Targeting PKCα alleviates iron overload in diabetes and hemochromatosis through the inhibition of ferroportin.

ABSTRACT: Ferroportin (Fpn) is the only iron exporter, playing a crucial role in systemic iron homeostasis. Fpn is negatively regulated by its ligand hepcidin, but other potential regulators in physiological and disease conditions remain poorly understood. Diabetes is a metabolic disorder that develops body iron loading with unknown mechanisms. By using diabetic mouse models and human duodenal specimens, we demonstrated that intestinal Fpn expression was increased in diabetes in a hepcidin-independent manner. Protein kinase C (PKC) is hyperactivated in diabetes. We showed that PKCα was required to sustain baseline Fpn expression and diabetes-induced Fpn upregulation in the enterocytes and macrophages. Knockout of PKCα abolished diabetes-associated iron overload. Mechanistically, activation of PKCα increased the exocytotic trafficking of Fpn and decreased the endocytic trafficking of Fpn in the resting state. Hyperactive PKCα also suppressed hepcidin-induced ubiquitination, internalization, and degradation of Fpn. We further observed that iron loading in the enterocytes and macrophages activated PKCα, acting as a novel mechanism to enhance Fpn-dependent iron efflux. Finally, we demonstrated that the loss-of-function of PKCα and pharmacological inhibition of PKC significantly alleviated hereditary hemochromatosis-associated iron overload. Our study has highlighted, to our knowledge, for the first time, that PKCα is an important positive regulator of Fpn and a new target in the control of iron homeostasis.

Speckle-correlation imaging through a kaleidoscopic multimode fiber.

Speckle-correlation imaging techniques are widely used for noninvasive imaging through complex scattering media. While light propagation through multimode fibers and scattering media share many analogies, reconstructing images through multimode fibers from speckle correlations remains an unsolved challenge. Here, we exploit a kaleidoscopic memory effect emerging in square-core multimode fibers and demonstrate fluorescence imaging with no prior knowledge on the fiber. Experimentally, our approach simply requires to translate random speckle patterns at the input of a square-core fiber and to measure the resulting fluorescence intensity with a bucket detector. The image of the fluorescent object is then reconstructed from the autocorrelation of the measured signal by solving an inverse problem. This strategy does not require the knowledge of the fragile deterministic relation between input and output fields, which makes it promising for the development of flexible minimally invasive endoscopes.

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells.

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.

Multimodal mapping of regional brain vulnerability to focal cortical dysplasia.

Focal cortical dysplasia (FCD) type II is a highly epileptogenic developmental malformation and a common cause of surgically treated drug-resistant epilepsy. While clinical observations suggest frequent occurrence in the frontal lobe, mechanisms for such propensity remain unexplored. Here, we hypothesized that cortex-wide spatial associations of FCD distribution with cortical cytoarchitecture, gene expression and organizational axes may offer complementary insights into processes that predispose given cortical regions to harbour FCD. We mapped the cortex-wide MRI distribution of FCDs in 337 patients collected from 13 sites worldwide. We then determined its associations with (i) cytoarchitectural features using histological atlases by Von Economo and Koskinas and BigBrain; (ii) whole-brain gene expression and spatiotemporal dynamics from prenatal to adulthood stages using the Allen Human Brain Atlas and PsychENCODE BrainSpan; and (iii) macroscale developmental axes of cortical organization. FCD lesions were preferentially located in the prefrontal and fronto-limbic cortices typified by low neuron density, large soma and thick grey matter. Transcriptomic associations with FCD distribution uncovered a prenatal component related to neuroglial proliferation and differentiation, likely accounting for the dysplastic makeup, and a postnatal component related to synaptogenesis and circuit organization, possibly contributing to circuit-level hyperexcitability. FCD distribution showed a strong association with the anterior region of the antero-posterior axis derived from heritability analysis of interregional structural covariance of cortical thickness, but not with structural and functional hierarchical axes. Reliability of all results was confirmed through resampling techniques. Multimodal associations with cytoarchitecture, gene expression and axes of cortical organization indicate that prenatal neurogenesis and postnatal synaptogenesis may be key points of developmental vulnerability of the frontal lobe to FCD. Concordant with a causal role of atypical neuroglial proliferation and growth, our results indicate that FCD-vulnerable cortices display properties indicative of earlier termination of neurogenesis and initiation of cell growth. They also suggest a potential contribution of aberrant postnatal synaptogenesis and circuit development to FCD epileptogenicity.

Imaging through a square multimode fiber by scanning focused spots with the memory effect.

The existence of a shift-shift memory effect in square waveguides, whereby any translation of the input field induces translations in the output field in four symmetrical directions, has been previously observed by correlation measurements. Here we demonstrate that this memory effect is also observed in real space and can be put to use for imaging purposes. First, a focus is created at the output of a square-core multimode fiber, by wavefront shaping based on feedback from a guide-star. Then, because of the memory effect, four symmetrical spots can be scanned at the fiber output by shifting the wavefront at the fiber input. We demonstrate that this property can be exploited to perform fluorescence imaging through the multimode fiber, without requiring the measurement of a transmission matrix.

Low-Temperature Gas-Phase Kinetics of Ethanol-Methanol Heterodimer Formation.

The structures of gas-phase noncovalently bound clusters have long been studied in supersonic expansions. This method of study, while providing a wealth of information about the nature of noncovalent bonds, precludes observation of the formation of the cluster, as the clusters form just after the orifice of the pulsed valve. Here, we directly observe formation of ethanol-methanol dimers via microwave spectroscopy in a controlled cryogenic environment. Time profiles of the concentration of reagents in the cell yielded gas-phase reaction rate constants of kMe-g = (2.8 ± 1.4) × 10-13 cm3 molecule-1 s-1 and kMe-t = (1.6 ± 0.8) × 10-13 cm3 molecule-1 s-1 for the pseudo-second-order ethanol-methanol dimerization reaction at 8 K. The relaxation cross section between the gauche and trans conformers of ethanol was also measured using the same technique. In addition, thermodynamic relaxation between conformers of ethanol over time allowed for selection of conformer stoichiometry in the ethanol-methanol dimerization reaction, but no change in the ratio of dimer conformers was observed with changing ethanol monomer stoichiometry.

Inter-individual variability in dorsal stream dynamics during word production.

The current standard model of language production involves a sensorimotor dorsal stream connecting areas in the temporo-parietal junction with those in the inferior frontal gyrus and lateral premotor cortex. These regions have been linked to various aspects of word production such as phonological processing or articulatory programming, primarily through neuropsychological and functional imaging group studies. Most if not all the theoretical descriptions of this model imply that the same network should be identifiable across individual speakers. We tested this hypothesis by quantifying the variability of activation observed across individuals within each dorsal stream anatomical region. This estimate was based on electrical activity recorded directly from the cerebral cortex with millisecond accuracy in awake epileptic patients clinically implanted with intracerebral depth electrodes for pre-surgical diagnosis. Each region's activity was quantified using two different metrics-intra-cerebral evoked related potentials and high gamma activity-at the level of the group, the individual and the recording contact. The two metrics show simultaneous activation of parietal and frontal regions during a picture naming task, in line with models that posit interactive processing during word retrieval. They also reveal different levels of between-patient variability across brain regions, except in core auditory and motor regions. The independence and non-uniformity of cortical activity estimated through the two metrics push the current model towards sub-second and sub-region explorations focused on individualized language speech production. Several hypotheses are considered for this within-region heterogeneity.

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

Optical nanotopography of fluorescent surfaces by axial position modulation.

We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely control the distance to an object of interest. The accuracy of this method is theoretically analyzed and the axial-locking accuracy is experimentally evaluated. Topographic imaging is demonstrated on fluorescently coated beads and fixed cells. This microscope allows for nanometric topography or tracking of dynamic fluorescent surfaces.

Multilobar unilateral hypoplasia with emphasis on the posterior quadrant and severe epilepsy in children with FCD ILAE Type 1A.

