Podcasts about vesicle

"What on earth is a Death Vesicle, Dr. K?" you ask. We'll learn all about this, about biofilms and so much more in today's episode. Music by Muzaproduction from Pixabayhttps://www.instagram.com/sciencewithdr_k/ https://www.youtube.com/channel/UC2EttB4pjdZ4WrU3-z4RqPghttps://www.sciencedaily.com/releases/2024/03/240305134236.htmhttps://www.sciencedirect.com/science/article/pii/S259020752400008X?via%3Dihub

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551580v1?rss=1 Authors: Mahapatra, S., Takahashi, T. Abstract: After exocytosis, release sites are cleared of vesicular residues to be replenished with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this mechanism. However, physiological significance of the site-clearance mechanism among diverse central synapses remains unknown. Here, we tested this using action-potential evoked EPSCs in mouse brainstem and hippocampal slices in physiologically optimized condition. Pharmacological block of endocytosis enhanced synaptic depression at brainstem calyceal fast synapses, whereas it attenuated synaptic facilitation at hippocampal CA1 slow synapses. Block of scaffold protein activity likewise enhanced synaptic depression at calyceal synapses but had no effect at hippocampal synapses. At calyceal synapses, enhancement of synaptic depression by blocking endocytosis or scaffold activity occurred at nearly identical time courses with a time constant of several milliseconds starting immediately after the stimulation onset. Neither endocytic nor scaffold inhibitors prolonged the recovery from short-term depression. We conclude that endocytic release-site clearance can be a universal phenomenon supporting vesicle replenishment across fast and slow synapses, whereas presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550521v1?rss=1 Authors: Dou, D., Aiken, J., Holzbaur, E. L. F. Abstract: Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1B{beta} complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549017v1?rss=1 Authors: Bera, M., Grushin, K., Kalyana Sundaram, V., Shahanoor, Z., Chatterjee, A., Radhakrishnan, A., Lee, S., Padmanarayana, M., Coleman, J., Pincet, F., Rothman, J. E., Dittman, J. S. Abstract: The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca2+-triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.11.548513v1?rss=1 Authors: Han, Y., Li, M., Zhao, B., Wang, H., Liu, Y., Liu, Z., Xu, J., Yang, R. Abstract: Microtubule based vesicle transport is an essential way for protein delivery. Selective transport of vesicles to soma-dendritic area or to axon is critical for maintaining neuron polarity. Although neuronal axon is full filled with plus-end-out microtubules, vesicles containing dendritic protein, like TfR labeled vesicles, barely been transported to the axon. How does neuron maintain the transport of TfR labeled vesicles dendritically? Using live cell imaging and loss of function assay, we found that KIF13A is a major transporter for TfR vesicles at soma-dendritic area. Over-expression of KIF13A drives dendritic TfR vesicles to axon. We further found that MARK2 binds to and phosphorylates KIF13A at 14-3-3 binding motif inhibiting its kinesin activity at the axon initial segment (AIS), which is critical for maintaining TfR vesicle at the soma-dendritic area. Overall, our results suggested a novel kinesin-based mechanism for dendritic-selective transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547926v1?rss=1 Authors: Pauwels, J., Van de Steene, T., Van de Velde, J., Eyckerman, S., Gevaert, K. Abstract: Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain protein concentrations that vastly exceed those of the EV proteome and contain particles in the same size and density range (e.g. protein aggregates and apolipoprotein particles). Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion and precipitation methods. Here, we describe an approach based on 300 kDa MWCO filtration, which allows processing of multiple samples in parallel within a reasonable timeframe and at moderate cost. We demonstrate that our strategy is capable of quantitatively retaining EV particles on filters, whilst allowing extensive washing with relatively high percentages of the mild detergent TWEEN-20. In addition, we provide evidence that the retained EVs can be recuperated from the filter for qualitative studies or can be directly lysed on the filter for the recovery of the EV protein cargo for proteome analysis. Applying this strategy on MCF7 conditioned medium using different percentages of serum, we observed dramatic changes in the EV proteome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547997v1?rss=1 Authors: Suzuki, S., West, M., Zhang, Y., Fan, J. S., Roberts, R. T., Odorizzi, G., Emr, S. D. Abstract: Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intralumenal vesicles (ILVs) at endosomes by Endosomal Sorting Complex Required for Transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.05.547834v1?rss=1 Authors: Bera, M., Radhakrishnan, A., Coleman, J., Kalyana Sundaram, R. V., Ramakrishnan, S., Pincet, F., Rothman, J. E. Abstract: The synaptic vesicle protein Synaptophysin has long been known to form a complex with the v-SNARE VAMP, but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully-defined reconstitution and single-molecule measurements, we now report that Synaptophysin functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Synaptophysin directs the assembly of 12 plus-or-minus sign 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 plus-or-minus sign 1 and 5 plus-or-minus sign 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Synaptophysin is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547658v1?rss=1 Authors: Hernandez-Diaz, S., Martinez-Olondo, P., Sanchez-Mirasierra, I., Montecinos-Oliva, C., Ghimire, S., Soukup, S. Abstract: Synapses are specialized neuronal compartments essential for brain communication. Neuronal communication mostly relies on the adequate supply and renovation of synaptic vesicles that fuse with the plasma membrane and release neurotransmitters in response to action potentials. Autophagy is an evolutionary conserved cellular mechanism essential for homeostasis that can be locally regulated in the neuronal synapse. However, the precise mechanisms controlling synaptic autophagy, especially during neuronal communication and pathological scenarios, remain elusive. Here, we report that neuronal activity and amino-acid deprivation regulate synaptic autophagy via distinct molecular mechanisms. We show that Synaptogyrin, a highly abundant presynaptic protein found in synaptic vesicles, is a novel negative regulator of synaptic autophagy in response to neuronal activity without affecting autophagy induction via amino-acid deprivation. We demonstrate that loss of Synaptogyrin modifies the localization of the autophagy protein Atg9 and boosts autophagosome formation at the synapse. Furthermore, activation of synaptic autophagy by loss of Synaptogyrin, but not by amino acid deprivation, leads to the degradation of synaptic vesicle components via autophagy. Reducing the levels of Synaptogyrin results in the degradation of synaptic TAU via autophagy and restores autophagy dysfunction observed in a Drosophila Tau model of Frontotemporal Dementia (FTD). Our data provide novel and valuable information to understand how autophagy is regulated at the synapse in response to neuronal activity and how this process participates in neuronal (dys)function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547132v1?rss=1 Authors: Cotter, R., Hellums, M., Grey, D., Batista, D., Pfitzer, J., Reed, M. N., Gramlich, M. W. Abstract: We have shown that tauopathy models display early-stage hyperexcitability due to increased presynaptic glutamate release that is mediated by an increase in vesicular glutamate transporter-1 (VGlut1). This hyperexcitability increases energy demand which in turn would increase demand on mitochondria. It is unclear how early-stage presynaptic changes in glutamate release are supported by or influence the function of mitochondria. Using Large Area Scanning Electron Microscopy (LA-SEM) and fluorescence microscopy, we demonstrate that mitochondrial changes in morphology, structure, and function in CA1/CA3 hippocampal neurons decrease resting mitochondrial membrane potential in P301L mice. However, P301L mitochondria maintain a high membrane potential during levels of high activity, suggesting that they can support increased energy demand during hyperexcitability. These activity-dependent differences in membrane potential can be rescued by inhibiting ATP-dependent VGlut1 vesicle refilling. This indicates that the increased VGlut1 per vesicle observed in P301L mice contributes to the differences in mitochondria membrane potential. Notably, the mitochondrial dysfunction in P301L mice occurs before any observable alterations in presynaptic release mechanics, suggesting these changes may represent early therapeutic targets. Finally, we propose a model of increased glutamate-mediated changes in mitochondrial morphology and function in P301L neurons that represents a potentially targetable pathway to reduce or arrest neurodegeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546143v1?rss=1 Authors: Bucher, M. L., Dunn, A. R., Bradner, J. M., Egerton, K. S., Burkett, J. P., Johnson, M. A., Miller, G. W. Abstract: Dopaminergic neurons of the substantia nigra exist in a persistent state of vulnerability resulting from high baseline oxidative stress, high energy demand, and broad unmyelinated axonal arborizations. Impairments in the storage of dopamine compound this stress due to cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant, and this toxicity is thought to contribute to the dopamine neuron degeneration that occurs Parkinson's disease. We have previously identified synaptic vesicle glycoprotein 2C (SV2C) as a modifier of vesicular dopamine function, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Here, we adapted a previously published in vitro assay utilizing false fluorescent neurotransmitter 206 (FFN206) to visualize how SV2C regulates vesicular dopamine dynamics and determined that SV2C promotes the uptake and retention of FFN206 within vesicles. In addition, we present data indicating that SV2C enhances the retention of dopamine in the vesicular compartment with radiolabeled dopamine in vesicles isolated from immortalized cells and from mouse brain. Further, we demonstrate that SV2C enhances the ability of vesicles to store the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) and that genetic ablation of SV2C results in enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced vulnerability in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants, and helps maintain the integrity of dopaminergic neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Glen Hodge is a life long geek and education advocate. He has type 1 diabetes but boy-oh-boy that ain't what he's here to talk about. Glen pops by the studio for a little surprise story for the fellas. They have no idea about what Glen is here to speak about which makes for a hilariously shocking episode. It all started on a date when Glen felt a pain in his lower abdomen followed by some blood in his ejaculate. It all ended with a trip to the max-chill urologist when he found out he is passing stones through his semen. Tune in for one of the wildest/most fascinating stories we've heard to date. Join the post-episode conversation over on Discord! https://discord.gg/expeUDN

Glen Hodge is a life long geek and education advocate. He has type 1 diabetes but boy-oh-boy that ain't what he's here to talk about. Glen pops by the studio for a little surprise story for the fellas. They have no idea about what Glen is here to speak about which makes for a hilariously shocking episode. It all started on a date when Glen felt a pain in his lower abdomen followed by some blood in his ejaculate. It all ended with a trip to the max-chill urologist when he found out he is passing stones through his semen. Tune in for one of the wildest/most fascinating stories we've heard to date. Join the post-episode conversation over on Discord! https://discord.gg/expeUDN

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537227v1?rss=1 Authors: Jimenez, A. J., Perez, F. Abstract: Cellular processes are regulated by the formation of specific membrane domains with different lipid and protein compositions. Small GTPases play a role in symmetry breaking and compartmentalization, with early endosomes presenting Rab5-enriched domains that regulate vesicle tethering and fusion. Rabaptin5, which binds activated Rab5 and forms dimers, is essential for endosome fusion and promotes Rab5 recruitment to membranes. Liquid-liquid phase separation (LLPS) is a mechanism for the biogenesis and maintenance of membrane-less organelles and intracellular organization. We show that several partners of Rab proteins, such as Rabaptin5, have phase-separation properties. In particular, Rabaptin5 form condensates close to membranes, while promoting the enrichment of several Rabs involved in early steps of endocytosis. We propose that phase separation of Rab partners ensures efficient recruitment of their respective Rabs and domain formation and maintenance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535716v1?rss=1 Authors: Montgomery, A. C., Mendoza, C. S., Garbouchian, A., Quinones, G. B., Bentley, M. Abstract: Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, both dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon- selective trafficking. We conclude that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.20.533583v1?rss=1 Authors: Song, S.-H., Augustine, G. J. Abstract: Synapsins cluster synaptic vesicles (SVs) to provide a reserve pool (RP) of SVs that maintains synaptic transmission during sustained activity. However, it is unknown how synapsins cluster SVs. Here we show that either liquid-liquid phase separation (LLPS) or tetramerization-dependent cross-linking can cluster SVs, depending upon whether a synapse is excitatory or inhibitory. Cell-free reconstitution revealed that both mechanisms can cluster SVs, with tetramerization bring more effective. At inhibitory synapses, perturbing synapsin-dependent LLPS impairs SV clustering and synchronization of GABA release, while perturbing synapsin tetramerization does not. At glutamatergic synapses, the opposite is true: synapsin tetramerization enhances clustering of glutamatergic SVs and mobilization of these SVs from the RP, while synapsin LLPS does not. Comparison of inhibitory and excitatory transmission during prolonged synaptic activity revealed that synapsin LLPS serves as a brake to limit GABA release, while synapsin tetramerization enables rapid mobilization of SVs from the RP to sustain glutamate release. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.26.530068v1?rss=1 Authors: Nadiminti, S. S. P., Dixit, S. B., Ratnakaran, N., Hegde, S., Swords, S., Grant, B. D., Koushika, S. P. Abstract: Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin- to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin- acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.23.529257v1?rss=1 Authors: Bebelman, M. P., Crudden, C., Snieder, B., Thanou, E., Langedijk, C. J. M., Viola, M., Eleonora, S., Baginska, U., Cotugno, O., Bebelman, J. P. M., van Eijndhoven, M. A. J., Bosch, L., Li, K. W., Smit, M. J., van Niel, G., Smit, A. B., Verweij, F. J., Pegtel, D. M. Abstract: Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad-spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIII{beta} as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.14.528546v1?rss=1 Authors: Stoops, E. H., Ferrin, M. A., Jorgens, D. M., Drubin, D. G. Abstract: Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actin's role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast WASP, an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin tails. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Dr. Mike Blatt is the Regius Professor of Botany at the University of Glasgow and Adjunct Professor at Pennsylvania State University. Mike is a cell biologist and physiologist who studies cells to understand how the parts fit together to accomplish important functions in plants. He is also passionate about electronics, and he has built much of the equipment they use for their work. Mike loves winter sports, especially downhill and cross country skiing. In fact, he has skied throughout most of his life is currently looking forward to an upcoming ski trip to the Alps with his father who is still hitting the slopes in his nineties! He conducted his undergraduate studies at Simon Fraser University in Vancouver and at the University of Wisconsin, Madison where he received his BS with honors in Botany and Biochemistry. Next, Mike was awarded a PhD in Plant Biology from Stanford University while working in the Department of Plant Biology at the Carnegie Institution of Washington. During his graduate work, Mike received a Fullbright-Hays Graduate Fellowship to study at the University of Nürnberg. Afterwards, Mike traveled to Yale University Medical School to accept an NRSA Postdoctoral Fellowship and then to the University of Cambridge to accept a NATO Postdoctoral Fellowship. He has served on the faculty at the University of London and Imperial College London prior to joining the faculty at the University of Glasgow. Mike has received many awards and honors throughout his career, including being named a Fellow of the Royal Society of Biology, the John Simon Guggenheim Memorial Foundation, the James Hutton Institute, and the Royal Society of Edinburgh. He is also Editor-in-Chief of the premier international journal Plant Physiology. In this interview, Mike discusses his experiences in life and science.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.06.523037v1?rss=1 Authors: Kanie, T., Ng, R., Abbott, K. L., Pongs, O., Jackson, P. K. Abstract: The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation. The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing proper localization to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.31.522373v1?rss=1 Authors: Coly, P.-M., Chatterjee, S., Mezine, F., El Jekmek, C., Devue, C., Nipoti, T., Lara Corona, M., Dingli, F., Loew, D., van Niel, G., Boulanger, C. M. Abstract: Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, high shear stress (HSS) has atheroprotective effects on the endothelium. Endothelial cell-derived extracellular vesicles have been shown to regulate inflammation, senescence and angiogenesis and therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between hemodynamic forces and extracellular vesicle release, the exact consequences of shear stress on the release and uptake of endothelial EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours. Large and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by TEM, Western blot analysis of EV markers, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labeled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in HUVEC conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Additionally, endothelial LSS-EVs appeared to have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins such as PECAM1, MCAM, which were involved in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be involved in elevating reactive oxygen species levels in recipient cells. These findings suggest that endothelial shear stress has a significant impact during EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.23.521775v1?rss=1 Authors: Vanherle, S., Guns, J., Loix, M., Mingneau, F., Dierckx, T., Vangansewinkel, T., Wolfs, E., Pincela Lins, P., Bronckaers, A., Lambrichts, I., Dehairs, J., Swinnen, J. V., Verberk, S. G. S., Haidar, M., Hendriks, J. J. A., Bogie, J. F. J. Abstract: Macrophages play major roles in the pathophysiology of various neurological disorders, being involved in seemingly opposing processes such as lesion progression and resolution. Yet, the molecular mechanisms that drive their harmful and benign effector functions remain poorly understood. Here, we demonstrate that extracellular vesicles (EVs) secreted by repair-associated macrophages (RAMs) enhance remyelination ex vivo and in vivo by promoting the differentiation of oligodendrocyte precursor cells (OPCs). Guided by lipidomic analysis and applying cholesterol depletion and enrichment strategies, we find that EVs released by RAMs show markedly elevated cholesterol levels and that cholestserol abundance controls their reparative impact on OPC maturation and remyelination. Mechanistically, EV-associated cholesterol was found to promote OPC differentiation through direct membrane fusion. Collectively, our findings highlight that EVs are essential for cholesterol trafficking in the brain and that changes in cholesterol abundance dictate the reparative impact of EVs released by macrophages in the brain, potentially having broad implications for therapeutic strategies aimed at promoting repair in neurodegenerative disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.20.521193v1?rss=1 Authors: Seidenthal, M., Janosi, B., Rosenkranz, N., Schuh, N., Elvers, N., Willoughby, M., Zhao, X., Gottschalk, A. Abstract: pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pHsensitive proteins. Neurotransmission is generally stimulated by electrophysiology, which is not feasible in small, intact animals, thus limiting the approach to cell culture regimes. Previous in vivo approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pHsensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact Caenorhabditis elegans nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.18.520925v1?rss=1 Authors: Subkhangulova, A., Gonzalez-Lozano, M. A., Groffen, A. J. A., van Weering, J. R. T., Smit, A. B., Toonen, R. F., Verhage, M. Abstract: Tomosyn is a large, non-canonical SNARE protein proposed to act as a competitive inhibitor of SNARE complex formation in vesicle exocytosis. In the brain, tomosyn inhibits fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model allowing conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l), and an assay that detects DCV exocytosis with single vesicle resolution in primary hippocampal neurons. Surprisingly, loss of both tomosyns did not affect DCV exocytosis but resulted in a strong reduction of intracellular levels of many DCV cargos, most prominently brain-derived neurotrophic factor (BDNF), granin VGF and prohormone convertase PCSK1. Reduced levels of DCV cargos were paralleled by decreased DCV size and impaired mRNA expression of the corresponding genes. We conclude that tomosyns regulate neuropeptide and neurotrophin secretion via control of DCV cargo production, and not at the step of cargo release. Our findings suggest a differential effect of tomosyn on the two main secretory pathways in mammalian neurons and argues against a conserved role of tomosyn as competitive inhibitor of SNARE complex formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.16.520731v1?rss=1 Authors: Bartlome, S., Xiao, Y., Ross, E., Dalby, M. J., Berry, C. C. Abstract: Breast cancer is the leading cause of cancer mortality in women worldwide and commonly metastasizes to the bone marrow, drastically reducing patient prognosis and survival. In the bone marrow niche, metastatic cells can enter into a dormant state, thereby evading immune surveillance and treatment, and can be reactivated to enter a proliferative state due to poorly understood cues. Mesenchymal stromal cells (MSCs) maintain cells in this niche partly by secreting extracellular matrix and paracrine factors and by responding to regenerative cues. MSCs also produce extracellular vesicles (EVs) that carry a range of cargoes, some of which are implicated in cell signalling. Here, we investigate if the changing metabolic state of MSCs alters the cargoes they package into EVs, and how these changing cargoes act on dormant breast cancer cells (BCCs) using an in vitro BCC spheroid model and a scratch assay to create a regenerative demand on MSCs. Our findings show that EVs produced by standard MSCs contain glycolytic metabolites that maintain BCC dormancy. When MSCs are placed under a regenerative demand and increase their respiration to fuel differentiation, these metabolites disappear from the EV cargo and their absence encourages rapid growth in the BCC spheroids. This work implicates EVs in cancer cell dormancy in the bone marrow niche and indicates that pressures on the niche, such as regeneration, can be a driver of BCC activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.15.520209v1?rss=1 Authors: Schmidt, S., Wichers-Misterek, J. S., Behrens, H. M., Birnbaum, J., Henshall, I., Jonscher, E., Flemming, S., Castro-Pena, C., Spielmann, T. Abstract: Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to Artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in ART resistance. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins are among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by identifying four novel proteins at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these, and previously identified proteins, showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance and endocytosis, strengthening the link to endocytosis. Despite this, most showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation. A second group of proteins did not influence endocytosis or ART resistance and was characterised by a lack of vesicle trafficking domains. We here identified the first essential protein of the second group and showed that it is needed in late-stage parasites. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of the endocytosis in malaria parasites. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.22.517322v1?rss=1 Authors: Pust, S., Brech, A., Wegner, C. S., Stenmark, H., Haglund, K. Abstract: Cellular abscission is the final step of cytokinesis that leads to the physical separation of the two daughter cells. The scaffold protein ALIX and the ESCRT-I protein TSG101 contribute to recruiting ESCRT-III to the midbody, which orchestrates the final membrane scission of the intercellular bridge. Here, we addressed by which mechanisms ALIX and the ESCRT-III subunit CHMP4B are transported to the midbody. Structured illumination microscopy revealed gradual accumulation of ALIX at the midbody, resulting in the formation of spiral-like structures extending from the midbody to the abscission site, which strongly co-localized with CHMP4B. Live-cell microscopy uncovered that ALIX appeared together with CHMP4B in vesicular structures, whose motility was microtubule-dependent. Depletion of ALIX led to structural alterations of the midbody and delayed recruitment of CHMP4B, resulting in delayed abscission. Likewise, depletion of the kinesin-1 motor KIF5B reduced the motility of ALIX-positive vesicles and caused delayed recruitment of ALIX, TSG101 and CHMP4B to the midbody, accompanied by impeded abscission. We propose that ALIX, TSG101 and CHMP4B are associated with endosomal vesicles transported along microtubules by kinesin-1, leading to their directional transport to the cytokinetic bridge and midbody, thereby contributing to their function in abscission. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.10.515864v1?rss=1 Authors: von Lersner, A. K., Fernandes, F. C. L., Ozawa, P. M. M., Vagner, T., Lima, S. M., Sung, B. H., Wehbe, M., Franze, K., Wilson, J. T., Irish, J. M., Weaver, A., Di Vizio, D., Zijlstra, A. Abstract: Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that impact human biology by contributing to intercellular communication. To resolve EV heterogeneity and define the EV populations associated with specific biological processes, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multi-parametric data collected by quantitative single-EV flow cytometry. After validating this method against synthetic standards, the EV Fingerprinting analysis of highly purified EVs enabled a much more granular resolution of biochemically distinct EV populations than previously established methods. The analysis of EVs produced after molecular perturbation of EV biogenesis through ablation of the GTPase Rab27a and overexpression of the tetraspanin CD63 revealed that EV Fingerprinting reflects the molecular state of a cell. Subsequent analysis of human plasma demonstrates the capacity of EV Fingerprinting to resolve EV populations in complex biological samples and detect tumor-cell derived EVs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514532v1?rss=1 Authors: Aljiboury, A. A., Ingram, E., Krishnan, N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Da Silva, J., Freshour, J., Hehnly, H. Abstract: An essential process for cilia formation during epithelialization is the movement of the centrosome to dock with the cell's nascent apical membrane. Our study examined centrosome positioning during the development of Danio rerio's left-right organizer (Kupffer's Vesicle, KV). We found that when KV mesenchymal-like cells transition into epithelial cells that are organizing into a rosette-like structure, KV cells move their centrosomes from random intracellular positions to the forming apical membrane in a Rab11 and Rab35 dependent manner. During this process, centrosomes construct cilia intracellularly that associated with Myo-Va while the centrosomes repositioned towards the rosette center. Once the centrosomes with associated cilia reach the rosette center, the intracellular cilia recruit Arl13b until they extend into the forming lumen. This process begins when the lumen reaches an area of approximately 300 m2. Using optogenetic and depletion strategies, we identified that the small GTPases, Rab11 and Rab35, regulate not only cilia formation, but lumenogenesis, whereas Rab8 was primarily involved in regulating cilia length. These studies substantiate both conserved and unique roles for Rab11, Rab35, and Rab8 function in cilia formation during lumenogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514202v1?rss=1 Authors: Koppensteiner, P., Bhandari, P., Önal, C., Borges-Merjane, C., Le Monnier, E., Nakamura, Y., Sadakata, T., Sanbo, M., Hirabayashi, M., Brose, N., Jonas, P., Shigemoto, R. Abstract: GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation induces a transition from tonic to phasic release accompanied by a 4-fold increase in readily releasable pool (RRP) size in MHb terminals, mirrored by a similar increase in the docked vesicle number at the presynaptic active zone (AZ). The tonic and phasic release vesicles have distinct coupling distances. We identified two vesicle-associated molecules, synaptoporin and CAPS2, selectively involved in tonic and phasic release, respectively. Synaptoporin mediates augmentation of tonic release and CAPS2 stabilizes readily releasable vesicles during phasic release. A newly developed Flash and Freeze-fracture method revealed selective recruitment of CAPS2 to the AZ during phasic release. Thus, we propose a novel two-pool mechanism underlying the GBR-mediated potentiation of release from MHb terminals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.18.512792v1?rss=1 Authors: Sun, L., York, S. B., Pate, B., Zhang, Y., Meckes, D. G. Abstract: Current extracellular vesicle (EV) isolation methods depend on large expensive equipment like ultracentrifuges and are laborious and time consuming. There is also currently no method available for high throughput isolation to meet clinical demands. Here, we present a method that combines our previous published ExtraPEG method and magnetic beads. Western blot and nanoparticle tracking analysis (NTA) of the purified EVs revealed higher or equivalent recovery and purity with this method compared to ExtraPEG or size exclusion chromatography (SEC) methods. With this newly developed workflow and automated liquid handling instrument, we have successfully isolated up to 96 EV samples from 5 L pre-cleared serum in 45 minutes. Moreover, DNA / small RNA / protein purification and profiling steps could be seamlessly integrated into the isolation workflow. To profile EV protein markers, EVs were lysed from the binding step and covalently bound to the surface of the beads. TotalSeq or ELISA antibody can be applied with under a standard protocol. With this extended protocol, researchers can easily complete EV isolation and protein profiling experiment within 8 hours. Taken together, we provide a high throughput method for EV isolation and molecular analyses that may be used for sensitive biomarker detection from biological fluids. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.04.510646v1?rss=1 Authors: Stephens, A. D., Fernandez-Villegas, A., Chung, C. W., Vanderpoorten, O., Pinotsi, D., Mela, I., Ward, E. N., McCoy, T. M., Cubitt, R., Routh, A. F., Kaminski, C. F., Schierle, G. S. K. Abstract: Monomeric alpha-synuclein (aSyn) is a well characterised as a lipid binding protein. aSyn is known to form amyloid fibrils which are also localised with lipids and organelles in so called Lewy bodies, insoluble structures found in Parkinson s disease patient s brains. It is still unclear under which conditions the aSyn-lipid interaction can start to become pathological. Previous work to address pathological interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes which not only have a more complex lipid composition, but also contain lipid interacting proteins. Here, we investigate how either monomeric or fibrillar aSyn interact with physiological synaptic vesicles (SV) isolated from rodent brain. Using small angle neutron scattering and high-resolution imaging we observe that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV. Furthermore, SV enhance the aggregation rate of aSyn, however increasing the SV:aSyn ratio causes a reduction in aggregation propensity. SV lipids appear as an integrated part of aSyn fibrils and while the fibril morphology differs to aSyn fibrils alone, the core fibril structure remains the same. We finally demonstrate that lipid-associated aSyn fibrils are more easily taken up into cortical i3Neurons derived from induced pluripotent stem cells. Our study sheds light on differences between interactions of aSyn with synthetic lipid vesicles and physiological SV. We show how aSyn fibrils may enhance pathology by disintegrating SV, which in turn may have fatal consequences for neurons. Furthermore, disease burden may additionally be impacted by an increased uptake of lipid-associated aSyn by neurons, leading to more SV damage and enhancing aSyn aggregation. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.08.25.505298v1?rss=1 Authors: Willows, J. W., Gunsch, G., Paradi, E., Blaszkiewicz, M., Tonniges, J. R., Pino, M. F., Smith, S. R., Sparks, L. M., Townsend, K. L. Abstract: Peripheral neuropathy is a pathophysiological state of nerve degeneration and loss of tissue innervation. The most prominent cause of small fiber neuropathy is diabetes which can be demyelinating in nature, but this has not yet been explored in adipose tissue. Both demyelinating neuropathies and axonopathies implicate Schwann cells (SCs), the peripheral glial required for nerve myelination and regeneration after injury. Here, we perform a comprehensive assessment of SCs and myelination patterns of subcutaneous white adipose tissue (scWAT) nerves, including changes that occur with obesity and other imbalanced energy states in mice and humans. We found that mouse scWAT is densely innervated by both myelinated and unmyelinated sensory and sympathetic nerves. Accordingly, scWAT is home to both myelinating and non-myelinating SCs; the greater proportion of which are myelinating. Furthermore, SCs were found closely associated with synaptic vesicle-containing nerve terminals in scWAT. Obese BTBR ob/ob mice exhibit diabetic peripheral neuropathy in scWAT, and display concordant demyelination specific to small fibers, which was also associated with a decrease in the pan-SC marker Sox10 and compensatory increase in Krox20 gene expression. Together this suggests that adipose SCs may be involved in regulating the plasticity or the neuropathy of adipose tissue nerves. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390179v1?rss=1 Authors: Pohorille, A., Wei, C. Abstract: To understand the transition from inanimate matter to life, we studied a process that directly couples simple metabolism to evolution via natural selection, demonstrated experimentally by Adamala and Szostak (Nat. Chem. 2013, 5, 495-501). In this process, dipeptides synthesized inside precursors of cells promote absorption of fatty acid micelles to vesicles inducing their preferential growth and division at the expense of other vesicles. The process is explained on the basis of coarse-grained molecular dynamics simulations, each extending for tens of microseconds, carried out to model fusion between a micelle and a membrane, both made of fatty acids in the absence and presence of hydrophobic dipeptides. In all systems with dipeptides, but not in their absence, fusion events were observed. They involve the formation of a stalk made by hydrophobic chains from the micelle and the membrane, similar to that postulated for vesicle-vesicle fusion. The emergence of a stalk is facilitated by transient clusters of dipeptides, side chains of which formed hydrophobic patches at the membrane surface. Committor probability calculations indicate that the size of a patch is a suitable reaction coordinate and allow for identifying the transition state for fusion. Free energy barrier to fusion is greatly reduced in the presence of dipeptides to only 4-5 kcal/mol, depending of the hydrophobicity of side chains. The mechanism of mediated fusion, which is expected to apply to other small peptides and hydrophobic molecules, provides a robust means by which a nascent metabolism can confer evolutionary advantage to precursors of cells. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390419v1?rss=1 Authors: Carnazza, K. E., Komer, L., Pineda, A., Na, Y., Ramlall, T., Buchman, V. L., Eliezer, D., Sharma, M., Burre, J. Abstract: -Synuclein (Syn), {beta}-synuclein ({beta}Syn), and {gamma}-synuclein ({gamma}Syn) are abundantly expressed in the vertebrate nervous system. Syn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of {beta}Syn and {gamma}Syn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that {beta}Syn and {gamma}Syn have a reduced affinity towards membranes compared to Syn, and that direct interaction of {beta}Syn or {gamma}Syn with Syn results in reduced membrane binding of Syn. Our data suggest that all three synucleins affect synapse function, but only Syn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while {beta}Syn and {gamma}Syn modulate the activity of Syn through regulating its binding to synaptic vesicles. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384602v1?rss=1 Authors: Vanhille Campos, C., Saric, A. Abstract: We study the effects of osmotic shocks on lipid vesicles via coarse-grained molecular dynamics simulations by explicitly considering the solute in the system. We find that depending on their nature (hypo- or hypertonic) such shocks can lead to bursting events or engulfing of external material into inner compartments, among other morphology transformations. We characterize the dynamics of these processes and observe a separation of time scales between the osmotic shock absorption and the shape relaxation. Our work consequently provides an insight into the dynamics of compartmentalization in vesicular systems as a result of osmotic shocks, which can be of interest in the context of early proto-cell development and proto-cell compartmentalisation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.385575v1?rss=1 Authors: Pulido, C., Ryan, T. A. Abstract: The human brain is a uniquely vulnerable organ as interruption in fuel supply leads to acute cognitive impairment on rapid time scales. The reasons for this vulnerability are not well understood, but nerve terminals are likely loci of this vulnerability as they do not store sufficient ATP molecules and must synthesize them on-demand during activity or suffer acute degradation in performance. The requirements for on-demand ATP synthesis however depends in part on the magnitude of resting metabolic rates. We show here that, at rest, synaptic vesicle (SV) pools are a major source of presynaptic basal energy consumption. This basal metabolism arises from SV-resident V-ATPases compensating for a hidden resting H+ efflux from the SV lumen. We show that this steady-state H+ efflux is 1) mediated by vesicular neurotransmitter transporters, 2) independent of the SV cycle, 3) accounts for ~half of the resting synaptic energy consumption and 4) contributes to nerve terminal intolerance of fuel deprivation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.13.337881v1?rss=1 Authors: Ter-Ovanesyan, D., Norman, M., Trieu, W., Lee, J.-H., Lazarovits, R., Church, G., Walt, D. R. Abstract: Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs and using them for diagnostics is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive assays to quantify EVs by immuno-isolating and detecting EV transmembrane proteins in microwell arrays. We developed single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays to rapidly optimize EV isolation using SEC from plasma and CSF. Our results highlight the utility of quantifying EVs using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids. Copy rights belong to original authors. Visit the link for more info

Dr. Franklin West is an Associate Professor in the Regenerative Biosciences Center at the University of Georgia. In the lab, Franklin and his team are developing and testing stem cell therapies to treat stroke and traumatic brain injury (TBI). They use induced pluripotent stem cells, which are cells that can be reprogrammed to develop into any kind of cell in the body. Traveling is a passion for Franklin. Though he had to cancel his trip to Seoul, South Korea planned for this summer, he is looking forward to his next big travel adventure. In the meantime, Franklin has been having fun taking care of his garden at home. He received his Bachelor of Science degree in biology from Morehouse College and was awarded his Ph.D. in stem cell biology from the University of Georgia. Franklin then worked as an Assistant Research Scientist at the University of Georgia for a few years before joining the faculty there in 2010. He received the University of Georgia College of Agricultural and Environmental Science’s Young Alumni Award in 2019 and recently received the University of Georgia Alumni Award “40 Under 40” distinction. Franklin was also named an “Emerging Scholar” in 2012 by Diverse Magazine and among the “Top 40 Under 40: Georgia’s Best and Brightest” by Georgia Trend Magazine. In our interview, Franklin will share more about his life and science.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.28.271593v1?rss=1 Authors: Almeida, R. G., Williamson, J. M., Madden, M. E., Early, J. J., Voas, M. G., Talbot, W. S., Bianco, I. H., Lyons, D. A. Abstract: To study activity-regulated myelination, we imaged synaptic vesicle fusion along single axons in living zebrafish, and found, surprisingly, that axonal synaptic vesicle fusion is driven by myelination. This myelin-induced axonal vesicle fusion was enriched along the unmyelinated domains into which newly-formed sheaths grew, and was promoted by neuronal activity, which in turn accelerated sheath growth. Our results indicate that neuronal activity consolidates sheath growth along axons already selected for myelination. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.20.260307v1?rss=1 Authors: Welsh, J. A., Killingswroth, B., Kepley, J., Traynor, T., McKinnon, K., Savage, J., Appel, D., Aldape, K., Camphausen, K., Berzofsky, J. A., Ivanov, A. R., Ghiran, I. H., Jones, J. C. Abstract: Evidence continues to increase of the clinical utility extracellular vesicles (EVs) can provide as translational biomarkers. While a wide variety of EV isolation and purification methods have been implemented, few techniques are high-throughput and scalable for removing excess fluorescent reagents (e.g. dyes, antibodies). EVs are too small to be recovered from routine cell-processing procedures, such as filtration or centrifugation. The lack of suitable methods for removing unbound labels, especially in optical assays, is a major roadblock to accurate EV phenotyping and utilization of EV assays in a translational or clinical setting. Therefore, we developed a method for using a multi-modal resin, referred to as EV-Clean, to remove unbound labels from EV samples, and we demonstrate improvement in flow cytometric EV analysis with the use of this EV-Clean method. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.17.254821v1?rss=1 Authors: Li, F., Sundaram, V. K., Gatta, A. T., Coleman, J., Krishnakumar, S., Pincet, F., Rothman, J. E. Abstract: Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.14.251322v1?rss=1 Authors: Huson, V., Meijer, M., Dekker, R., ter Veer, M., Ruiter, M., van Weering, J., Verhage, M., Cornelisse, L. N. Abstract: Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1 dependent and independent mechanisms that lower the energy barrier independently in an additive manner to produce PTP by multiplication of release rates. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.23.217018v1?rss=1 Authors: Kaneda, H., Ida, Y., Kuwahara, R., Sato, I., Nakano, T., Tokuda, H., Sato, T., Murakoshi, T., Honke, K., Kotani, N. Abstract: Extracellular vesicles (EVs) have been investigated for use in clinical testing in recent years. Specific EV surface proteins provide distinguishing characteristics, but are insufficient for more detailed classification of EVs. Here, we suggest a novel "Bimolecular surface antigen expressed in EV (BiEV)" as a potential indicator for more efficient EV screening. A BiEV can be identified using a proximity labeling method applicable to EV membrane proteins. We examined the screening of BiEV in cancer cell-secreted EV included in serum EVs from a model mouse for lung cancer, showing that the CHL1-SLC4A1 BiEV was a significant candidate. ELISA quantification of CHL1-SLC4A1 BiEV appeared to suggest a potential for cancer screening of these mice. Using the same protocols, we found that CHL1-caspase 14 BiEV was significantly elevated in lung cancer patients. A BiEV strategy may be able to make a contribution to more effective EV screening, resulting in novel clinical applications. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.214023v1?rss=1 Authors: Gramlich, M. W., Balseiro Gomez, S., Tabei, S. M. A., Parkes, M., Yogev, S. Abstract: Axonal motor driven cargo utilizes the microtubule cytoskeleton in order to direct cargo, such as presynaptic vesicle precursors, to where they are needed. This transport requires vesicles to travel up to microns in distance. It has recently been observed that finite microtubule lengths can act as roadblocks inhibiting vesicles and increasing the time required for transport. Vesicles reach the end of a microtubule and pause until they can navigate to a neighboring microtubule in order to continue transport. The mechanism by which axonal vesicles navigate the end of a microtubule in order to continue mobility is unknown. In this manuscript we model experimentally observed vesicle pausing at microtubule ends in C. elegans. We show that a single rate-constant model reproduces the time vesicles pause at MT-ends. This model is based on the time a vesicle must detach from its current microtubule and re-attach to a neighboring microtubule. We show that vesicle pause times are different for anterograde and retrograde motion, suggesting that vesicles utilize different proteins at plus and minus end sites. Last, we show that vesicles do not likely utilize a tug-of-war like mechanism and reverse direction in order to navigate microtubule ends. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.17.208132v1?rss=1 Authors: Jimenez-Marin, A., Diez, I., Labayru, G., Sistiaga, A., Sepulcre, J., Lopez de Munain, A., Cortes, J. M. Abstract: Despite significant research, the biological mechanisms underlying the brain degeneration in Myotonic Dystrophy Type I (DM1) remain largely unknown. Here we have assessed brain degeneration by measuring the volume loss (VL) and cognitive deficits (CD) in two cohorts of DM1 patients, and associating them to the large-scale brain transcriptome maps provided by the Allen Human Brain Atlas (AHBA). From a list of preselected hypothesis-driven genes, three of them appear to play a major role in degeneration: dystrophin (DMD), alpha-synuclein (SNCA) and the microtubule-associated protein tau (MAPT). Moreover, a purely data-driven strategy identified gene clusters enriched for key biological processes in the central nervous system, such as synaptic vesicle recycling, localization, endocytosis and exocytosis, and the serotonin and dopamine neurotransmitter pathways. Therefore, by combining large-scale transcriptome interactions with brain imaging and cognitive function, we provide a new more comprehensive understanding of DM1 that might help define future therapeutic strategies and research into this condition. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.12.147975v1?rss=1 Authors: Kavalali, E. T., Afuwape, O., Chanaday, N., Kasap, M., Monteggia, L. M. Abstract: Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Using the fluorescent reporter vGluT1-pHluorin, we observed that compensatory endocytosis after 20 Hz stimulation was arrested in ~40% of presynaptic boutons, while remaining synapses showed only a modest effect suggesting the existence of a dynamin-independent endocytic pathway in central synapses. Surprisingly, we found that the retrieval of single synaptic vesicles, after either evoked or spontaneous fusion, was largely impervious to disruption of dynamins. Overall, our results suggest that classical dynamindependent endocytosis is not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.29.069344v1?rss=1 Authors: Vargas, K. J., Colosi, P. L., Girardi, E., Chandra, S. S. Abstract: -Synuclein plays a central role in Parkinson disease (PD); hence, elucidating its normal physiological function(s) is important. -Synuclein and family members {beta}-, and {gamma}-synuclein, are presynaptically enriched proteins. Synucleins sense and generate membrane curvature, properties consistent with their described roles in synaptic vesicle (SV) cycling. We have previously shown SV endocytosis (SVE) deficits in {beta}{gamma}-synuclein knockout (KO) neurons. Here, we investigate which steps of SVE are regulated by -synuclein. Immuno-electron microscopy (EM) of synaptosomes reveals that -synuclein relocalizes from SVs to the synaptic membrane upon stimulation, allowing -synuclein to function there during or after stimulation. Using membrane recruitment assays, we show that -synuclein is co-localized with clathrin patches. We also observe that recruitment of clathrin and its adaptor, AP180, to synaptic membranes is altered in the absence of synucleins. Visualizing clathrin assembly on membranes in an in vitro reconstitution system reveal that synucleins increase clathrin patch size and curvature, facilitating clathrin coated pit maturation during the early steps of SVE. Copy rights belong to original authors. Visit the link for more info

