Simultaneous quantification of actin monomer and filament dynamics with modelling assisted analysis of photoactivation

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments

Using microscopy to manipulate and visualize signal transduction in living cells

Signaling events in cells are often localized and transient. Understanding how these pathways are regulated in space and time requires the development of new tools that reveal spatiotemporal transduction. Subcellular events can be visualized in real-time by high-resolution light microscopy. Technological advances in live-cell imaging have offered the means to not only observe the phenotypic consequences of signaling events, but to visualize and manipulate the activity of their components. This dissertation describes four studies in which microscopy is implemented to manipulate or visualize signal transduction in living cells. The first study demonstrates contributions to Chromophore Assisted Laser Inactivation, a light-mediated loss of function tool. The second study describes the generation of a new probe to visualize the activation of Src-family kinases. The third study utilizes a biosensor for the GTPase RhoA to reveal novel information about how this signaling component is spatiotemporally regulated in neurons. Finally, the fourth study describes a new computational method for the automated identification and tracking of protein structures called focal adhesions. Together these studies demonstrate the power of using microscopy to gain key insights to the spatiotemporal details of signal transduction

Examination of the Sanctuary: Uses of a Meditative Space in a Women's Health Clinic

Abortion, much like pregnancy, can play a significant role in the course of a woman's life. Even so, abortion is complicated by a number of barriers - legal, financial, emotional, spiritual, religious, and physical -- with which women and men struggle beyond the mere complexity of undergoing a surgical procedure. Although abortion can be pivotal in a woman's life, every individual is remarkably different, and for that reason so is every abortion experience. For over 30 years, Allegheny Reproductive Health Center (ARHC) has embraced a holistic approach that seeks to understand and address the needs of women and men seeking termination services. In 2008, ARHC erected a space, the Sanctuary, designed specifically to encourage personal reflection and quiet meditation among patients and abortion providers coping with the complex issues surrounding abortion. Thus, in order to understand the impact of the Sanctuary, this exploratory study investigates the various uses of the meditative space. Qualitative research was conducted through the use of semi-structured interviews with clinic workers and by asking visitors to the meditative space to respond to a short questionnaire, which upon completion was placed in a secure box installed within the Sanctuary. From the results, four overarching themes were identified: 1. To work within the abortion field is both complex and important; 2. The next step in abortion care is a holistic approach that addresses a patient's specific needs; 3. The development of the Sanctuary is a step toward advancing abortion care for women and men; and 4. To establish an understanding of how the space is used by patients and clinic workers. These results imply that applying a holistic approach in abortion care, through the use of a space such as the Sanctuary, is respectful of the varability in women's and men's experiences related to pregnancy termination. Moreover, the findings indicate that participants support the placement and use of the Sanctuary, for themseleves as well as patients. In terms of public health relevance, professionals within the field can utilize this research to advance the understanding of how to assist women and men in managing the complexities of abortion

Chromosome Fragmentation after Induction of a Double-Strand Break Is an Active Process Prevented by the RMX Repair Complex

Chromosome aberrations are common outcomes of exposure to DNA-damaging agents or altered replication events and are associated with various diseases and a variety of carcinomas, including leukemias, lymphomas, sarcomas, and epithelial tumors 1 and 2. The incidence of aberrations can be greatly increased as a result of defects in DNA repair pathways [3]. Although there is considerable information about the molecular events associated with the induction and repair of a double-strand break (DSB), little is known about the events that ultimately lead to translocations or deletions through the formation of chromosome breaks or the dissociation of broken ends. We describe a system for visualizing DNA ends at the site of a DSB in living cells. After induction of the break, DNA ends flanking the DSB site in wild-type cells remained adjacent. Loss of a functional RMX complex (Rad50/Mre11/Xrs2) or a mutation in the Rad50 Zn-hook structure resulted in DNA ends being dispersed in approximately 10%–20% of cells. Thus, the RMX complex holds broken ends together and counteracts mitotic spindle forces that can be destructive to damaged chromosomes

Chromophore-assisted laser inactivation in cell biology

Chromophore-assisted laser inactivation (CALI) is a technique whereby engineered proteins and dye molecules that produce substantial amounts of reactive oxygen species upon absorption of light are used to perturb biological systems in a spatially and temporally defined manner. CALI is an important complement to conventional genetic and pharmacological manipulations. In this review, we examine the applications of CALI to cell biology and discuss the underlying photochemical mechanisms that mediate this powerful technique

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics

To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model FRAP and chromophore assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (~0.1 s−1) than that measured in vitro (0.01–0.0004 s−1). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anti-capping ability. We included the VASP protein as the anti-capping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs

Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia

Chromophore-assisted laser inactivation (CALI) was developed to instantly and specifically inactivate cofilin in cells. Simultaneous CALI and live imaging revealed that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow in lamellipodia.Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow

CellGeo: A computational platform for the analysis of shape changes in cells with complex geometries

The open source MATLAB application CellGeo is a user-friendly computational platform that allows simultaneous, automated tracking and analysis of dynamic changes in cell shape, including protrusions ranging from filopodia to lamellipodia to growth cones.Cell biologists increasingly rely on computer-aided image analysis, allowing them to collect precise, unbiased quantitative results. However, despite great progress in image processing and computer vision, current computational approaches fail to address many key aspects of cell behavior, including the cell protrusions that guide cell migration and drive morphogenesis. We developed the open source MATLAB application CellGeo, a user-friendly computational platform to allow simultaneous, automated tracking and analysis of dynamic changes in cell shape, including protrusions ranging from filopodia to lamellipodia. Our method maps an arbitrary cell shape onto a tree graph that, unlike traditional skeletonization algorithms, preserves complex boundary features. CellGeo allows rigorous but flexible definition and accurate automated detection and tracking of geometric features of interest. We demonstrate CellGeo’s utility by deriving new insights into (a) the roles of Diaphanous, Enabled, and Capping protein in regulating filopodia and lamellipodia dynamics in Drosophila melanogaster cells and (b) the dynamic properties of growth cones in catecholaminergic a–differentiated neuroblastoma cells

The efficacy of adding short-term psychodynamic psychotherapy to antidepressants in the treatment of depression:A systematic review and meta-analysis of individual participant data

Contains fulltext : 220185.pdf (Publisher’s version ) (Closed access)Purpose: We examined the efficacy of adding short-term psychodynamic psychotherapy (STPP) to antidepressants in the treatment of depression by means of a systematic review and meta-analysis of individual participant data, which is currently considered the most reliable method for evidence synthesis. Results: A thorough systematic literature search resulted in 7 studies comparing combined treatment of antidepressants and STPP versus antidepressant mono-therapy (n = 3) or versus antidepressants and brief supportive psychotherapy (n = 4). Individual participant data were obtained for all these studies and totaled 482 participants. Across the total sample of studies, combined treatment of antidepressants and STPP was found significantly more efficacious in terms of depressive symptom levels at both post-treatment (Cohen's d = 0.26, SE = 0.10, p = .01) and follow-up (d = 0.50, SE = 0.10, p < .001). This effect was most apparent at follow-up and in studies examining STPP's specific treatment efficacy. Effects were still apparent in analyses that controlled for risk of bias and STPP quality in the primary studies. Conclusions: These findings support the evidence-base of adding STPP to antidepressants in the treatment of depression. However, further studies are needed, particularly assessing outcome measures other than depression and cost-effectiveness, as well as examining the relative merits of STPP versus other psychotherapies as added to antidepressants.10 p

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics