Fast cell membrane displacements in B lymphocytes Modulation by dihydrocytochalasin B and colchicine

AbstractA novel type of cell membrane movement was characterized in B lymphocytes. Local submicron cell membrane displacements, within the frequency range 0.3–15 Hz, were registered in a murine lymphoma B cell line by a novel optical method based on point dark field microscopy. The cell membrane displacements were measured by monitoring changes in light scattering from very small illuminated areas (0.25 μm2) at the edge of the cell surface. B lymphocytes manifest a relative change in light scattering of 7.7 ± 1.3% (mean ± SD) which corresponds to cell membrane transverse displacement of 131 ± 22 nm. The confinement of cell membrane displacements to microdomains (≤0.2 μm2) emerged from the observed dependence of the displacement amplitude on the area size from which it is monitored. Colchicine (1 μM) decreased membrane fluctuations down to a value of 88 ± 14 nm, whereas dihydrocytochalasin B (2 μM) increased the amplitude of membrane displacements up to 184 ± 31 nm. These findings demonstrate the existence of a dynamic mechanical interaction between the cytoskeleton and the cell membrane in the frequency range of 0.3–15 Hz. The modulation of these interactions by the disruption of microfilaments or microtubules is explained in terms of the induced strain changes imposed on the cell membrane

Condensin I recruitment and uneven chromatin condensation precede mitotic cell death in response to DNA damage

Mitotic cell death (MCD) is a prominent but poorly defined form of death that stems from aberrant mitosis. One of the early steps in MCD is premature mitosis and uneven chromatin condensation (UCC). The mechanism underlying this phenomenon is currently unknown. In this study, we show that DNA damage in cells with a compromised p53-mediated G2/M checkpoint triggers the unscheduled activation of cyclin-dependent kinase 1 (Cdk1), activation and chromatin loading of the condensin I complex, and UCC followed by the appearance of multimicronucleated cells, which is evidence of MCD. We demonstrate that these processes engage some of the players of normal mitotic chromatin packaging but not those that drive the apoptotic chromatin condensation. Our findings establish a link between the induction of DNA damage and mitotic abnormalities (UCC) through the unscheduled activation of Cdk1 and recruitment of condensin I. These results demonstrate a clear distinction between the mechanisms that drive MCD-associated and apoptosis-related chromatin condensation and provide mechanistic insights and new readouts for a major cell death process in treated tumors

Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD