11 research outputs found
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
Limited intermixing of synaptic vesicle components upon vesicle recycling.
No full textSynaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing similar to 19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH-sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin-tagged proteins. While the native proteins had a clustered distribution, the GFP-tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin-tagged vesicle proteins are less well integrated in clusters
Endosomal sorting of readily releasable synaptic vesicles.
No full textNeurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.</jats:p
Untersuchung von endosomalem Recycling von synaptischen Vesikeln
No full textWährend der Kommunikation in chemischen Synapsen verschmelzen kleine mit Neurotransmitter gefüllte synaptische Vesikel, die sich in der prä-synaptischen Nervenendigung (Bouton) befinden, mit der Zellmembran (Exozytose) and entlassen ihren Neurotransmitter in den synaptischen Spalt. Die Vesikel werden zurück in das Bouton gestülpt (Endozytose), wo sie mit neu befüllt werden und für weitere Exozytose-Runden in den Vesikel Pool integriert werden (Vesikel Recycling). Wie synaptische Vesikel wiederverwertet werden wird bereits seit mehr als drei Jahrzehnten diskutiert. Hier haben wir die Beteiligung von Endosomen beim Recycling von synaptischen Vesikeln anhand einer Vielzahl an Bildgebungsverfahren untersucht. Erstens haben wir drei neue Exozytose-Reporter generiert, indem wir eine pH-abhängige GFP Variante (pHluorin) mit der lumenalen Domäne der endosomalen Proteine Vti1a, Syntaxin 13 und Syntaxin 6 gekoppelt haben. Wir haben diese Reporter in Hippocampus Neuronen exprimiert und ihre Zirkulation während verschiedener Stimulationen verglichen. Dabei haben wir gefunden, dass sie vorzugsweise durch eine readily releasable pool Stimulation (RRP; 20Hz / 2 Sekunden) mobilisiert werden. Zweitens, indem wir Photo-oxidations Elektronen Mikroskopie von FM Farbstoff-gefärbten RRP Vesikeln verwendet haben, haben wir das vorübergehende Aufkommen großer gefärbter Organellen 10 Sekunden nach der Stimulation beobachtet, was auf die Verschmelzung von endozytierten Vesikeln mit Endosomen hindeutete. Als nächstes haben wir hochauflösende Dünnschnitt STED Mikroskopie und Video-geschwindigkeits Live-STED Mikroskopie angewendet um zu bestätigen, dass RRP Vesikel mit Endosomen (gekennzeichnet durch Rab5-GFP) verschmelzen. Interessanterweise schien stärkere recycling pool Stimulation (20 Hz / 30 Sekunden) keine bessere Kolokalisation von Vesikeln und Endosomen hervorzurufen. Drittens haben wir zytosolische Syntaxin 13 Fragmente exprimiert um die Bildung von endosomalen Fusionskomplexen, und somit die Verschmelzung von Vesikeln und Endosomen, zu blockieren. Auffallenderweise zog diese Störung eine drastische ~60% Reduktion der RRP Größe nach sich, was die Wichtigkeit endosomaler Funktion für den RRP hervorhebt. Viertens zeigten 2-Farben STED Messungen, dass synaptische Vesikel in der Zellmembran als mehr-Komponenten Verbünde bestehen, während der Vergleich von allgemeinem Pool und kürzlich endozytiertem Pool isolierter Vesikel aufdeckte, dass Endozytose unreine Vesikel zurückgewinnt und so endosomales Aufreinigen bedingt. Dies ist eine der ersten Untersuchungen, die einen molekularen und mechanistischen Bestimmungsfaktor von RRP Vesikeln vorschlägt. Wir schlussfolgern, dass die RRP Vesikel in einem hochgradig Verschmelzungs-kompetenten Zustand erhalten werden da sie durch Endosomen geschleust werden, so dass sie die privilegierten Vesikel sind, die während physiologischer Aktivität benutzt werden.During communication at chemical synapses, small neurotransmitter-filled synaptic vesicles, housed in the pre-synaptic nerve terminal (bouton), fuse with the plasma membrane (exocytosis) and release their neurotransmitter content into the synaptic cleft. The fused synaptic vesicles are retrieved into the bouton (endocytosis), where they are refilled with neurotransmitter and re-integrated into the synaptic vesicle pool for further rounds of exocytosis (vesicle recycling). Through which pathways the synaptic vesicles recycle has been a matter of debate for more than three decades. Here we investigated the involvement of endosomes in the recycling of synaptic vesicles by resorting to a variety of novel imaging approaches. First, we generated three novel exocytosis reporters by fusing a pH-sensitive GFP variant (pHluorin) to the lumenal domain of the early endosomal fusion proteins Vti1a, Syntaxin 13 and Syntaxin 6. We compared the recycling of these reporters expressed in hippocampal neurons upon different stimulation paradigms and found them to be preferentially mobilized during readily releasable pool stimulation (RRP; 20 Hz/ 2 seconds). Second, using photo-oxidation electron microscopy of FM dye-labeled RRP vesicles, we observed a transient appearance of larger labeled organelles at 10 seconds after stimulation, indicative of endosomal fusion of endocytosed vesicles. We next used high-resolution thin-section STED microscopy and video-rate live-STED microscopy, to confirm that endocytosed RRP vesicles fuse with bona fide endosomes (identified by Rab5-GFP). Interestingly, stronger recycling pool stimulation (20 Hz/30 seconds) did not seem to result in higher colocalization of vesicles and endosomes in these experiments. Third, we used expression of soluble Syntaxin 13 fragment to block formation of endosomal fusion complexes and therefore endosomal fusion of synaptic vesicles. Strikingly, this perturbation resulted in a drastic ~60% decrease in RRP size, underscoring the importance of endosomal function for the RRP. Fourth, 2-color STED measurements indicated that synaptic vesicles persist as multi-component clusters in the plasma membrane, while comparison of the composition of general pool and recently endocytosed isolated synaptic vesicles revealed that endocytosis causes retrieval of dirty vesicles, thus necessitating endosomal sorting. This is one of the first studies proposing a molecular and mechanistic determinant of RRP vesicles. We conclude that the RRP vesicles are maintained in a highly fusion-competent state by recycling through endosomes, making them the privileged vesicles drawn upon during physiological activity
Hydra: a scalable proteomic search engine which utilizes the Hadoop distributed computing framework.
No full textpeer reviewedBACKGROUND: For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. RESULTS: We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. CONCLUSION: The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources
Author Correction: Lifespan-extending interventions induce consistent patterns of fatty acid oxidation in mouse livers.
No full textIntegration of time-series meta-omics data reveals how microbial ecosystems respond to disturbance
No full textpeer reviewedThe development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive
phenotypic plasticity and niche complementarity in oleaginous microbial populations from a
biological wastewater treatment plant. We perform meta-omics analyses (metagenomics,
metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months
at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover
four distinct fundamental niche types. Throughout the time-series, we observe a major,
transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst
community members. Ex situ bioreactor experiments confirm that responses occur within
five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our
results show that community resistance and resilience are a function of phenotypic plasticity
and niche complementarity, and set the foundation for future ecological engineering efforts
Comparative integrated omics: identification of key functionalities in microbial community-wide metabolic networks
No full textpeer reviewedBACKGROUND: Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships is essential for ultimately driving these systems towards desired outcomes, e.g., the enrichment in organisms capable of accumulating valuable resources during BWWT.
METHODS: A comparative integrated omic analysis including metagenomics, metatranscriptomics and metaproteomics was carried out to elucidate functional differences between seasonally distinct oleaginous mixed microbial communities (OMMCs) sampled from an anoxic BWWT tank. A computational framework for the reconstruction of community-wide metabolic networks from multi-omic data was developed. These provide an overview of the functional capabilities by incorporating gene copy, transcript and protein abundances. To identify functional genes, which have a disproportionately important role in community function, we define a high relative gene expression and a high betweenness centrality relative to node degree as gene-centric and network topological features, respectively.
RESULTS: Genes exhibiting high expression relative to gene copy abundance include genes involved in glycerolipid metabolism, particularly triacylglycerol lipase, encoded by known lipid accumulating populations, e.g., Candidatus Microthrix parvicella. Genes with a high relative gene expression and topologically important positions in the network include genes involved in nitrogen metabolism and fatty acid biosynthesis, encoded by Nitrosomonas spp. and Rhodococcus spp. Such genes may be regarded as ‘keystone genes’ as they are likely to be encoded by keystone species.
CONCLUSION: The linking of key functionalities to community members through integrated omics opens up exciting possibilities for devising prediction and control strategies for microbial communities in the future
Community-integrated omics links dominance of a microbial generalist to fine-tuned resource usage
No full textpeer reviewedMicrobial communities are complex and dynamic systems that are primarily structured
according to their members’ ecological niches. To investigate how niche breadth (generalist
versus specialist lifestyle strategies) relates to ecological success, we develop and apply an
integrative workflow for the multi-omic analysis of oleaginous mixed microbial communities
from a biological wastewater treatment plant. Time- and space-resolved coupled metabolomic
and taxonomic analyses demonstrate that the community-wide lipid accumulation
phenotype is associated with the dominance of the generalist bacterium Candidatus Microthrix
spp. By integrating population-level genomic reconstructions (reflecting fundamental
niches) with transcriptomic and proteomic data (realised niches), we identify finely tuned
gene expression governing resource usage by Candidatus Microthrix parvicella over time.
Moreover, our results indicate that the fluctuating environmental conditions constrain the
accumulation of genetic variation in Candidatus Microthrix parvicella likely due to fitness
trade-offs. Based on our observations, niche breadth has to be considered as an important
factor for understanding the evolutionary processes governing (microbial) population sizes
and structures in situ
