Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis

Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo

Apoptosis of cells and their subsequent removal via efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. Here, we developed a genetically encoded fluorescent reporter, CharON, that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. To confront high rate of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment, with some efferocytic macrophages carrying high corpse burden. However, overburdened macrophages were compromised in clearing wound debris, revealing an inherent vulnerability. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo

Selective endocytosis of Ca(2+)-permeable AMPARs by the Alzheimer's disease risk factor CALM bidirectionally controls synaptic plasticity

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca(2+)-permeable (CP) versus heteromeric Ca(2+)-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain

Quantifying Exocytosis by Combination of Membrane Capacitance Measurements and Total Internal Reflection Fluorescence Microscopy in Chromaffin Cells

Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques

Analyse des signaux pour un dispositif de mesure et de stimulation du système nerveux central

- Un des enjeux actuels en Neurosciences est de pouvoir enregistrer simultanément les activités d'un grand nombre de cellules au sein de grands réseaux de neurones, et de pouvoir stimuler de manière dynamique ces réseaux afin d'en contrôler les activités. Le but du projet Neurocom est de réaliser un système multiélectrode haute densité intégré sur silicium, permettant d'enregistrer et de stimuler de grands réseaux de neurones in vitro. Ce dispositif sera constitué d'une microstructure d'électrodes stérilisable hybridée sur un circuit analogique intégré (préamplification, filtrage, multiplexage, stimulation), lui-même interfacé via une carte numérique de commande et acquisition reliée à un PC. Afin de pouvoir mieux appréhender les phénomènes bioélectriques et électrochimiques à l'interface capteur et donc mieux spécifier le cahier des charges et l'architecture du système, la maquette de test NEUROCOM1 a été conçue en électronique discrète et est actuellement utilisée pour conduire différents tests

Forty years on: clathrin-coated pits continue to fascinate

Clathrin mediated endocytosis (CME) is a fundamental process in cell biology and has been extensively investigated throughout the last several decades. Every cell biologist learns about it at some point during their education and the beauty of this process has led many of us to go deeper and make it the topic of our own research. Great progress has been made towards elucidating the mechanisms of CME and the field is becoming increasingly complex with several hundred new publications every year. This makes it easy to get lost in the vast amount of literature and to forget about the fundamentals of the field, based on the careful interpretation of simple observations made over 40 years ago. A study performed by Anderson, Brown and Goldstein in 1977 (Anderson et al., 1977) is a prime example of this. We therefore want to take a step back and examine how this seminal study was pivotal to our understanding of CME and its progression into ever increasing complexity over the last four decades

In Vivo Determination of Fluctuating Forces during Endosome Trafficking Using a Combination of Active and Passive Microrheology

BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes

Polyamine sensitivity of gap junctions is required for skin pattern formation in zebrafish

Gap junctions allow the direct and bidirectional transfer of small molecules between cells. Polyamine sensitivity, which has been observed for a certain gap junction in vitro, confers rectification property to gap junction. Here we report that the polyamine sensitivity of gap junctions in vivo is crucial for skin pattern formation in zebrafish. Transgenic experiments have revealed that several connexin genes were able to rescue the spot phenotype of mutant zebrafish. Mutational analyses of the N-terminal region of connexins revealed that the ExxxE motif, a hypothetical polyamine-binding site, was important for connexin's role in pattern formation. Ectopic expression of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme, also caused stripe pattern changes, which further indicates that the polyamine sensitivity of gap junctions is crucial. This is the first report to show that polyamine sensitivity has a physiologically relevant function and is related to skin pattern formation in animals

Fast Homeostatic Plasticity of Inhibition via Activity-Dependent Vesicular Filling

Synaptic activity in the central nervous system undergoes rapid state-dependent changes, requiring constant adaptation of the homeostasis between excitation and inhibition. The underlying mechanisms are, however, largely unclear. Chronic changes in network activity result in enhanced production of the inhibitory transmitter GABA, indicating that presynaptic GABA content is a variable parameter for homeostatic plasticity. Here we tested whether such changes in inhibitory transmitter content do also occur at the fast time scale required to ensure inhibition-excitation-homeostasis in dynamic cortical networks. We found that intense stimulation of afferent fibers in the CA1 region of mouse hippocampal slices yielded a rapid and lasting increase in quantal size of miniature inhibitory postsynaptic currents. This potentiation was mediated by the uptake of GABA and glutamate into presynaptic endings of inhibitory interneurons (the latter serving as precursor for the synthesis of GABA). Thus, enhanced release of inhibitory and excitatory transmitters from active networks leads to enhanced presynaptic GABA content. Thereby, inhibitory efficacy follows local neuronal activity, constituting a negative feedback loop and providing a mechanism for rapid homeostatic scaling in cortical circuits

The Mechanochemistry of Endocytosis

An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo