FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity.

To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in Dictyostelium and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.

FLN-2 functions in parallel to LINC complexes and Cdc42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and pro-inflammatory responses. Studies performed in tissue culture cells have implicated LINC (linker of nucleoskeleton and cytoskeleton) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In C. elegans larvae, 6 pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function, this and structural predictions suggest that FLN-2 is not a divergent filamin. The immunoglobulin (Ig)-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.

In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific to Ras, Rac or Rho small GTPases of the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components of signal transduction networks which link upstream G-protein coupled receptors to downstream cytoskeletal components and help cells migrate through their dynamic microenvironment. Conventional approaches to perturb and examine these signaling and cytoskeletal networks, such as gene knockout or overexpression, are protracted which allows networks to readjust through gene expression changes. Moreover, these tools lack spatial resolution to probe the effects of local network activations. To overcome these challenges, blue light-inducible cryptochrome- and LOV domain-based dimerization systems have been recently developed to control signaling or cytoskeletal events in a spatiotemporally precise manner. We illustrate that, within minutes of global membrane recruitment of full-length GEFs or their catalytic domains only, widespread increases or decreases in F-actin rich protrusions and cell size occur, depending on the particular node in the networks targeted. Additionally, we demonstrate localized GEF recruitment as a robust assay system to study local network activation-driven changes in polarity and directed migration. Altogether, these optical tools confirmed GEFs of Ras superfamily GTPases as regulators of cell shape, actin dynamics, and polarity. Furthermore, this optogenetic toolbox may be exploited in perturbing complex signaling interactions in varied physiological contexts including mammalian embryogenesis.

[Exploring the Interplay between communication of the Gospel und popular religion. A case study on the church-launched YouTube channel "Jana believes"]. / Kommunikation des Evangeliums und Populäre Religion. Annäherung an ein Spannungsverhältnis am Beispiel des YouTube-Kanals "Jana glaubt".

The relationship between religion and popular culture has attracted considerable attention in the field of the sociology of religion. However, church-based religious communication has rarely been explored in this perspective. This in mind, the paper explores the YouTube channel "Jana believes", a pioneering and also controversial project sponsored by the Evangelical Church in Germany.The argumentation proceeds in five steps. First, Hubert Knoblauch's concept of "popular religion" is interlinked with the practical-theological perspective of "communicating the Gospel" as well as with more recent approaches to the mediatization of religion. Then characteristic elements and video genres of YouTube communication as part of popular culture are explicated. Against this background, the distinct profile of the YouTube channel "Jana believes" is outlined, with specific regard to the vlog genre. Subsequently, the interplay between popular religion, communicating the Gospel and mediatization of religion is explored in a more in depth-analysis of two exemplary video sequences.Finally, the summary brings together both sides of the interplay in question: On the one hand, the channel videos are designed according to established standards of personalized YouTube communication. In line with the concept of popular religion, the boundaries between private and public become fluid. Public communication of the Gospel is constituted by sharing what is private in life and faith. On the other hand, in the case of this channel the subjective enactment of faith is also shaped and transformed by the involvement of the church. Particularly regarding questions of authenticity and authority, the tension between popular religion and communicating the Gospel at times leads to potentially conflictual negotiation processes.

Using Live-Cell Imaging and Synthetic Biology to Probe Directed Migration in Dictyostelium.

For decades, the social amoeba Dictyostelium discoideum has been an invaluable tool for dissecting the biology of eukaryotic cells. Its short growth cycle and genetic tractability make it ideal for a variety of biochemical, cell biological, and biophysical assays. Dictyostelium have been widely used as a model of eukaryotic cell motility because the signaling and mechanical networks which they use to steer and produce forward motion are highly conserved. Because these migration networks consist of hundreds of interconnected proteins, perturbing individual molecules can have subtle effects or alter cell morphology and signaling in major unpredictable ways. Therefore, to fully understand this network, we must be able to quantitatively assess the consequences of abrupt modifications. This ability will allow us better control cell migration, which is critical for development and disease, in vivo. Here, we review recent advances in imaging, synthetic biology, and computational analysis which enable researchers to tune the activity of individual molecules in single living cells and precisely measure the effects on cellular motility and signaling. We also provide practical advice and resources to assist in applying these approaches in Dictyostelium.

Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner.

The spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. Each mammalian kinetochore binds many microtubules, but how many attached microtubules are required to turn off the checkpoint, and how the kinetochore monitors microtubule numbers, are not known and are central to understanding SAC mechanisms and function. To address these questions, here we systematically tune and fix the fraction of Hec1 molecules capable of microtubule binding. We show that Hec1 molecules independently bind microtubules within single kinetochores, but that the kinetochore does not independently process attachment information from different molecules. Few attached microtubules (20% occupancy) can trigger complete Mad1 loss, and Mad1 loss is slower in this case. Finally, we show using laser ablation that individual kinetochores detect changes in microtubule binding, not in spindle forces that accompany attachment. Thus, the mammalian kinetochore responds specifically to the binding of each microtubule and counts microtubules as a single unit in a sensitive and switch-like manner. This may allow kinetochores to rapidly react to early attachments and maintain a robust SAC response despite dynamic microtubule numbers.

The mammalian kinetochore-microtubule interface: robust mechanics and computation with many microtubules.

The kinetochore drives chromosome segregation at cell division. It acts as a physical link between chromosomes and dynamic microtubules, and as a signaling hub detecting and processing microtubule attachments to control anaphase onset. The mammalian kinetochore is a large macromolecular machine that forms a dynamic interface with the many microtubules that it binds. While we know most of the kinetochore's component parts, how they work together to give rise to its robust functions remains poorly understood. Here we highlight recent findings that shed light on this question, driven by an expanding physical and molecular toolkit. We present emerging principles that underlie the kinetochore's robust microtubule grip, such as redundancy, specialization, and dynamicity, and present signal processing principles that connect this microtubule grip to robust computation. Throughout, we identify open questions, and define simple engineering concepts that provide insight into kinetochore function.

Cyclin B2 is required for progression through meiosis in mouse oocytes.

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance.

Curriculum Evaluation Using Path Analysis.

The purpose of this study was to explore the application of path analysis to evaluate the curriculum model and provide guidance in course sequencing. Using statistical package R to add and subtract various path connections, the curriculum model was improved to a proposed curriculum model, which passed the exact-fit test (χ42 = 45.612, with P = .286 > .05). Path analysis provided an objective method to evaluate the curriculum model and course sequencing in the baccalaureate program under study, while informing possible placement of courses.

Spindle assembly checkpoint satisfaction occurs via end-on but not lateral attachments under tension.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. What signals the SAC monitors remains unclear. We do not know the contributions of different microtubule attachment features or tension from biorientation to SAC satisfaction nor how these possible cues change during attachment. In this study, we quantify concurrent Mad1 intensity and report on SAC silencing, real-time attachment geometry, occupancy, and tension at individual mammalian kinetochores. We show that Mad1 loss from the kinetochore is switch-like with robust kinetics and that tension across sister kinetochores is established just before Mad1 loss events at the first sister. We demonstrate that CenpE-mediated lateral attachment of the second sister can persistently generate this metaphase-like tension before biorientation, likely stabilizing sister end-on attachment, yet cannot induce Mad1 loss from that kinetochore. Instead, Mad1 loss begins after several end-on microtubules attach. Thus, end-on attachment provides geometry-specific molecular cues or force on specific kinetochore linkages that other attachment geometries cannot provide.

Imaging and physically probing kinetochores in live dividing cells.

The kinetochore mediates chromosome segregation at cell division. It is the macromolecular machine that links chromosomes to spindle microtubules, and is made of more than 100 protein species in mammalian cells. Molecular tools are presently revealing the biochemical interactions and regulatory mechanisms that ensure proper kinetochore function. Here, we discuss two approaches for imaging and physically probing kinetochores despite mitotic cell rounding and rapid kinetochore dynamics. First, we describe how mild spindle compression can improve kinetochore imaging and how stronger compression can mechanically perturb the spindle and kinetochores. Second, we describe how simultaneously imaging two-colored kinetochore reporter probes at subpixel resolution can report on kinetochore structural dynamics under cellular forces. We hope that the experimental details we provide here will make these two approaches broadly accessible and help move forward our understanding of kinetochore function--and make these approaches adaptable to the study of other cellular structures.

