Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation.

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.Herchel Smith Fellowship, Funai Foundation scholarship, Austrian Science Fun

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways

Filopodyan: An open-source pipeline for the analysis of filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/ VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.J.L. Gallop and V. Urbančič are supported by the Wellcome Trust (WT095829AIA). J. Mason and B. Richier are supported by the European Research Council (281971). C.E. Holt is supported by the Wellcome Trust (program grant 085314) and the European Research Council (advanced grant 322817). The Gurdon Institute is funded by the Wellcome Trust (203144) and Cancer Research UK (C6946/A24843)

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Rôle des gènes clock et clockwork orange dans l'horloge circadienne chez Drosophila melanogaster

La plupart des êtres vivants possèdent une horloge interne pour anticiper le jour et la nuit. Cette horloge, dite circadienne, contrôle un grand nombre de fonctions, sur une période de 24 heures. Elle persiste dans des conditions environnementales constantes, indiquant qu elle fonctionne de façon autonome. L horloge moléculaire est composée de gènes d horloge, organisés en boucles de régulation transcriptionnelle négative interconnectées autour d un centre activateur de transcription, le complexe CLK-CYC. CLK-CYC active à la fois les protéines inhibitrices de son activité et la protéine répresseur de l expression de CLK. Nous avons étudié le gène clockwork orange (cwo), potentielle cible de CLK-CYC, de la famille des facteurs de transcription bHLH-Orange. cwo est un nouveau gène d horloge, il forme une nouvelle boucle de l horloge moléculaire, encore centrée autour de CLK-CYC. L expression de cwo est activée par CLK-CYC. CWO favorise l expression des cibles de CLK-CYC et permet de maintenir des oscillations transcriptionnelles robustes. Les rythmes circadiens du comportement de la drosophile sont contrôlés par une horloge cérébrale. Le neuropeptide PDF est exprimé spécifiquement dans certains des neurones d horloge, les s-LNvs. Le développement des s-LNvs, qui commence à la fin de l embryogenèse, est contrôlé par CLK et CYC. Leur absence induit d importants défauts morphologiques des s-LNvs pendant le développement et ils finissent par disparaître au cours de la métamorphose. CLK et CYC contrôlent donc doublement à l horloge circadienne : ils contrôlent à la fois la construction de l horloge cérébrale et le fonctionnement de cette horloge.Most organisms have an internal clock that allows anticipation of night and day transitions. This clock, called circadian clock, controls many functions with a 24 hours period. It persists in constant conditions, showing that it works in an autonomous way. Clock genes organised in interconnected transcriptional negative feedback loops compose the molecular clock. The core of the molecular loops is a transcriptional activator complex, the CLOCK-CYCLE complex. CLK-CYC active the expression of its own inhibitors and the repressor of clock gene expression. We have studied the function of a new gene, clockwork orange (cwo), potential transcriptional target of CLK-CYC. Cwo is a transcription factor from the bHLH-Orange domains family. We show here that cwo is a new clock gene, it forms a new loop centred on the CLK-CYC complex. The cwo expression is activated by CLK-CYC. CWO facilitates CLK-CYC targets activation and allows maintaining robust transcriptional oscillations. A cerebral clock consisting of a network of different clock neurons groups controls drosophila activity rhythms. The Neuropeptide PDF is specifically expressed in some of these clock neurons, the s-LNvs. The s-LNvs development begins at the end of embryogenesis and is controlled by CLK and CYC. CLK or CYC absence induces many morphological defects of s-LNvs during development and the neurons disappear during metamorphosis. CLK and CYC thus control both the construction of the cerebral clock during development and the functioning of the molecular clock within this cerebral clock.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Integrin signaling downregulates filopodia in muscle-tendon attachment

Cells need to sense their environment to ensure accurate targeting to specific destinations. This occurs in developing muscles, which need to attach to tendon cells before muscle contractions can begin. Elongating myotube tips form filopodia, which are presumed to have sensory roles, and are later suppressed upon building the attachment site. Here, we use live imaging and quantitative image analysis of lateral transverse (LT) myotubes in Drosophila to show that filopodia suppression occurs as a result of integrin signaling. Loss of the integrin subunits PS2 and PS increased filopodia number and length at stages when they are normally suppressed. Conversely, inducing integrin signaling, achieved by expression of constitutively dimerised PS cytoplasmic domain (di), prematurely suppressed filopodia. We discovered that the integrin signal is transmitted through the protein Git (G-protein receptor coupled interacting protein) and its downstream kinase Pak (p21-activated kinase). Absence of these proteins causes profuse filopodia and prevents filopodial inhibition by di. Thus, integrin signaling terminates the exploratory behaviour of myotubes seeking tendons, enabling the actin machinery to focus on forming a strong attachment and assembling the contractile apparatus

Production of dissolved organic carbon by Arctic plankton communities: Responses to elevated carbon dioxide and the availability of light and nutrients

The extracellular release of dissolved organic carbon (DOC) by phytoplankton is a potentially important source of labile organic carbon for bacterioplankton in pelagic ecosystems. In the context of increasing seawater partial pressure of CO2 (pCO2), via the oceanic absorption of elevated atmospheric CO2 (ocean acidification), several previous studies have reported increases to the relative amount of carbon fixed into particulates, via primary production (PP), and dissolved phases (DOC). During the summer of 2012 we measured DOC production by phytoplankton communities in the Nordic seas of the Arctic Ocean (Greenland, Norwegian and Barents Sea) from both in situ sampling and during three bioassay experiments where pCO2 levels (targets ~550 µatm, ~750 µatm, ~1000 µatm) were elevated relative to ambient conditions. Measurements of DOC production and PP came from 24 h incubations and therefore represent net DOC production rates, where an unknown portion of the DOC released has potentially been utilised by heterotrophic organisms. Production of DOC (net pDOC) by in situ communities varied from 0.09 to 0.64 mmol C m−3 d−1 (average 0.25 mmol C m−3 d−1), with comparative rates in two of the experimental bioassays (0.04–1.23 mmol C m−3 d−1) and increasing dramatically in the third (up to 5.88 mmol C m−3 d−1). When expressed as a fraction of total carbon fixation (i.e., PP plus pDOC), percentage extracellular release (PER) was 14% on average (range 2–46%) for in situ measurements, with PER in the three bioassays having a very similar range (2–50%). A marked increase in pDOC (and PER) was only observed in one of the bioassays where nutrient levels (nitrate, silicic acid) dropped dramatically relative to starting (ambient) concentrations; no pCO2 treatment effect on pDOC (or PER) was evident across the three experiments. Examination of in situ net pDOC (and PER) found significant correlations with decreasing silicic acid and increasing euphotic zone depth, indicating that nutrient and light availability were strong drivers of the partitioning of primary production between particulate and dissolved phases. Furthermore, the third bioassay experiment had relatively high levels of diatom biomass as well as a strong response to nitrate and silicic acid depletion, and we suggest that nutrient starved or light limited diatom communities may be strong producers of DOC in Arctic ecosystems