring proteins. Finally, a rapamycin-inducible dimerization system was established in C. elegans using FRB and FKBP-12 domains of mTOR signaling pathways. In future, this protein dimerization tool can be used to target Aurora A and TPXL-1 to the plasma membrane and determine whether ectopic localization of TPXL-1 and Aurora A, without inhibiting PP6, can result in anillin localization defects. In summary, high-throughput RNAi screen revealed 18 new regulators of cytokinesis, two of which (Sept7 and PKN2) were further validated in HeLa cells and C. elegans one-cell embryos. In addition, Aurora A was shown to restrict the localization of contractile ring proteins to the cell equators in both the model systems

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions

Simultaneous occurrence of chronic myeloid leukemia and chronic lymphocytic leukemia: Report of an unusual case

The coexistence of chronic myeloid leukaemia(CML) and chronic lymphocytic leukemia(CLL) has been reported occasionally in literature, with only seven cases of simultaneous occurrence of these two diseases. We present here a case of 57 yr male patient where a complete blood count and differential done using volume conductivity scatter (VCS) technology suggested a diagnosis of CML with CLL. It was further confirmed by immunophneotyping and cytogenetic analysis. The patient was started on tyrosine kinase inhibitor, 400 mg once daily. Four months after the treatment, patient is doing fine with a count of 22 × 10 9 /L and 64% lymphocytes