777 research outputs found
Synaptic membrane proteins form stable microdomains in early endosomes.
No full textIn the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein-protein and protein-lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission-depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl-beta-cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein-free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling. Microsc. Res. Tech. 73:606-617, 2010. (C) 2009 Wiley-Liss, Inc
Endosomal fusion upon SNARE knockdown is maintained by residual SNARE activity and enhanced docking.
No full textSNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically similar to 10%) were sufficient for a substantial amount of SNARE-SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery
The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation
No full textSoluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites
SNARE function is not involved in early endosome docking.
No full textDocking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F(ab) fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function
ULF vlny/fluktuace ve forešoku: Statistický přístup
No full textTitle: ULF waves/fluctuations in the foreshock: Statistical approach Author: Anna Mita Department: Department of Surface and Plasma Science Supervisor: Prof. RNDr. Jana Šafránková, DrSc., Department of Surface and Plasma Science Abstract: The thesis deals with ultra-low frequency (ULF) wave activity in the terrestrial foreshock, a turbulent region upstream of the bow shock that plays a crucial role in the interaction of the solar wind with the magnetosphere. In the thesis, we analyze some aspects of the behavior of ULF waves/fluctuations of the magnetic field, using a statistical point of view. We focused (i) on the boundary between the pure solar wind and wave activity region represented by enhanced ULF fluctuations, and on the changes of the boundary position with solar wind conditions and interplanetary magnetic field orientation; and (ii) on growth rates of foreshock ULF fluctuations at different distances from the bow shock with a special highlight on a region close to the Moon. In both statistical studies, we use THEMIS/ARTEMIS data and describe the behavior of fluctuations by the standard deviations in the ULF range of 0.0017-0.17 Hz. We present a novel coordinate system, determine so-called "foreshock ULF boundary" and report its realistic shape that is consistent with the theory of the foreshock...Název práce: ULF vlny/fluktuace ve forešoku: Statistický přístup Autor: Anna Mita Katedra: Katedra fyziky povrchů a plazmatu Vedoucí disertační práce: Prof. RNDr. Jana Šafránková, DrSc., Katedra fyziky povrchů a plazmatu Abstrakt: Předložená práce se zabývá ULF (Ultra Low Frequency) fluktuacemi v turbulentní oblasti před rázovou vlnou (forešok), která hraje klíčovou roli v interakci slunečního větru s magnetosférou Země. V práci analyzujeme některé aspekty chování ULF vln/fluktuací magnetického pole s využitím statistického zpracování dat naměřených dvěma družicemi projektu THEMIS/ARTEMIS. Zaměřili jsme se (i) na hranici mezi neporušeným slunečním větrem a oblastí charakterizovanou vysokými ULF fluktuacemi a na změny polohy této hranice v závislosti na podmínkách ve slunečním větru a na orientaci meziplanetárního magnetického pole; a (ii) na rychlost růstu nebo tlumení ULF vln/fluktuací v různých vzdálenostech od rázové vlny se zvláštním zřetelem na oblast blízko Měsíce. Ve statistických studiích popisujeme chování fluktuací pomocí směrodatných odchylek v rozsahu 0,0017-0,17 Hz. V práci představujeme nový souřadnicový systém umožňující určit tzv. "foreshock ULF boundary", diskutujeme nalezený tvar a ukazujeme, že je v souladu s teorií formování forešoku. Ve druhé studii se soustřeďujeme na růst (případně...Katedra fyziky povrchů a plazmatuDepartment of Surface and Plasma ScienceFaculty of Mathematics and PhysicsMatematicko-fyzikální fakult
ULF waves/fluctuations in the foreshock: Statistical approach
No full textTitle: ULF waves/fluctuations in the foreshock: Statistical approach Author: Anna Mita Department: Department of Surface and Plasma Science Supervisor: Prof. RNDr. Jana Šafránková, DrSc., Department of Surface and Plasma Science Abstract: The thesis deals with ultra-low frequency (ULF) wave activity in the terrestrial foreshock, a turbulent region upstream of the bow shock that plays a crucial role in the interaction of the solar wind with the magnetosphere. In the thesis, we analyze some aspects of the behavior of ULF waves/fluctuations of the magnetic field, using a statistical point of view. We focused (i) on the boundary between the pure solar wind and wave activity region represented by enhanced ULF fluctuations, and on the changes of the boundary position with solar wind conditions and interplanetary magnetic field orientation; and (ii) on growth rates of foreshock ULF fluctuations at different distances from the bow shock with a special highlight on a region close to the Moon. In both statistical studies, we use THEMIS/ARTEMIS data and describe the behavior of fluctuations by the standard deviations in the ULF range of 0.0017-0.17 Hz. We present a novel coordinate system, determine so-called "foreshock ULF boundary" and report its realistic shape that is consistent with the theory of the foreshock..