Early Indicators of NCLEX-RN performance.

Health Education Systems Incorporated (HESI(™)) test results, course grades, and National Council Licensure Examination-Registered Nurse (NCLEX-RN(®)) outcomes of students in an associate degree nursing program at a midwestern public university were investigated. Statistical analysis revealed that introductory Fundamentals HESI test scores, more than either comprehensive HESI Exit Exam scores or other specialty HESI test scores, significantly predicted NCLEX-RN outcomes in this study (p < 0.05), while controlling for grade point average and high school percentile rank. In addition, of the general education courses and the nursing courses in the associate nursing program examined, Pediatric Nursing, Medical-Surgical Nursing, and Maternity Nursing course grades were found most statistically significantly influential of all the HESI test scores (p < 0.01).

toca-1 is in a novel pathway that functions in parallel with a SUN-KASH nuclear envelope bridge to move nuclei in Caenorhabditis elegans.

Moving the nucleus to an intracellular location is critical to many fundamental cell and developmental processes, including cell migration, differentiation, fertilization, and establishment of cellular polarity. Bridges of SUN and KASH proteins span the nuclear envelope and mediate many nuclear positioning events, but other pathways function independently through poorly characterized mechanisms. To identify and characterize novel mechanisms of nuclear migration, we conducted a nonbiased forward genetic screen for mutations that enhanced the nuclear migration defect of unc-84, which encodes a SUN protein. In Caenorhabditis elegans larvae, failure of hypodermal P-cell nuclear migration results in uncoordinated and egg-laying-defective animals. The process of P-cell nuclear migration in unc-84 null animals is temperature sensitive; at 25° migration fails in unc-84 mutants, but at 15° the migration occurs normally. We hypothesized that an additional pathway functions in parallel to the unc-84 pathway to move P-cell nuclei at 15°. In support of our hypothesis, forward genetic screens isolated eight emu (enhancer of the nuclear migration defect of unc-84) mutations that disrupt nuclear migration only in a null unc-84 background. The yc20 mutant was determined to carry a mutation in the toca-1 gene. TOCA-1 functions to move P-cell nuclei in a cell-autonomous manner. TOCA-1 is conserved in humans, where it functions to nucleate and organize actin during endocytosis. Therefore, we have uncovered a player in a previously unknown, likely actin-dependent, pathway that functions to move nuclei in parallel to SUN-KASH bridges. The other emu mutations potentially represent other components of this novel pathway.

Pretest online discussion groups to augment teaching and learning.

Tests and final examination scores of three semesters of control students in a nursing foundation course were compared with tests and final examination scores of three semesters of participating students. Participating students were offered access to an asynchronous pretest online discussion activity with a faculty e-moderator. While the simplified Bloom's revised taxonomy assisted in creating appropriate preparatory test and final examination questions for pretest online discussion, Salmon's five-stage online method provided direction to the e-moderator on how to encourage students to achieve Bloom's higher-order thinking skills during the pretest online discussions. Statistical analysis showed the pretest online discussion activity had a generally positive impact on tests and final examination scores, when controlling for a number of possible confounding variables, including instructor, cumulative grade point average, age, and credit hours.

The tryptophan pathway genes of the Sargasso Sea metagenome: new operon structures and the prevalence of non-operon organization.

BACKGROUND: The enormous database of microbial DNA generated from the Sargasso Sea metagenome provides a unique opportunity to locate genes participating in different biosynthetic pathways and to attempt to understand the relationship and evolution of those genes. In this article, an analysis of the Sargasso Sea metagenome is made with respect to the seven genes of the tryptophan pathway. RESULTS: At least 5% of all the genes that are related to amino acid biosynthesis are tryptophan (trp) genes. Many contigs and scaffolds contain whole or split operons that are similar to previously analyzed trp gene organizations. Only two scaffolds discovered in this analysis possess a different operon organization of tryptophan pathway genes than those previously known. Many marine organisms lack an operon-type organization of these genes or have mini-operons containing only two trp genes. In addition, the trpB genes from this search reveal that the dichotomous division between trpB_1 and trpB_2 also occurs in organisms from the Sargasso Sea. One cluster was found to contain trpB sequences that were closely related to each other but distinct from most known trpB sequences. CONCLUSION: The data show that trp genes are widely dispersed within this metagenome. The novel organization of these genes and an unusual group of trpB_1 sequences that were found among some of these Sargasso Sea bacteria indicate that there is much to be discovered about both the reason for certain gene orders and the regulation of tryptophan biosynthesis in marine bacteria.

Twin pair resemblance for psychiatric hospitalization in the Swedish Twin Registry: a 32-year follow-up study of 29,602 twin pairs.

BACKGROUND: Allgulander et al. (Allgulander C, Nowak J, Rice JP (1991) Acta Psychiatr Scand 83, 12) published twin pair analyses of psychiatric hospitalization for like-sex pairs from the Swedish Twin Registry born 1926-1958. As noted in a subsequent letter (Allgulander C, Nowak J, Rice JP (1992) Acta Psychiatr Scand 86, 421), several features of the original study resulted in under-ascertainment of cases and underestimated heritability, particularly for alcoholism. The present report updates the prior results by using 17 additional years of follow-up, including members of opposite-sex twin pairs, and addressing biases arising from cohort effects and from excluding pairs with unknown zygosity. METHODS: Registry records for 29,602 twin pairs born 1926-1958 were matched against national databases of psychiatric and medical hospitalizations from 1972-2000 to obtain ICD diagnostic codes. Zygosity was known for 10,903 opposite-sex pairs and 15,401 like-sex pairs who participated previously in research. Twin-pair resemblance and genetic and environmental variance proportions were estimated for hospitalization for alcoholism, affective disorders, psychosis, and (in females) anxiety disorders. RESULTS: Hospitalization rates during the ascertainment window were: alcoholism: males = 3.67%, females = 0.94%; affective disorders: males = 1.99%, females = 2.75%; anxiety disorders: males = 0.46%, females = 0.74%; and psychotic disorders: males = 1.70%, females = 1.96%. Twins from like-sex pairs with unknown zygosity had significantly higher prevalences than those with known zygosity. Tetrachoric correlations and heritability estimates were affected by the method used to model unknown zygosity and cohort effects. CONCLUSIONS: Inclusion of additional follow-up information, opposite-sex twin pairs, age-adjustment, and use of current ICD definitions yielded higher heritability estimates for alcoholism, anxiety disorders, and psychosis than previously published for this nationally-representative sample of twins from Sweden. The results show that relatively small selection biases can alter twin study results and underscore the importance of addressing under-ascertainment of cases in genetic research based on volunteers.

Detection of Salmonella by bacteriophage Felix 01.

Salmonellae are mammalian pathogens that are transmitted mainly through foodstuffs and their handlers. Rapid detection requires both specificity and sensitivity in samples containing other bacteria. A solution to this problem is the use of the great specificity conferred by bacteriophages. After implanting reporter genes in a phage genome, the reporter gene products can be measured with great sensitivity when a bacterial host is present. Bacteriophage Felix 01 infects almost all Salmonella strains and has been manipulated to contain the lux genes specifying bacterial luciferase, an enzyme that converts chemical energy to visible light. A widely applicable methodology for preventing the escape of such recombinant phage has also been developed.

Visualization of active biomass distribution in a BGAC fluidized bed reactor using GFP tagged Pseudomonas putida F1.

A favorable microenvironment for biofilm growth on GAC particles was shown using green fluorescent protein (GFP) as a marker for a phenol degrading bacterium, Pseudomonas putida F1. The dispersion of P. putida F1 in a biofilm covering granulated activated carbon (GAC) particles was monitored and compared to a biofilm on non-activated granular carbon particles. Laser scanning confocal microscopy (LSCM) micrographs of the biofilms taken from two fluidized bed reactors operating under identical conditions, showed higher fluorescent green areas in the GAC biofilm, especially close to the GAC surface. Quantitative analysis of the biofilm by COMSTAT, a three-dimensional biofilm structure analysis program, showed higher biomass concentration and higher viability in the GAC covered biofilm vs. the non-activated carbon biofilm. In addition, better effluent quality was measured for the BGAC reactor, which strongly suggests a significantly larger biofilm surface area available to the substrate, as opposed to that of the non-activated carbon carrier reactor.