Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia.

Background: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.

Using an ImageJ-based script to detect replication stress and associated cell cycle exit from G2 phase by fluorescence microscopy.

Replication stress risks genomic integrity. Depending on the level, replication stress can lead to slower progression through S phase and entry into G2 phase with DNA damage. In G2 phase, cells either recover and eventually enter mitosis or permanently withdraw from the cell cycle.

Spatial organization-dependent EphA2 transcriptional responses revealed by ligand nanocalipers.

Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated.

Neutralization of the Positive Charges on Histone Tails by RNA Promotes an Open Chromatin Structure.

RNA associates extensively with chromatin and can influence its structure; however, the potential role of the negative charges of RNA on chromatin structure remains unknown. Here, we demonstrate that RNA prevents precipitation of histones and can attenuate electrostatic interactions between histones and DNA, thereby loosening up the chromatin structure. This effect is independent of the sequence of RNA but dependent on its single-stranded nature, length, concentration, and negative charge.

DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation.

ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration.

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle.

Cell Cycle Dynamics of Proteins and Post-translational Modifications Using Quantitative Immunofluorescence.

Immunofluorescence can be a powerful tool to detect protein levels, intracellular localization, and post-translational modifications. However, standard immunofluorescence provides only a still picture and thus lacks temporal information. Here, we describe a method to extract temporal information from immunofluorescence images of fixed cells.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via insulin-like growth factor-1 receptor-independent mechanism.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest.

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition.

During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear.

Translocation of surface-localized effectors in type III secretion.

Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells.

Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors.

One important feature of Yersinia pseudotuberculosis that enables resistance against the host immune defence is delivery of the antiphagocytic effectors YopH and YopE into phagocytic cells. The tyrosine phosphatase YopH influences integrin signalling, and YopE impairs cytoskeletal dynamics by inactivating Rho GTPases. Here, we report the impact of these effectors on internalization by dendritic cells (DCs), which internalize antigens to orchestrate host immune responses.

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress.

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.