OBJECTIVE: Focal cortical dysplasia (FCD) Type 1 and its three subtypes have yet not been fully characterized at the clinical, anatomopathological, and molecular level (International League Against Epilepsy [ILAE] FCD classification from 2011). We aimed to describe the clinical phenotype of patients with histopathologically confirmed FCD1A obtained from a single epilepsy center between 2002 and 2016. METHODS: Medical records were retrieved from the hospital's archive. Results from electroencephalography (EEG) video recordings, neuroimaging, and histopathology were reevaluated. Magnetic resonance imaging (MRI) post-processing was retrospectively performed in nine patients. DNA methylation studies were carried out from archival surgical brain tissue in 11 patients. RESULTS: Nineteen children with a histopathological diagnosis of FCD1A were included. The average onset of epilepsy was 0.9 years (range 0.2-10 years). All children had severe cognitive impairment and one third had mild motor deficits, yet fine finger movements were preserved in all patients. All patients had daily seizures, being drug resistant from disease onset. Interictal electroencephalography revealed bilateral multi-regional epileptiform discharges. Interictal status epilepticus was observed in 8 and countless subclinical seizures in 11 patients. Regional continuous irregular slow waves were of higher lateralizing and localizing yield than spikes. Posterior background rhythms were normal in 16 of 19 children. Neuroimaging showed unilateral multilobar hypoplasia and increased T2-FLAIR signals of the white matter in 18 of 19 patients. All children underwent tailored multilobar resections, with seizure freedom achieved in 47% (Engel class I). There was no case with frontal involvement without involvement of the posterior quadrant by MRI and histopathology. DNA methylation profiling distinguished FCD1A samples from all other epilepsy specimens and controls. SIGNIFICANCE: We identified a cohort of young children with drug resistance from seizure onset, bad EEG with posterior emphasis, lack of any focal neurological deficits but severe cognitive impairment, subtle hypoplasia of the epileptogenic area on MRI, and histopathologically defined and molecularly confirmed by DNA methylation analysis as FCD ILAE Type 1A.

Atlas of lesion locations and postsurgical seizure freedom in focal cortical dysplasia: A MELD study.

OBJECTIVE: Drug-resistant focal epilepsy is often caused by focal cortical dysplasias (FCDs). The distribution of these lesions across the cerebral cortex and the impact of lesion location on clinical presentation and surgical outcome are largely unknown. We created a neuroimaging cohort of patients with individually mapped FCDs to determine factors associated with lesion location and predictors of postsurgical outcome. METHODS: The MELD (Multi-centre Epilepsy Lesion Detection) project collated a retrospective cohort of 580 patients with epilepsy attributed to FCD from 20 epilepsy centers worldwide. Magnetic resonance imaging-based maps of individual FCDs with accompanying demographic, clinical, and surgical information were collected. We mapped the distribution of FCDs, examined for associations between clinical factors and lesion location, and developed a predictive model of postsurgical seizure freedom. RESULTS: FCDs were nonuniformly distributed, concentrating in the superior frontal sulcus, frontal pole, and temporal pole. Epilepsy onset was typically before the age of 10 years. Earlier epilepsy onset was associated with lesions in primary sensory areas, whereas later epilepsy onset was associated with lesions in association cortices. Lesions in temporal and occipital lobes tended to be larger than frontal lobe lesions. Seizure freedom rates varied with FCD location, from around 30% in visual, motor, and premotor areas to 75% in superior temporal and frontal gyri. The predictive model of postsurgical seizure freedom had a positive predictive value of 70% and negative predictive value of 61%. SIGNIFICANCE: FCD location is an important determinant of its size, the age at epilepsy onset, and the likelihood of seizure freedom postsurgery. Our atlas of lesion locations can be used to guide the radiological search for subtle lesions in individual patients. Our atlas of regional seizure freedom rates and associated predictive model can be used to estimate individual likelihoods of postsurgical seizure freedom. Data-driven atlases and predictive models are essential for evidence-based, precision medicine and risk counseling in epilepsy.

Associations of patient-reported care satisfaction with symptom burden and healthcare use in hospitalized patients with cancer.

