Estimation of free-living walking cadence from wrist-worn sensor accelerometry data and its association with SF-36 quality of life scores.

Objective. We evaluate the stride segmentation performance of the Adaptive Empirical Pattern Transformation (ADEPT) for subsecond-level accelerometry data collected in the free-living environment using a wrist-worn sensor.Approach. We substantially expand the scope of the existing ADEPT pattern-matching algorithm. Methods are applied to subsecond-level accelerometry data collected continuously for 4 weeks in 45 participants, including 30 arthritis and 15 control patients. We estimate the daily walking cadence for each participant and quantify its association with SF-36 quality of life measures.Main results. We provide free, open-source software to segment individual walking strides in subsecond-level accelerometry data. Walking cadence is significantly associated with the role physical score reported via SF-36 after adjusting for age, gender, weight and height.Significance. Methods provide automatic, precise walking stride segmentation, which allows estimation of walking cadence from free-living wrist-worn accelerometry data. Results provide new evidence of associations between free-living walking parameters and health outcomes.

A Thorough Examination of Morning Activity Patterns in Adults with Arthritis and Healthy Controls Using Actigraphy Data.

BACKGROUND: Wearable sensors allow researchers to remotely capture digital health data, including physical activity, which may identify digital biomarkers to differentiate healthy and clinical cohorts. To date, research has focused on high-level data (e.g., overall step counts) which may limit our insights to whether people move differently, rather than how they move differently. OBJECTIVE: This study therefore aimed to use actigraphy data to thoroughly examine activity patterns during the first hours following waking in arthritis patients (n = 45) and healthy controls (n = 30). METHODS: Participants wore an Actigraph GT9X Link for 28 days. Activity counts were analysed and compared over varying epochs, ranging from 15 min to 4 h, starting with waking in the morning. The sum, and a measure of rate of change of cumulative activity in the period immediately after waking (area under the curve [AUC]) for each time period, was calculated for each participant, each day, and individual and group means were calculated. Two-tailed independent t tests determined differences between the groups. RESULTS: No differences were seen for summed activity counts across any time period studied. However, differences were noted in the AUC analysis for the discrete measures of relative activity. Specifically, within the first 15, 30, 45, and 60 min following waking, the AUC for activity counts was significantly higher in arthritis patients compared to controls, particularly at the 30 min period (t = -4.24, p = 0.0002). Thus, while both cohorts moved the same amount, the way in which they moved was different. CONCLUSION: This study is the first to show that a detailed analysis of actigraphy variables could identify activity pattern changes associated with arthritis, where the high-level daily summaries did not. Results suggest discrete variables derived from raw data may be useful to help identify clinical cohorts and should be explored further to determine if they may be effective clinical biomarkers.

Comparing the Usability and Acceptability of Wearable Sensors Among Older Irish Adults in a Real-World Context: Observational Study.

BACKGROUND: Wearable devices are valuable assessment tools for patient outcomes in contexts such as clinical trials. To be successfully deployed, however, participants must be willing to wear them. Another concern is that usability studies are rarely published, often fail to test devices beyond 24 hours, and need to be repeated frequently to ensure that contemporary devices are assessed. OBJECTIVE: This study aimed to compare multiple wearable sensors in a real-world context to establish their usability within an older adult (>50 years) population. METHODS: Eight older adults wore seven devices for a minimum of 1 week each: Actigraph GT9x, Actibelt, Actiwatch, Biovotion, Hexoskin, Mc10 Biostamp_RC, and Wavelet. Usability was established through mixed methods using semistructured interviews and three questionnaires, namely, the Intrinsic Motivation Inventory (IMI), the System Usability Scale (SUS), and an acceptability questionnaire. Quantitative data were reported descriptively and qualitative data were analyzed using deductive content analysis. Data were then integrated using triangulation. RESULTS: Results demonstrated that no device was considered optimal as all scored below average in the SUS (median, IQR; min-max=57.5, 12.5; 47.5-63.8). Hexoskin was the lowest scored device based on the IMI (3.6; 3.4-4.5), while Biovotion, Actibelt, and Mc10 Biostamp_RC achieved the highest median results on the acceptability questionnaire (3.6 on a 6-point Likert scale). Qualitatively, participants were willing to accept less comfort, less device discretion, and high charging burdens if the devices were perceived as useful, namely through the provision of feedback for the user. Participants agreed that the purpose of use is a key enabler for long-term compliance. These views were particularly noted by those not currently wearing an activity-tracking device. Participants believed that wrist-worn sensors were the most versatile and easy to use, and therefore, the most suitable for long-term use. In particular, Actiwatch and Wavelet stood out for their comfort. The convergence of quantitative and qualitative data was demonstrated in the study. CONCLUSIONS: Based on the results, the following context-specific recommendations can be made: (1) researchers should consider their device selection in relation to both individual and environmental factors, and not simply the primary outcome of the research study; (2) if researchers do not wish their participants to have access to feedback from the devices, then a simple, wrist-worn device that acts as a watch is preferable; (3) if feedback is allowed, then it should be made available to help participants remain engaged; this is likely to apply only to people without cognitive impairments; (4) battery life of 1 week should be considered as a necessary feature to enhance data capture; (5) researchers should consider providing additional information about the purpose of devices to participants to support their continued use.

Autoencoder as a New Method for Maintaining Data Privacy While Analyzing Videos of Patients With Motor Dysfunction: Proof-of-Concept Study.

BACKGROUND: In chronic neurological diseases, especially in multiple sclerosis (MS), clinical assessment of motor dysfunction is crucial to monitor the disease in patients. Traditional scales are not sensitive enough to detect slight changes. Video recordings of patient performance are more accurate and increase the reliability of severity ratings. When these recordings are automated, quantitative disability assessments by machine learning algorithms can be created. Creation of these algorithms involves non-health care professionals, which is a challenge for maintaining data privacy. However, autoencoders can address this issue. OBJECTIVE: The aim of this proof-of-concept study was to test whether coded frame vectors of autoencoders contain relevant information for analyzing videos of the motor performance of patients with MS. METHODS: In this study, 20 pre-rated videos of patients performing the finger-to-nose test were recorded. An autoencoder created encoded frame vectors from the original videos and decoded the videos again. The original and decoded videos were shown to 10 neurologists at an academic MS center in Basel, Switzerland. The neurologists tested whether the 200 videos were human-readable after decoding and rated the severity grade of each original and decoded video according to the Neurostatus-Expanded Disability Status Scale definitions of limb ataxia. Furthermore, the neurologists tested whether ratings were equivalent between the original and decoded videos. RESULTS: In total, 172 of 200 (86.0%) videos were of sufficient quality to be ratable. The intrarater agreement between the original and decoded videos was 0.317 (Cohen weighted kappa). The average difference in the ratings between the original and decoded videos was 0.26, in which the original videos were rated as more severe. The interrater agreement between the original videos was 0.459 and that between the decoded videos was 0.302. The agreement was higher when no deficits or very severe deficits were present. CONCLUSIONS: The vast majority of videos (172/200, 86.0%) decoded by the autoencoder contained clinically relevant information and had fair intrarater agreement with the original videos. Autoencoders are a potential method for enabling the use of patient videos while preserving data privacy, especially when non-health-care professionals are involved.

Setwise comparison: efficient fine-grained rating of movement videos using algorithmic support - a proof of concept study.

Purpose: Clinical ordinal rating scales of movements, e.g., the Expanded Disability Status Scale, have poor intra- and interrater reliability, are insensitive to subtle differences and result in coarse-grained ratings compared to relative comparative rating methods. We therefore established video-based setwise comparison as a fine-grained, reliable and efficient rating method of motor dysfunction using algorithmic support.Materials and methods: Eight neurologists rated a set of 40 multiple sclerosis patient videos of the Finger-to-Nose-Test using both the newly developed setwise comparison and the established pairwise comparison techniques, which result in a continuous rating scale. Reliability was assessed by the intra-class correlation coefficient. Construct validity was estimated as Pearson's correlation between the continuous scale and severity ratings according to the Neurostatus scale for upper-extremity tremor/dysmetria and the Nine-hole-peg-test. Comparing the time needed for ratings assessed efficiency.Results: Intra-class correlation coefficient was 0.83 for setwise and 0.7 for pairwise comparison. Correlation to the tremor/dysmetria score of the Neurostatus was 0.86 for both rating procedures and correlation to the Nine-hole-peg-test was 0.64 (setwise) and 0.66 (pairwise). The time needed to rate 40 videos was 22.9 ± 6.9 minutes (setwise) and 77.8 ± 14.5 minutes (pairwise).Conclusions: Setwise comparison is an efficient, valid and reliable method for fine-grained rating of motor dysfunction that can be applied to larger datasets. It is substantially more efficient than pairwise comparison.Implications for rehabilitationDisability rating is crucial in clinical neurorehabilitation and in clinical trials.Humans are naturally inconsistent in rating items on ordinal scales leading to poor intra- and interrater reliability, insensitivity to subtle differences and coarse-grained ratings.Video-based setwise comparison is a new rating method enabling fine-grained, reliable and efficient ratings of motor dysfunction using algorithmic support.

"It's Not as Simple as Just Looking at One Chart": A Qualitative Study Exploring Clinician's Opinions on Various Visualisation Strategies to Represent Longitudinal Actigraphy Data.

BACKGROUND: Data derived from wearable activity trackers may provide important clinical insights into disease progression and response to intervention, but only if clinicians can interpret it in a meaningful manner. Longitudinal activity data can be visually presented in multiple ways, but research has failed to explore how clinicians interact with and interpret these visualisations. In response, this study developed a variety of visualisations to understand whether alternative data presentation strategies can provide clinicians with meaningful insights into patient's physical activity patterns. OBJECTIVE: To explore clinicians' opinions on different visualisations of actigraphy data. METHODS: Four visualisations (stacked bar chart, clustered bar chart, linear heatmap and radial heatmap) were created using Matplotlib and Seaborn Python libraries. A focus group was conducted with 14 clinicians across 2 hospitals. Focus groups were audio-recorded, transcribed and analysed using inductive thematic analysis. RESULTS: Three major themes were identified: (1) the importance of context, (2) interpreting the visualisations and (3) applying visualisations to clinical practice. Although clinicians saw the potential value in the visualisations, they expressed a need for further contextual information to gain clinical benefits from them. Allied health professionals preferred more granular, temporal information compared to doctors. Specifically, physiotherapists favoured heatmaps, whereas the remaining members of the team favoured stacked bar charts. Overall, heatmaps were considered more difficult to interpret. CONCLUSION: The current lack of contextual data provided by wearables hampers their use in clinical practice. Clinicians favour data presented in a familiar format and yet desire multi-faceted filtering. Future research should implement user-centred design processes to identify ways in which all clinical needs can be met, potentially using an interactive system that caters for multiple levels of granularity. Irrespective of how data is displayed, unless clinicians can apply it in a manner that best supports their role, the potential of this data cannot be fully realised.

Observational Study of a Wearable Sensor and Smartphone Application Supporting Unsupervised Exercises to Assess Pain and Stiffness.

