Partial inhibition of Cdk1 in G₂phase overrides the SAC and decouples mitotic events

Abstract

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G2 phase onwards. Addition of low doses of RO3306 in G2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

Deformability of Human Mesenchymal Stem Cells Is Dependent on Vimentin Intermediate Filaments

“…Notably, vimentin has also been shown to be downregulated in MSCs 17 of osteoarthritic patients 27 , which raises questions about the potential efficacy of 18 autologous stem cell therapies for treatment of OA. 19 20 The role of vimentin is still being examined using a variety of techniques to decrease, 21 disrupt, or collapse vimentin IFs, but it is clear that they are involved in modulating the 22 mechanical properties of cells. In fibroblasts, mutations resulting in vimentin deficiency 23 have been linked to not only impaired migration, but also reduction of mechanical 1 stability and stiffness of the cytoplasm 13,34 .…”

Interferon‐alpha‐induced hepatitis C virus clearance restores p53 tumor suppressor more than direct‐acting antivirals

“…The immunostaining and immunofluorescence images were quantified using a computer image analysis software package (ImageJ; National Institutes of Health, Bethesda, MD) 12, 13. All measurements were made at least in triplicate (n = 3).…”

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits

“…The formula used was CTCF D Integrated Density -(Area of selected cell X mean fluorescence of background reading), as described in the literature. 38 …”