On this week's The Sci-Files, your hosts Chelsie and Danny interview Victoria Toomajian. Victoria is a third-year Ph.D. candidate in the MSU Biomedical Engineering department, in the Contag Lab. She studies the use of extracellular vesicles as a delivery tool, focusing on the heart post-myocardial infarction as a therapeutic target. Extracellular vesicles (EVs) are small, membrane-bound particles released from cells, which play a role in cell to cell communication. They naturally contain a variety of molecular cargo including proteins and nucleic acids, and it has been shown that they can be used to deliver drugs, RNA, and DNA in vivo and in vitro. EVs are believed to have some advantages over synthetic delivery tools due to their natural biocompatibility, potential natural targeting abilities, and ability to cross natural barriers. Victoria's work uses immune cell-derived EVs, which have been used as delivery vehicles to areas of inflammation, to delivery therapeutic cargo to the heart, which is inflamed after injury. She will also be examining the mechanisms behind immune cell-derived EVs trafficking to areas of inflammation and EV distribution.If you're interested in talking about your MSU research on the radio or nominating a student, please email Chelsie and Danny at scifiles@impact89fm.org. You can ask questions about future episodes here. Check The Sci-Files out on Twitter, Facebook, Instagram, and YouTube! 

As a Ph.D. student at Oxford University, Scott Bonner's work aims to examine extracellular vesicle (EV) heterogeneity and what it might teach us about the therapeutic function of EVs. He explains the following: How many EVs one cell can produce, and why it is difficult albeit possible to examine singular vesicle phenotypes How significant a role EVs play in communication between cells, and what other methods cells use for intercellular communication How certain EV purification methods might disrupt the integrity of an EV itself by altering its shape and/or therapeutic potential Extracellular vesicles hold great potential as a therapeutic delivery platform and might provide therapy for everything from broken bones to complicated disease processes like cancer. In addition, they could be used to package and deliver drugs to very specific regions in the body without running the risk of being hindered by the immune system, thereby providing greater efficacy than what's currently seen with drugs administered conventionally. Scott Bonner shares what compelled him to pursue a career in EV-based research, and how his interest was jump-started by his time as a research assistant for Evox Therapeutics, a company that is now well-known in the field of exosome and EV-based therapeutics. Bonner's current research aims to better understand vesicle heterogeneity and involves the creation of single-cell clones of a particular cell type that are grown separate from all other cells and cell types. Over time, the expectation is that the phenotypes of these cells will drift apart—even if only slightly—and that this could provide insight into how differences in EV phenotype affect EV function. Ultimately, the findings could provide the industry with valuable information about the physical characteristics of EVs that hold the potential to therapeutically affect specific disease processes, such as breast cancer. A number of interesting topics are explored, so tune in, and email your questions or comments to scott.bonner@wolfson.ox.ac.uk.