Dream on, but with your eyes open: An obituary for Ulf Kadritzke (1943-2020)
No full textUlf Kadritzke war der PROKLA als Autor und Mitglied des Redaktionsbeirats viele Jahre eng verbunden. Mit seinen Ideen und seinem Engagement hat er nicht nur die PROKLA bereichert und geprägt. Am 14. November 2020 verstarb Ulf Kadritzke überraschend.Ulf Kadritzke was closely associated with PROKLA for many years as an author and a member of the editorial advisory board. With his ideas and commitment, he enriched and shaped not only PROKLA. Ulf Kadritzke passed away unexpectedly on November 14, 2020
Ulf eta Cornelia: Berlingo maitasun istorio bat
No full text“Ulf and Cornelia: A Berlin love story” is part of the PhD thesis Berlin fin de millenium: An Experiment in Corporeal Ethnography, carried out between 1998 and 2003. In this article, the author puts into practice his investigative endeavor as a discovery process, narrating his encounters with Ulf and Cornelia, two bodies in transit in a Berlin characterized by its dramatic urban transformation. The focus of the ethnography is Ulf’s enamored experience. Using wonder as a knowledge technique, we will dive in the realm previous to the formation of stable identities, meaning the domain of corporeal stimulus and unconscious impulses. The subjects that appear in the scenes of this article, the author himself and the city where they live are in construction.“Ulf y Cornelia: una historia de amor berlinesa” es un apartado que pertenece a la tesis Berlín fin de milenio: un experimento en la etnografía corporal, realizada entre 1998 y 2003. En este artículo, el autor lleva a cabo una labor de investigación puesta en práctica como un proceso de descubrimientos, relatando sus encuentros con Ulf y Cornelia, dos cuerpos en tránsito en un Berlín sumido en una dramática transformación urbana. La etnografía está enfocada particularmente en la experiencia de enamoramiento de Ulf. Utilizando la capacidad de asombro como técnica para el conocimiento, nos sumergiremos en el ámbito anterior a la formación de identidades estables, es decir, en el dominio de estímulos corporales e impulsos inconscientes. Se trata, pues, de un artículo donde tanto los sujetos en escena como el propio autor y la ciudad en la que viven están en proceso de construcción.“Ulf eta Cornelia: maitasun istorio bat Berlinen”, 1999-2003 urteen artean garatutako Berlin fin de millenium: gorputz etnografíaren gaineko esperimentu bat tesiaren ataletako bat da. Artikulu honetan, autoreak, Ulfekin eta Corneliarekin, Berlin behin behineko bizilekutzat duten bi gorputzekin, bizitako uneak kontatzen dizkigu. Zehazki, Ulfen maitemintzea izango da etengabeko aurkikuntza prozesuan oinarritutako etnografía honen ardatza. Lilurarako gaitasuna ezagutzarako teknika bezala erabiliaz, nortasun egonkorrak eratu aurretiko eremura eramango gaitu, hau da, gorputz-estimulu eta bulkada inkontzienteen mundura. Azpimarratu behar da, artikulu honetan protagonista diren pertsonak, autorea bera eta bizi diren hiria eraikuntza prozesuan daudela
A functional characterisation of early endosomal SNARE-proteins
No full textEin Großteil des intrazellulären Transports erfolgt über das Abschnüren, den Transport und die anschließende Fusion von Vesikeln, wobei dieser letzte Schritt von SNARE-Proteinen bewerkstelligt wird. Neben deren Funktion in der Fusion wird schon seit einiger Zeit auch über eine Beteiligung an dem, der Fusion vorausgehenden Prozess des Docking, d.h. der stabilen Verknüpfung der Organellen spekuliert. Diese Frage wurde in der vorliegenden Arbeit an frühen Endosomen untersucht. Frühe Endosomen bilden ein zentrales Sortierungskompartiment, in dem die meisten Endozytoserouten