BACKGROUND: Evaluation of pain and stiffness in patients with arthritis is largely based on participants retrospectively reporting their self-perceived pain/stiffness. This is subjective and may not accurately reflect the true impact of therapeutic interventions. We now have access to sensor-based systems to continuously capture objective information regarding movement and activity. OBJECTIVES: We present an observational study aimed to collect sensor data from participants monitored while performing an unsupervised version of a standard motor task, known as the Five Times Sit to Stand (5×STS) test. The first objective was to explore whether the participants would perform the test regularly in their home environment, and do so in a correct and consistent manner. The second objective was to demonstrate that the measurements collected would enable us to derive an objective signal related to morning pain and stiffness. METHODS: We recruited a total of 45 participants, of whom 30 participants fulfilled pre-defined criteria for osteoarthritis, rheumatoid arthritis, or psoriatic arthritis and 15 participants were healthy volunteers. All participants wore accelerometers on their wrists, day and night for about 4 weeks. The participants were asked to perform the 5×STS test in their own home environment at the same time in the morning 3 times per week. We investigated the relationship between pain/stiffness and measurements collected during the 5×STS test by comparing the 5×STS test duration with the patient-reported outcome (PRO) questionnaires, filled in via a smartphone. RESULTS: During the study, we successfully captured accelerometer data from each participant for a period of 4 weeks. The participants performed 56% of the prescribed 5×STS tests. We observed that different tests made by the same participants were performed with subject-specific characteristics that remained consistent throughout the study. We showed that 5×STS test duration (the time taken to complete the 5×STS test) was significantly and robustly associated with the pain and stiffness intensity reported via the PROs, particularly the questions asked in the morning. CONCLUSIONS: This study demonstrates the feasibility and usefulness of regular, sensor-based, monitored, unsupervised physical tests to objectively assess the impact of disease on function in the home environment. This approach may permit remote disease monitoring in clinical trials and support the development of novel endpoints from passively collected actigraphy data.

Deregulated ERK1/2 MAP kinase signaling promotes aneuploidy by a Fbxw7ß-Aurora A pathway.

Aneuploidy is a common feature of human solid tumors and is often associated with poor prognosis. There is growing evidence that oncogenic signaling pathways, which are universally dysregulated in cancer, contribute to the promotion of aneuploidy. However, the mechanisms connecting signaling pathways to the execution of mitosis and cytokinesis are not well understood. Here, we show that hyperactivation of the ERK1/2 MAP kinase pathway in epithelial cells impairs cytokinesis, leading to polyploidization and aneuploidy. Mechanistically, deregulated ERK1/2 signaling specifically downregulates expression of the F-box protein Fbxw7ß, a substrate-binding subunit of the SCF(Fbxw7) ubiquitin ligase, resulting in the accumulation of the mitotic kinase Aurora A. Reduction of Aurora A levels by RNA interference or pharmacological inhibition of MEK1/2 reverts the defect in cytokinesis and decreases the frequency of abnormal cell divisions induced by oncogenic H-Ras(V12). Reciprocally, overexpression of Aurora A or silencing of Fbxw7ß phenocopies the effect of H-Ras(V12) on cell division. In vivo, conditional activation of MEK2 in the mouse intestine lowers Fbxw7ß expression, resulting in the accumulation of cells with enlarged nuclei. We propose that the ERK1/2/ Fbxw7ß/Aurora A axis identified in this study contributes to genomic instability and tumor progression.

A theoretical model of cytokinesis implicates feedback between membrane curvature and cytoskeletal organization in asymmetric cytokinetic furrowing.

During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in theCaenorhabditis eleganszygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane-cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity.

Mitotic chromosome length scales in response to both cell and nuclear size.

Multicellular development requires that cells reduce in size as a result of consecutive cell divisions without increase in embryo volume. To maintain cellular integrity, organelle size adapts to cell size throughout development. During mitosis, the longest chromosome arm must be shorter than half of the mitotic spindle for proper chromosome segregation. Using high-resolution time-lapse microscopy of living Caenorhabditis elegans embryos, we have quantified the relation between cell size and chromosome length. In control embryos, chromosome length scaled to cell size. Artificial reduction of cell size resulted in a shortening of chromosome length, following a trend predicted by measurements from control embryos. Disturbing the RAN (Ras-related nuclear protein)-GTP gradient decoupled nuclear size from cell size and resulted in chromosome scaling to nuclear size rather than cell size; smaller nuclei contained shorter chromosomes independent of cell size. In sum, quantitative analysis relating cell, nuclear, and chromosome size predicts two levels of chromosome length regulation: one through cell size and a second in response to nuclear size.