Veronika Kralj-Iglic, PhD is a physicist and Chair of Biomechanics at the University of Ljubljana, Slovenia who has done a variety of work in the field of medicine, classical physics, quantum physics, and orthopedics, but who says her favorite work has revolved around extracellular vesicle research. Why? According to Dr. Kralj-Iglic, it's because of the amount of potential that extracellular vesicles hold for the future of medicine and biology. "These little vesicles present a hope that maybe we will get to an understanding of the function of living systems,” she says, commenting on the many questions that remain regarding the mechanism underlying the causes of cancer. She shares insight from years' worth of research, and touches on the following: The details of two clinical trials involving EVs that she is currently working on How cells have the ability to use parts of their cell membrane to create vesicles that help catalyze their actions How the behavior of EVs is similar to artificial and biological membranes

A brief preview of the upcoming full episode 53, featuring upcoming topics that include tips for amplifying learning in the A&P course, updates regarding the role of exosomes in the spread of cancer and how heart shape relates to human activity. There's more... some word dissections and Kevin's recommendation for The A&P Professor Book Club. If you cannot see or activate the audio player click here. Questions & Feedback: 1-833-LION-DEN (1-833-546-6336) Follow The A&P Professor on Twitter, Facebook, Blogger, Nuzzel, Tumblr, or Instagram!       Topics 1 minute Strategies to amplify learning in the A&P course The role of exosomes in the spread of cancer How heart shape relates to type of activity Word Dissections 8.5 minutes Metastasis Extracellular vesicle (EV) Exosome Oncosome Transcytosis Book Club 3.5 minutes Prime Mover:  A Natural History of Muscle by Steve Vogel amzn.to/30jcKcm Special opportunity Contribute YOUR book recommendation for A&P teachers! First five submitted and used will be in a drawing for a Kindle Fire HD 10 tablet amzn.to/2WwLZvb Any contribution used will receive a $25 gift certificate The best contribution is one that you have recorded in your own voice (or in a voicemail at 1-833-LION-DEN) Check out The A&P Professor Book Club   If the hyperlinks here are not active, go to TAPPradio.org to find the episode page. More details at the episode page. Transcript available at the script page. Listen to any episode on your Alexa device. Need help accessing resources locked behind a paywall? Check out this advice from Episode 32 to get what you need! https://youtu.be/JU_l76JGwVw?t=440   Sponsors   Transcript and captions for this episode are supported by the  American Association for Anatomy. anatomy.org     The Human Anatomy & Physiology Society  also provides marketing support for this podcast.  theAPprofessor.org/haps     Distribution of this episode is supported by  NYCC's online graduate program in  Human Anatomy & Physiology Instruction (HAPI)  nycc.edu/hapi   Clicking on sponsor links  helps let them know you appreciate their support of this podcast!   Referrals also help defray podcasting expenses.  Amazon TextExpander Snagit & Camtasia The A&P Professor Logo Items   Follow The A&P Professor on  Twitter, Facebook, Blogger, Nuzzel, Tumblr, or Instagram!   The A&P Professor® and Lion Den® are registered trademarks of Lion Den Inc. (Kevin Patton)  

Just around the corner is the publication of Exosomes in Health and Disease, an enormous compilation of data on exosomes and their role in almost every disease you can imagine. What's an exosome, you might ask? At just a fraction of the size of even the smallest bacteria, exosomes are tiny vesicles communicating with and carrying information to cells anywhere in the body. We've known about them for decades, but a recent discovery holds huge implications for our understanding of epigenetics, epigenetic inheritance, and the causes of diseases. Denis Noble, physiologist, researcher, and former Chair of Cardiovascular Physiology at the University of Oxford joins the podcast today to offer a fascinating conversation about the presence of epigenetic data within exosomes, their ability to control the genome of other cells by simply communicating with them, and the profile components that could indicate cancer or a particular disease state. He also discusses the techniques used to extract such small vesicles from blood plasma and the challenges that this process has brought about, the transmission of epigenetic information by exosomes through the germline, symbiogenesis, and more. Tune in for all the details.