BACKGROUND: Hospitalized patients with cancer often experience a high symptom burden, which may impact care satisfaction and healthcare utilization. METHODS: We prospectively enrolled patients with cancer and unplanned hospitalizations from September 2014 to April 2017. Upon admission, we assessed patients' care satisfaction (FAMCARE items: satisfaction with care coordination and speed with which symptoms are treated) and physical (Edmonton Symptom Assessment System [ESAS]) and psychological (Patient Health Questionnaire-4 [PHQ-4]) symptoms. We used regression models to identify factors associated with care satisfaction and associations of satisfaction with symptom burden and hospital length of stay (LOS). RESULTS: Among 1,576 participants, most reported being "satisfied"/ "very satisfied" with care coordination (90%) and speed with which symptoms are treated (89%). Older age (coordination: B < 0.01, P = 0.02, speed: B = 0.01, P < 0.01) and admission to a dedicated oncology service (B = 0.20, P < 0.01 for each) were associated with higher satisfaction. Higher satisfaction with care coordination was associated with lower ESAS-physical (B = - 1.28, P < 0.01), ESAS-total (B = - 2.73, P < 0.01), PHQ4-depression (B = - 0.14, P = 0.02), and PHQ4-anxiety (B = - 0.16, P < 0.01) symptoms. Higher satisfaction with speed with which symptoms are treated was associated with lower ESAS-physical (B = - 1.32, P < 0.01), ESAS-total (B = - 2.46, P < 0.01), PHQ4-depression (B = - 0.14, P = 0.01), and PHQ4-anxiety (B = - 0.17, P < 0.01) symptoms. Satisfaction with care coordination (B = - 0.48, P = 0.04) and speed with which symptoms are treated (B = - 0.44, P = 0.04) correlated with shorter LOS. CONCLUSIONS: Hospitalized patients with cancer report high care satisfaction, which correlates with older age and admission to a dedicated oncology service. Significant associations among higher care satisfaction, lower symptom burden, and shorter hospital LOS highlight the importance of improving symptom management and care coordination in this population.

Optical memory effect in square multimode fibers.

We demonstrate experimentally the existence of a translational optical memory effect in square-core multimode fibers. We found that symmetry properties of square-core waveguides lead to speckle patterns shifting along four directions at the fiber output for any given shift direction at the input. A simple theoretical model based on a perfectly reflective square waveguide is introduced to predict and interpret this phenomenon. We report experimental results obtained with 532-nm coherent light propagating through a square-core step-index multimode fiber, demonstrating that this translational memory effect can be observed for shift distances up to typically 10 µm after propagation through several centimeters of fiber.

Quantitative analysis of hidden particles diffusing behind a scattering layer using speckle correlation.

Speckle-correlation imaging is a family of methods that makes use of the "memory effect" to image objects hidden behind visually opaque layers. Here, we show that a correlation analysis can be applied to quantitative imaging of an ensemble of dynamic fluorescent beads diffusing on a 2D surface. We use an epi-fluorescence microscope where both the illumination and detection light patterns are speckled, due to light scattering by a thin disordered layer. The spatio-temporal cross-correlation of the detection speckle pattern is calculated as a function of lag time and spatial shift and is used to determine the diffusion constant and number of fluorescent particles in the sample without requiring any phase retrieval procedure. It is worth to note that the "memory effect" range is not required to extend beyond a distance of few speckle grains, thus making our method potentially useful for nearly arbitrary values of the thickness of the scattering layer.

Value of 7T MRI and post-processing in patients with nonlesional 3T MRI undergoing epilepsy presurgical evaluation.

OBJECTIVE: Ultra-high-field 7-Tesla (7T) magnetic resonance imaging (MRI) offers increased signal-to-noise and contrast-to-noise ratios, which may improve visualization of cortical malformations. We aim to assess the clinical value of in vivo structural 7T MRI and its post-processing for the noninvasive identification of epileptic brain lesions in patients with pharmacoresistant epilepsy and nonlesional 3T MRI who are undergoing presurgical evaluation. METHODS: Sixty-seven patients were included who had nonlesional 3T MRI by official radiology report. Epilepsy protocols were used for the 3T and 7T acquisitions. Post-processing of the 7T T1-weighted magnetization-prepared two rapid acquisition gradient echoes sequence was performed using the morphometric analysis program (MAP) with comparison to a normal database consisting of 50 healthy controls. Review of 7T was performed by an experienced board-certified neuroradiologist and at the multimodal patient management conference. The clinical significance of 7T findings was assessed based on intracranial electroencephalography (ICEEG) ictal onset, surgery, postoperative seizure outcomes, and histopathology. RESULTS: Unaided visual review of 7T detected previously unappreciated subtle lesions in 22% (15/67). When aided by 7T MAP, the total yield increased to 43% (29/67). The location of the 7T-identified lesion was identical to or contained within the ICEEG ictal onset in 13 of 16 (81%). Complete resection of the 7T-identified lesion was associated with seizure freedom (P = .03). Histopathology of the 7T-identified lesions encountered mainly focal cortical dysplasia (FCD). 7T MAP yielded 25% more lesions (6/24) than 3T MAP, and showed improved conspicuity in 46% (11/24). SIGNIFICANCE: Our data suggest a major benefit of 7T with post-processing for detecting subtle FCD lesions for patients with pharmacoresistant epilepsy and nonlesional 3T MRI.