zusammenlaufen und können untereinander fusionieren, was sie zu einem viel genutzten Modellsystem gemacht hat. Um den Einfluss der SNARE-Proteine auf das Docking von frühen Endosomen analysieren zu können, musste zunächst ein geeignetes Assay entwickelt werden, da sich das für das vakuoläre System bekannte Aggregationsassay als nicht geeignet herausstellte. Mit dem neuen Assay konnte gezeigt werden, dass zwar EEA1 und Rab5 wie erwartet wichtig für das Docking von frühen Endosomen sind, dass dieses aber von einer SNARE-Funktion unabhängig ist, da weder das Verhindern der SNARE-Aktivierung durch mutiertes alphaSNAP, noch die Zugabe von löslichen SNARE-Fragmenten oder gegen die SNAREs gerichteter Fab-Fragmente einen großen Einfluss auf das Docking hatte, während die Fusion signifikant inhibiert wurde. Versuche, die SNARE-Regulation über die Rekonstitution der endosomalen SNAREs in Liposomen und anschließende Fusion mit frühen Endosomen zu analysieren, haben sich als sehr schwierig herausgestellt. Das Problem lag hierbei nicht etwa darin, dass keine Fusion erzielt worden wäre, sondern im Gegenteil daran, dass selbst proteinfreie Liposomen mit Endosomen fusionierten und zwar in vergleichbarem Umfang und praktisch identischer Kinetik wie SNARE-haltige Liposomen. Durch die Vergrößerung der Liposomen (und den dadurch reduzierte Krümmungsstress) stieg die Spezifität der Reaktion etwas an, so dass Proteoliposomen doppelt so stark mit Endosomen fusionierten wie proteinfreie Liposomen. Der vielversprechendste Weg scheint jedoch ein top-down Ansatz zu sein, d.h. Endosomen zu solubilisieren, einzelne Proteine über Immundepletion aus dieser Mischung zu entfernen und aus den verbleibenden Proteinen und Lipiden ueber Detergenzentfernug Proteoliposomen mit sehr endosomenähnlicher Zusammensetzung zu erzeugen. Für das Docking und die Fusion von Endosomen ist das Zusammenwirken von zahlreichen Faktoren erforderlich, welche in der Regel in spezialisierten Membrandomänen konzentriert vorliegen. Die Regulation dieser Domänen ist jedoch noch weitgehend unklar und wurde daher im letzten Teil der vorliegenden Arbeit für SNAREs und Synaptophysin untersucht. Bisher war es aufgrund der geringen Größe von Endosomen (ungefähr 200 nm Durchmesser) sehr schwer, Aussagen über unterschiedliche endosomale Domänen zu treffen, da sie im Bereich der Auflösungsgrenze konventioneller Lichtmikroskopie liegen. Für die vorliegende Studie konnte jedoch auf die neuentwickelte STED-Mikroskopie zurückgegriffen werden, wobei das verwendete kommerziell erhältliche Gerät eine Auflösung von 70-90 nm besitzt und die endosomale Membran dadurch einer Analyse zugänglich machte. Das im zweiten Abschnitt gewonnene Wissen über die Fusion von Liposomen mit Endosomen konnte hier genutzt werden, um über diese Fusion die Lip! idzusammensetzung der Endosomen zu verändern. Allerdings führten weder erhöhte Cholesterinkonzentrationen noch die Zufuhr von PI und PIP2 zu einer Veränderung in der Zahl der Cluster, was auf eine relative Stabilität dieser hindeutet. Interessanterweise scheinen auch die als Membranorganisatoren ins Spiel gebrachten Rab-Proteine keineswegs essentiell für diese Domänen zu sein, da die Extraktion der Rab-Proteine durch GDI praktisch keinen Einfluss auf die Domänenanzahl der untersuchten Proteine hatte.Most intracellular transport processes occur via budding and fusion of vesicles, where fusion is dependent on SNARE proteins. Fusion is preceded by docking, the close contact of vesicles which has been proposed to depend on SNARE proteins as well from work in C. elegans and S. Cerevesiae. As this is still heavily debated the role of SNAREs in docking was analysed for early endosomes. Early