Dr. Mike Blatt is the Regius Professor of Botany at the University of Glasgow and Adjuct Professor at Pennsylvania State University. He conducted his undergraduate studies at Simon Fraser University in Vancouver and at the University of Wisconsin, Madison where he received his BS with honors in Botany and Biochemistry. Next, Mike was awarded a PhD in Plant Biology from Stanford University while working in the Department of Plant Biology at the Carnegie Institution of Washington. During his graduate work, Mike received a Fullbright-Hays Graduate Fellowship to study at the University of Nürnberg. Afterwards, Mike traveled to Yale University Medical School to accept an NRSA Postdoctoral Fellowship and then to the University of Cambridge to accept a NATO Postdoctoral Fellowship. He has served on the faculty at the University of London and Imperial College London prior to joining the faculty at the University of Glasgow. Mike has received many awards and honors throughout his career, including being named a Fellow of the Royal Society of Biology, the John Simon Guggenheim Memorial Foundation, the James Hutton Institute, and the Royal Society of Edinburgh. He is also Editor-in-Chief of the premier international journal Plant Physiology. Mike joins us to discuss his experiences in life and science.

Takahiro Ochiya explains how bone marrow mesenchymal stem cells can cause metastatic breast cancer cells to become dormant.

A neurobiologist discusses the machinery that allows neurons to communicate

An autophagy receptor specifically targets Salmonella for degradation in infected cells.

Presented by George M. Langford, Dean of the College of Arts and Sciences and Professor of Cell Biology and Neuroscience at Syracuse University; first E. E. Just Professor and Director of E. E. Just Program at Dartmouth College

Patrik Verstreken, Department of Molecular and Developmental Genetics, K.U. Leuven, Centre for Human Genetics, La of Neuronal Communication, Leuven BELGIUM speaks on "Synaptic vesicle trafficking in health and neurological disease". This seminar has been recorded by ICGEB Trieste

Dr. Silvio Rizzoli sprach im TR 3-Seminar

Vesicle traffic in eukaryotic cells is a tightly organized process involving a multitude of regulatory proteins. Key regulators of this traffic are small GTPases called Rabs. With about 60 members in the human genome, they constitute the largest subgroup in the superfamily of Ras like monomeric GTPases. They recruit effector proteins to specific membranes and thus define the identity of organelles. Rabs switch between an active, GTP bound state and an inactive GDP bound state. Key regulators of this conversion are RabGAPs, which accelerate the hydrolysis of bound GTP. All RabGAPs are characterized by the presence of a TBC domain. In the human genome 40 RabGAPs were identified, most of which had not been studied so far. To assign them to their specific Rab proteins, a novel reverse yeast two-hybrid screening method was developed. This identified a GAP for Rab5 termed RabGAP-5. RabGAP-5 stimulated the GTPase activity of Rab5. Its expression inactivated Rab5 and redistributed the Rab5 effector EEA1 from early endosomes to the cytoplasm. RabGAP-5 also blocked the Rab5 dependent uptake of EGF and transferrin from the plasma membrane. When RabGAP-5 was depleted, the size of endosomes was increased, indicating elevated Rab5-GTP levels. Endocytosed EGF was unable to exit the endosome, indicating that trafficking through endosomes was also blocked. To identify GAPs and Rabs implicated in the regulation of early secretory events simultaneously, a second novel screening method was established. It involved the analysis of phenotypes caused by the inactivation of endogenous target Rabs via the overexpression of RabGAPs. Changes in Golgi morphology, ERGIC organisation and the proceeding of secretion were only observed with one candidate RabGAP, the highly conserved protein TBC1D20. TBC1D20 showed activity towards Rab1 and Rab2 in vitro, and acted primarily on Rab1 in vivo. In contrast to all other RabGAPs it has a transmembrane domain, which localises it to the ER. TBC1D20 interacts with RTN-1 on ER membranes. This interaction modulates the activity of TBC1D20. These data indicate a novel function for Rab1 in regulating ER exit, and thus extend the classical view of RabGAPs as regulators of active Rab lifetime.

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Wed, 1 Jan 1992 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7360/1/Gratzl_Manfred_7360.pdf Gratzl, Manfred; Gratzl, O.; Vereczkey, C.; Langley, K.; Lahr, G.

Wed, 1 Jan 1992 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3790/1/3790.pdf Sackmann, E.; Rädler, Joachim O. ddc:530, Physik

Gas vesicle formation and buoyancy regulation in Pelodictyon phaeoclathratiforme strain BU1 (Green sulfur bacteria) was investigated under various laboratory conditions. Cells formed gas vesicles exclusively at light intensities below 5 mol · m-2 · s-1 in the stationary phase. No effect of incubation temperature or nutrient limitation was observed. Gas space of gas vesicles occupied always less than 1.2% of the total cell volume. A maximum cell turgor pressure of 330 kPa was determined which is comparable to values determined for cyanobacterial species. Since a pressure of at least 485 kPa was required to collapse the weakest gas vesicles in Pelodictyon phaeoclathratiforme, short-term regulation of cell density by the turgor pressure mechanism can be excluded. Instead, regulation of the cell density is accomplished by the cease of gas vacuole production and accumulation of carbohydrate at high light intensity. The carbohydrate content of exponentially growing cells increased with light intensity, reaching a maximum of 35% of dry cell mass above 10 mol · m-2 · s-1. Density of the cells increased concomitantly. At maximum density, protein and carbohydrate together accounted for 62% of the total cell ballast. Cells harvested in the stationary phase had a significantly lower carbohydrate content (8–12% of the dry cell mass) and cell density (1010–1014 kg · m-3 with gas vesicles collapsed) which in this case was independent of light intensity. Due to the presence of gas vesicles in these cultures, the density of cells reached a minimum value of 998.5 kg · m-3 at 0.5 mol · m-2 · s-1. The cell volume during the stationary phase was three times higher than during exponential growth, leading to considerable changes in the buoyancy of Pelodictyon phaeoclathratiforme. Microscopic observations indicate that extracellular slime layers may contribute to these variations of cell volume.

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 pM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 pM (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 pM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 pM and after addition of 35 mM K+ a dissociation constant of 170 pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 pM and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+ for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+, points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate- polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed spectral changes were unaffected by the presence of Ca2+ (100 pM free), the sites interacting with the dye and Ca2+ must be regarded as different.

Tue, 1 Jan 1980 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7495/1/7495.pdf Gratzl, Manfred ddc:610, Medizin

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.22.056119v1?rss=1 Authors: Moya-Diaz, J., James, B., Lagnado, L. Abstract: Multivesicular release (MVR) allows retinal bipolar cells to transmit visual signals as changes in both the rate and amplitude of synaptic events. How do neuromodulators reguate this vesicle code? By imaging larval zebrafish, we find that the variability of calcium influx is a major source of synaptic noise. Dopamine increases synaptic gain up to 15-fold while Substance P reduces it 7-fold, both by acting on the presynaptic calcium transient to alter the distribution of amplitudes of multivesicular events. An increase in gain is accompanied by a decrease in the temporal precision of transmission and a reduction in the efficiency with which vesicles transfer visual information. The decrease in gain caused by Substance P was also associated with a shift in temporal filtering from band-pass to low-pass. This study demonstrates how neuromodulators act on the synaptic transformation of the visual signal to alter the way information is coded with vesicles. Copy rights belong to original authors. Visit the link for more info