Investigating the regulation of stem and progenitor cell mitotic progression by in situ imaging.

Genome stability relies upon efficacious chromosome congression and regulation by the spindle assembly checkpoint (SAC). The study of these fundamental mitotic processes in adult stem and progenitor cells has been limited by the technical challenge of imaging mitosis in these cells in situ. Notably, how broader physiological changes, such as dietary intake or age, affect mitotic progression in stem and/or progenitor cells is largely unknown. Using in situ imaging of C. elegans adult germlines, we describe the mitotic parameters of an adult stem and progenitor cell population in an intact animal. We find that SAC regulation in germline stem and progenitor cells is distinct from that found in early embryonic divisions and is more similar to that of classical tissue culture models. We further show that changes in organismal physiology affect mitotic progression in germline stem and progenitor cells. Reducing dietary intake produces a checkpoint-dependent delay in anaphase onset, and inducing dietary restriction when the checkpoint is impaired increases the incidence of segregation errors in mitotic and meiotic cells. Similarly, developmental aging of the germline stem and progenitor cell population correlates with a decline in the rate of several mitotic processes. These results provide the first in vivo validation of models for SAC regulation developed in tissue culture systems and demonstrate that several fundamental features of mitotic progression in adult stem and progenitor cells are highly sensitive to organismal physiological changes.

Usability and Acceptability of ASSESS MS: Assessment of Motor Dysfunction in Multiple Sclerosis Using Depth-Sensing Computer Vision.

BACKGROUND: Sensor-based recordings of human movements are becoming increasingly important for the assessment of motor symptoms in neurological disorders beyond rehabilitative purposes. ASSESS MS is a movement recording and analysis system being developed to automate the classification of motor dysfunction in patients with multiple sclerosis (MS) using depth-sensing computer vision. It aims to provide a more consistent and finer-grained measurement of motor dysfunction than currently possible. OBJECTIVE: To test the usability and acceptability of ASSESS MS with health professionals and patients with MS. METHODS: A prospective, mixed-methods study was carried out at 3 centers. After a 1-hour training session, a convenience sample of 12 health professionals (6 neurologists and 6 nurses) used ASSESS MS to capture recordings of standardized movements performed by 51 volunteer patients. Metrics for effectiveness, efficiency, and acceptability were defined and used to analyze data captured by ASSESS MS, video recordings of each examination, feedback questionnaires, and follow-up interviews. RESULTS: All health professionals were able to complete recordings using ASSESS MS, achieving high levels of standardization on 3 of 4 metrics (movement performance, lateral positioning, and clear camera view but not distance positioning). Results were unaffected by patients' level of physical or cognitive disability. ASSESS MS was perceived as easy to use by both patients and health professionals with high scores on the Likert-scale questions and positive interview commentary. ASSESS MS was highly acceptable to patients on all dimensions considered, including attitudes to future use, interaction (with health professionals), and overall perceptions of ASSESS MS. Health professionals also accepted ASSESS MS, but with greater ambivalence arising from the need to alter patient interaction styles. There was little variation in results across participating centers, and no differences between neurologists and nurses. CONCLUSIONS: In typical clinical settings, ASSESS MS is usable and acceptable to both patients and health professionals, generating data of a quality suitable for clinical analysis. An iterative design process appears to have been successful in accounting for factors that permit ASSESS MS to be used by a range of health professionals in new settings with minimal training. The study shows the potential of shifting ubiquitous sensing technologies from research into the clinic through a design approach that gives appropriate attention to the clinic environment.

Quantifying progression of multiple sclerosis via classification of depth videos.