Confocal fluorescence correlation spectroscopy through a sparse layer of scattering objects.

In the presence of strong light scattering, as often encountered in biological tissue, optical microscopy becomes challenging and technical demanding. Beside image quality, the quantitative determination of molecular properties is also strongly affected by scattering. We have carried out fluorescence correlation spectroscopy (FCS) experiments, in a solution of fluorophores, through a sparse scattering layer made of dielectric beads. We observe that the fluorescence signal steadily decreases as the focus is moved away from the scattering layer. By contrast, the estimated number of molecules recovers its normal value beyond a characteristic distance of about twice the bead diameters, below which it is strongly biased. Accompanying theoretical modeling demonstrates how diffraction and refraction by the scattering layer and their impact on FCS measurements depend on size and refractive index of the beads.

A fingerprint of the epileptogenic zone in human epilepsies.

Defining a bio-electrical marker for the brain area responsible for initiating a seizure remains an unsolved problem. Fast gamma activity has been identified as the most specific marker for seizure onset, but conflicting results have been reported. In this study, we describe an alternative marker, based on an objective description of interictal to ictal transition, with the aim of identifying a time-frequency pattern or 'fingerprint' that can differentiate the epileptogenic zone from areas of propagation. Seventeen patients who underwent stereoelectroencephalography were included in the study. Each had seizure onset characterized by sustained gamma activity and were seizure-free after tailored resection or laser ablation. We postulated that the epileptogenic zone was always located inside the resection region based on seizure freedom following surgery. To characterize the ictal frequency pattern, we applied the Morlet wavelet transform to data from each pair of adjacent intracerebral electrode contacts. Based on a visual assessment of the time-frequency plots, we hypothesized that a specific time-frequency pattern in the epileptogenic zone should include a combination of (i) sharp transients or spikes; preceding (ii) multiband fast activity concurrent; with (iii) suppression of lower frequencies. To test this hypothesis, we developed software that automatically extracted each of these features from the time-frequency data. We then used a support vector machine to classify each contact-pair as being within epileptogenic zone or not, based on these features. Our machine learning system identified this pattern in 15 of 17 patients. The total number of identified contacts across all patients was 64, with 58 localized inside the resected area. Subsequent quantitative analysis showed strong correlation between maximum frequency of fast activity and suppression inside the resection but not outside. We did not observe significant discrimination power using only the maximum frequency or the timing of fast activity to differentiate contacts either between resected and non-resected regions or between contacts identified as epileptogenic versus non-epileptogenic. Instead of identifying a single frequency or a single timing trait, we observed the more complex pattern described above that distinguishes the epileptogenic zone. This pattern encompasses interictal to ictal transition and may extend until seizure end. Its time-frequency characteristics can be explained in light of recent models emphasizing the role of fast inhibitory interneurons acting on pyramidal cells as a prominent mechanism in seizure triggering. The pattern clearly differentiates the epileptogenic zone from areas of propagation and, as such, represents an epileptogenic zone 'fingerprint'.awx306media15687076823001.

7T MR of intracranial pathology: Preliminary observations and comparisons to 3T and 1.5T.