endosomes form the first compartment reached by most routes of endocytosis and can undergo homotypic fusion which has rendered them a widely used model system. A new fluorescence based assay was developed which allowed to monitor their docking and fusion in parallel. Previous suggestions of EEA1 and Rab5 as important docking factors could be confirmed whereas no influence of SNARE proteins was found. Even though SNARE-function was blocked by three different means: competitive inhibition, antibodies and block of activation, docking remained intact whereas fusion was reduced significantly. A role of SNARE-function in early endosomal docking could therefore be ruled out. A different set of experiments aimed at the analysis of SNARE-regulation by use of a minimal system, i.e. proteoliposomes containing early endosomal SNARE proteins. Even though these proteoliposomes can fuse with each other in a SNARE-specific manner, fusion of proteoliposomes with endosomes was dependent on endosomal but not on liposomal protein content, i.e. trypsinated proteoliposomes reached similar fusion rates with endosomes as control proteoliposomes. The specificity of fusion could be increased when endosomes were solubilised completely and these mixtures were used for proteoliposome formation. Immunodepletion of selected proteins from these mixtures before reconstitution might therefore be used in the future to adress their role even though the assay has to be optimised further beforehand. Fusion and docking take place at specialised regions of the plasma membrane. Therefore, it was analysed how SNARE proteins and the vesicle marker synaptophysin distribute on endosomes. Domain analysis was performed with the recently developed superresolution STED-Microscope. The high fusion rate of liposomes with endosomes could finally be used in this context as a relatively non-invasive tool to change the lipid composition of early endosomes and thereby address the role of lipids in domain formation and stability. This was feasible because fusion was not dependent on a certain lipid composition so that liposomes with high concentrations for example of phosphatidyl inositol could be used to have a large impact on the endosomal lipid composition. Nevertheless the cluster of synaptophysin remained intact and were only destroyed after treatment with beta-methyl-cyclodextrin showing that its domains are rather stable. Interesting differences were observed between the distribution of syntaxin-1 on pure lipid membranes and endosomes as it formed much more clusters on the latter
Simulation of Bounce Resonance ULF Wave-Particle Interactions
No full textPoloidal mode ultra-low-frequency (ULF) waves with high azimuthal mode number (high-m) are common throughout Earth's magnetosphere. The electric fields in these waves are on the order of tens of millivolts per meter, and are large enough to energize ions and electrons via drift- and drift-bounce wave-particle resonance. In this article, we present full orbit test-particle simulations of drift-bounce resonance using a numerical model of ULF waves. The model uses a dipole magnetic field and can accommodate a realistic ionosphere with height-resolved Pedersen and Hall conductivity. It is demonstrated that second-harmonic poloidal mode waves can energize ions to tens of keV depending on the wave amplitude. The test-particle simulations also predict ion flux oscillations that have energy dispersion similar to many satellite observations. It will be shown that features in the energy dispersion are in agreement with theoretical considerations.CPCI-S(ISTP)[email protected]; [email protected]
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