This paper presents new learning-based techniques for measuring disease progression in Multiple Sclerosis (MS) patients. Our system aims to augment conventional neurological examinations by adding quantitative evidence of disease progression. An off-the-shelf depth camera is used to image the patient at the examination, during which he/she is asked to perform carefully selected movements. Our algorithms then automatically analyze the videos, assessing the quality of each movement and classifying them as healthy or non-healthy. Our contribution is three-fold: We i) introduce ensembles of randomized SVM classifiers and compare them with decision forests on the task of depth video classification; ii) demonstrate automatic selection of discriminative landmarks in the depth videos, showing their clinical relevance; iii) validate our classification algorithms quantitatively on a new dataset of 1041 videos of both MS patients and healthy volunteers. We achieve average Dice scores well in excess of the 80% mark, confirming the validity of our approach in practical applications. Our results suggest that this technique could be fruitful for depth-camera supported clinical assessments for a range of conditions.

Quantitative analysis of cytokinesis in situ during C. elegans postembryonic development.

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.

In situ imaging in C. elegans reveals developmental regulation of microtubule dynamics.

Microtubules (MTs) are cytoskeletal polymers that undergo dynamic instability, the stochastic transition between growth and shrinkage phases. MT dynamics are required for diverse cellular processes and, while intrinsic to tubulin, are highly regulated. However, little is known about how MT dynamics facilitate or are regulated by tissue biogenesis and differentiation. We imaged MT dynamics in a smooth muscle-like lineage in intact developing Caenorhabditis elegans. All aspects of MT dynamics change significantly as stem-like precursors exit mitosis and, secondarily, as they differentiate. We found that suppression, but not enhancement, of dynamics perturbs differentiated muscle function in vivo. Distinct ensembles of MT-associated proteins are specifically required for tissue biogenesis versus tissue function. A CLASP family MT stabilizer and the depolymerizing kinesin MCAK are differentially required for MT dynamics in the precursor or differentiated cells, respectively. All of these multidimensional phenotypic comparisons were facilitated by a data display method called the diamond graph.

S. cerevisiae chromosomes biorient via gradual resolution of syntely between S phase and anaphase.

Following DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis.

Octameric CENP-A nucleosomes are present at human centromeres throughout the cell cycle.

The presence of a single centromere on each chromosome that signals formation of a mitotic kinetochore is central to accurate chromosome segregation. The histone H3 variant centromere protein-A (CENP-A) is critical for centromere identity and function; CENP-A chromatin acts as an epigenetic mark to direct both centromere and kinetochore assembly. Interpreting the centromere epigenetic mark ensures propagation of a single centromere per chromosome to maintain ploidy. Thus, understanding the nature of CENP-A chromatin is crucial for all cell divisions. However, there are ongoing debates over the fundamental composition of centromeric chromatin. Here we show that natively assembled human CENP-A nucleosomes are octameric throughout the cell cycle. Using total internal reflection fluorescence (TIRF)-coupled photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells, we find that the majority of CENP-A nucleosomes contain CENP-A dimers. In addition, we detect the presence of H2B and H4 in these nucleosomes. Surprisingly, CENP-A associated with the chaperone HJURP can exist as either monomer or dimer, indicating possible assembly intermediates. Thus, our findings indicate that octameric CENP-A nucleosomes mark the centromeric region to ensure proper epigenetic inheritance and kinetochore assembly.

Phosphorylation of the eukaryotic translation initiation factor 4E-transporter (4E-T) by c-Jun N-terminal kinase promotes stress-dependent P-body assembly.

Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.

Evi5 promotes collective cell migration through its Rab-GAP activity.

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.

Impedance responses reveal ß2-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the ß2-adrenergic receptor (ß2AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the ß2AR signaling network revealed the differential contribution of G(s)-, G(i)- and Gßγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²âº in the impedance response and led to the discovery of a novel ß2AR-promoted Ca²âº mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of ß-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.