PURPOSE: There have been an increasing number of studies involving ultra-high-field 7T of intracranial pathology, however, comprehensive clinical studies of neuropathology at 7T still remain limited. 7T has the advantage of a higher signal-to-noise ratio and a higher contrast-to-noise ratio, compared to current low field clinical MR scanners. We hypothesized 7T applied clinically, may improve detection and characterization of intracranial pathology. MATERIALS AND METHODS: We performed an IRB-approved 7T prospective study of patients with neurological disease who previously had lower field 3T and 1.5T. All patients underwent 7T scans, using comparable clinical imaging protocols, with the aim of qualitatively comparing neurological lesions at 7T with 3T or 1.5T. To qualitatively assess lesion conspicuity at 7T compared with low field, 80-paired images were viewed by 10 experienced neuroradiologists and scored on a 5-point scale. Inter-rater agreement was characterized using a raw percent agreement and mean weighted kappa. RESULTS: One-hundred and four patients with known neurological disease have been scanned to date. Fifty-five patients with epilepsy, 18 patients with mild traumatic brain injury, 11 patients with known or suspected multiple sclerosis, 9 patients with amyotrophic lateral sclerosis, 4 patients with intracranial neoplasm, 2 patients with orbital melanoma, 2 patients with cortical infarcts, 2 patients with cavernous malformations, and 1 patient with cerebral amyloid angiopathy. From qualitative observations, we found better resolution and improved detection of lesions at 7T compared to 3T. There was a 55% raw inter-rater agreement that lesions were more conspicuous on 7T than 3T/1.5T, compared with a 6% agreement that lesions were more conspicuous on 3T/1.5T than 7T. CONCLUSION: Our findings show that the primary clinical advantages of 7T magnets, which include higher signal-to-noise ratio, higher contrast-to-noise ratio, smaller voxels and stronger susceptibility contrast, may increase lesion conspicuity, detection and characterization compared to low field 1.5T and 3T. However, low field which detects a plethora of intracranial pathology remains the mainstay for diagnostic imaging until limitations at 7T are addressed and further evidence of utility provided.

αvß3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site.

BACKGROUND INFORMATION: Integrins are key receptors that allow cells to sense and respond to their mechanical environment. Although they bind the same ligand, ß1 and ß3 integrins have distinct and cooperative roles in mechanotransduction. RESULTS: Using traction force microscopy on unconstrained cells, we show that deleting ß3 causes traction forces to increase, whereas the deletion of ß1 integrin results in a strong decrease of contractile forces. Consistently, loss of ß3 integrin also induces an increase in ß1 integrin activation. Using a genetic approach, we identified the phosphorylation of the distal NPXY domain as an essential process for ß3 integrin to be able to modulate traction forces. Loss of ß3 integrins also impacted cell shape and the spatial distribution of traction forces, by causing forces to be generated closer to the cell edge, and the cell shape. CONCLUSIONS: Our results emphasize the role of ß3 integrin in spatial distribution of cellular forces. We speculate that, by modulating its affinity with kindlin, ß3 integrins may be able to locate near the cell edge where it can control ß1 integrin activation and clustering. SIGNIFICANCE: Tensional homeostasis at the single cell level is performed by the ability of ß3 adhesions to negatively regulate the activation degree and spatial localization of ß1 integrins. By combining genetic approaches and new tools to analyze traction distribution and cell morphology on a population of cells we were able to identify the molecular partners involved in cellular forces regulation.

Optimizing the metric in sensorless adaptive optical microscopy with fluorescence fluctuations.

Adaptive optics (AO) strategies using optimization-based, sensorless approaches are widely used, especially for microscopy applications. To converge rapidly to the best correction, such approaches require that a quality metric and a set of modes be chosen optimally. Fluorescence fluctuations microscopy, a family of methods that provides quantitative measurements of molecular concentration and mobility in living specimen, is in particular need of adaptive optics, since its results can be strongly biased by optical aberrations. We examined two possible metrics for sensorless AO, measured in a solution of fluorophores diffusing in 3D: the fluorescence count rate and the molecular brightness (or number of photons detected per molecule in the observation volume). We studied their respective measurement noise and sensitivity to aberrations. Then, AO correction accuracy was experimentally assessed by measuring the residual aberration after correcting a known wavefront. We proposed a theoretical framework to predict the correction accuracy, knowing the metric measurement noise and sensitivity. In the small aberration range, the brightness allows more accurate corrections when fluorophores are few but bright, whereas the count rate performs better in more concentrated solutions. When correcting large aberrations, the count rate is expected to be a more reliable metric.