Pulsed Feedback Defers Cellular Differentiation

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle

Immunologically reactive M. leprae antigens with relevance to diagnosis and vaccine development

<p>Abstract</p> <p>Background</p> <p>Leprosy is a chronic infectious disease caused by <it>Mycobacterium leprae </it>that can manifest a wide variety of immunological and clinical outcomes ranging from potent humoral responses among borderline lepromatous (BL) and lepromatous (LL) patients to strong cellular responses among tuberculoid (TT) and borderline tuberculoid (BT) patients. Until recently, relatively little has been known about the immune responses to individual proteins of <it>M. leprae </it>recognized during leprosy.</p> <p>Methods</p> <p>The immune reactivity to a panel of 33 <it>M. leprae </it>recombinant proteins was evaluated among leprosy patients and controls from a high endemic area for leprosy (Goiania/GO, Central Brazil). Serum IgG responses were measured by ELISA (45 participants/group) and T cell responses (20 participants/group) were evaluated by IFN-gamma production in 24 hours whole blood cultures with antigen (whole blood assay-WBA). Study groups were newly diagnosed, untreated TT/BT and BL/LL leprosy patients classified by Ridley Jopling criteria and household contacts of BL/LL patients (HHC). Control groups were HIV-1 negative pulmonary tuberculosis patients (TB) and healthy individuals from the same endemic area (EC). In silico predictions indicated the level of identity of <it>M. leprae </it>proteins with homologues in other mycobacteria and the presence of T cell and B cell epitopes.</p> <p>Results</p> <p>Despite the prediction that all proteins would be reactive, 16 of 33 (48%) of the single proteins tested were immunogenic (recognized in WBA or ELISA) and seventeen were non-immunogenic (not recognized in either assay). Among the 16 immunogenic proteins, 9 were considered leprosy specific in WBA inducing cell-mediated IFN-gamma secretion from TT/BT patients and HHC. Three of these proteins were also leprosy specific in serology being recognized by serum IgG from LL/BL patients. Seven of the immunogenic proteins were not leprosy specific.</p> <p>Conclusions</p> <p>New <it>M. leprae </it>antigens recognized by antibody responses of BL/LL patients and cellular responses of TT/BT leprosy patients were identified. An improved serological diagnostic test for leprosy could be developed by incorporating these IgG-reactive antigens to the current PGL-I based tests. Moreover our data indicate that the WBA is a robust, relatively simple and user friendly format for a T cell based diagnostic test. The field use of these test formats in leprosy endemic countries could contribute to early leprosy diagnosis before the development of deformities and disabilities.</p

ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa

ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure “ReCLIP” (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes

Novel ATP-Independent RNA Annealing Activity of the Dengue Virus NS3 Helicase

The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or non-viral sequences. This new activity was found to be ATP-independent. It was determined that a mutated NS3 lacking ATPase activity retained full-RNA annealing activity. Using an ATP regeneration system and different ATP concentrations, we observed that NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. In addition, we observed that NS3 enhanced RNA-RNA interactions between molecules representing the ends of the viral genome that are known to be necessary for viral RNA synthesis. We propose that, according to the ATP availability, NS3 could function regulating the folding or unfolding of viral RNA structures

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Activity of the multikinase inhibitor dasatinib against ovarian cancer cells

BackgroundHere, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer.MethodsWe examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs.ResultsConcentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib.ConclusionsThese data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer

Selective Activation of p120ctn-Kaiso Signaling to Unlock Contact Inhibition of ARPE-19 Cells without Epithelial-Mesenchymal Transition

Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues