Myosin II contributes to cell-scale actin network treadmilling through network disassembly.

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton: protrusion of the leading edge requires assembly of a network of actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

Analysis of microtubule dynamic instability using a plus-end growth marker.

Regulation of microtubule dynamics is essential for many cell biological processes and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracked all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We introduce spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. We validated the algorithm by comparing the results to data for manually tracked, homogeneously labeled microtubules and by analyzing the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.

plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.

Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in Jaqaman et al. (2008). Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper, we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (Matov et al., 2010). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in Matov et al. (2010).

Examining the DSM-5 alternative personality disorder model operationalization of antisocial personality disorder and psychopathy in a male correctional sample.

For decades, it has been known that the Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnosis of Antisocial Personality Disorder (ASPD) is a nonadequate operationalization of psychopathy (Crego & Widiger, 2015). The DSM-5 alternative model of personality disorders provides an opportunity to rectify some of these long held concerns. The current study compared the Section III alternative model's trait-based conception of ASPD with the categorical model from the main diagnostic codes section of DSM-5 in terms of associations with differing models of psychopathy. We also evaluated the validity of the trait-based conception more broadly in relation to measures of antisocial tendencies as well as psychopathy. Participants were 200 male inmates who were administered a battery of self-report and interview-based researcher rating measures of relevant constructs. Analyses showed that Section III ASPD outperformed Section II ASPD in predicting scores on Hare's (2003) Psychopathy Checklist-Revised (PCL-R; r = .88 vs. .59). Additionally, aggregate scores for Section III ASPD performed well in capturing variance in differing ASPD and psychopathy measures. Finally, we found that the Section III ASPD impairment criteria added incrementally to the Section III ASPD traits in predicting PCL-R psychopathy and SCID-II ASPD. (PsycINFO Database Record

Can brief measures effectively screen for pain and somatic malingering? Examination of the Modified Somatic Perception Questionnaire and Pain Disability Index.

BACKGROUND CONTEXT: Recent rise in fraudulent disability claims in the United States has resulted in psychologists being increasingly called upon to use psychological tests to determine whether disability claims based on psychological or somatic/pain complaints are legitimate. PURPOSE: To examine two brief measures, Modified Somatic Perception Questionnaire (MSPQ) and the Pain Disability Index (PDI), and their ability to screen for malingering in relation to the Bianchini et al. criteria for malingered pain-related disability published in The Spine Journal (2005). STUDY DESIGN: Examined brief self-report measures between litigating and nonlitigating pain samples. PATIENT SAMPLE: We compared 144 disability litigants, predominantly presenting a history of musculoskeletal injuries with psychiatric overlay, with 167 nonlitigating pain patients who were predominantly in treatment for chronic back pain issues and other musculoskeletal conditions. OUTCOME MEASURES: Modified Somatic Perception Questionnaire, Pain Disability Index, Minnesota Multiphasic Personality Inventory-2 Restructured Form, Test of Memory Malingering, Letter Memory Test, Victoria Symptom Validity Test, Structured Interview of Reported Symptoms-second edition, Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders somatoform disorders module. METHODS: We examined a sample of 144 individuals undergoing compensation-seeking evaluations in relation to 167 nonlitigating pain patients. RESULTS: Group differences on both the MSPQ and PDI were calculated, as well as sensitivities, specificities, and positive and negative predictive powers for both measures at selected cutoffs. CONCLUSIONS: The results suggest that both the MSPQ and PDI are useful to screen for pain malingering in forensic evaluations, especially the MSPQ, which performed the best in differentiating between the groups.

Tumor suppressor NF2/Merlin is a microtubule stabilizer.

Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end.

Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.

Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.

Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis.

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end-tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II-mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.

Differential transmission of actin motion within focal adhesions.

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.