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I read from exocytosis to exopeptidase. The word of the episode is "exodus". Theme music from Jonah Kraut https://jonahkraut.bandcamp.com/ Merchandising! https://www.teepublic.com/user/spejampar "The Dictionary - Letter A" on YouTube "The Dictionary - Letter B" on YouTube "The Dictionary - Letter C" on YouTube "The Dictionary - Letter D" on YouTube "The Dictionary - Letter E" on YouTube Featured in a Top 10 Dictionary Podcasts list! https://blog.feedspot.com/dictionary_podcasts/ Backwards Talking on YouTube: https://www.youtube.com/playlist?list=PLmIujMwEDbgZUexyR90jaTEEVmAYcCzuq https://linktr.ee/spejampar dictionarypod@gmail.com https://www.facebook.com/thedictionarypod/ https://www.threads.net/@dictionarypod https://twitter.com/dictionarypod https://www.instagram.com/dictionarypod/ https://www.patreon.com/spejampar https://www.tiktok.com/@spejampar 917-727-5757
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550499v1?rss=1 Authors: O'Shaughnessy, E. C., Lam, M., Ryken, S. E., Wiesner, T., Lukasik, K., Zuchero, J. B., Leterrier, C., Adalsteinsson, D., Gupton, S. L. Abstract: Exocytosis is a fundamental process used by all eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, we have named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are much longer lasting than those in immature murine cortical neurons, and that clustering of exocytic events occurs across cell types. 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/2023.04.13.536686v1?rss=1 Authors: Veerabhadraswamy, P., Belekar, P., Kothegala, L., Gandasi, N. R. Abstract: Type-2 diabetes (T2D) is characterized by high blood glucose due to compromised insulin secretion from pancreatic beta-cells. Beta-cells primarily comprise insulin-secreting large-dense-core-vesicles/insulin-secretory-granules (ISGs) and also multivesicular-bodies (MVBs). MVBs are vesicles of endosomal origin containing intraluminal vesicles, which upon fusion with the plasma membrane, secrete exosomes. These play a significant role in the physiology and pathology of T2D via intercellular communication. The role of MVBs and their influence on ISGs of beta-cells or their characterization is yet to be uncovered. In our study, we characterized the role of MVBs by comparing them to largely well-characterized ISGs in beta-cells. We compared the density, localization, and exocytosis of MVBs with ISGs in beta-cells. For this, we developed a novel probe where we exploit the efficiency of tetraspanins CD63 and CD151 to label the MVBs in beta-cells. We showed that the beta-cells have a significantly higher density of ISGs than MVBs. MVBs and ISGs are spatially localized apart within beta-cells. The proteins that localize with MVBs are different from the ones that localize with ISGs. Exocytosis of ISGs occurs at the periphery of the beta-cells and takes significantly lesser time when compared to exosome release, which is non-peripheral and takes a longer duration. Further, we also observed a significant reduction in the density of ISGs and MVBs in T2D patients' islets compared to healthy controls. Studying the effect of MVBs on insulin secretion in physiological and T2D conditions has huge potential. This study provides a strong basis to open new avenues for such future studies. 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/2023.02.22.529485v1?rss=1 Authors: Panagiotou, S., Nguyen, P. M., Tan, K.-W., Mueller, A., Wendt, A., Eliasson, L., Tengholm, A., Solimena, M., Idevall-Hagren, O. Abstract: Insulin secretion is the process whereby insulin-containing granules fuse with the plasma membrane of {beta}-cells. Exocytosis is preceded by cargo loading, maturation and transport of the secretory granules; processes that require modification of both the protein and lipid composition of the granules. We recently identified phosphatidylinositol-4 phosphate (PI[4]P) dephosphorylation by INPP5F/Sac2 on the surface of insulin granules as a key step that precedes stable granule docking at the plasma membrane and that is required for normal insulin secretion. Here, we show that PI(4)P is used to target the lipid exchange protein oxysterol-binding protein (OSBP) to the granule surface where it is involved in PI(4)P/cholesterol exchange. Loss of Sac2 resulted in excess accumulation of cholesterol on insulin granules that was normalized when OSBP expression was reduced. Acute inhibition of OSBP resulted in dramatic cellular redistribution of OSBP to insulin granules where it colocalized with the ER-resident protein VAP-A at ER-granule contact sites. Stimulation of insulin secretion also resulted in translocation of OSBP to the insulin granule surface in a process that depended on Ca2+-induced acidification of the cytosol. Similar to Sac2 knockdown, inhibition of OSBP suppressed insulin secretion without affecting insulin production. In conclusion, we show that lipid exchange at ER-granule contacts sites is involved in the exocytic process, and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation. 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/2023.02.16.528874v1?rss=1 Authors: Rijal, R., Ismail, I., Jing, S., Gomer, R. H. Abstract: Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity, and this effect requires GrlD and Ppk1 but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, to both decrease energy expenditures and retain nutrients. 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/2023.01.09.523261v1?rss=1 Authors: Arabiatorre, A., Formanowicz, M., Bankaitis, V. A., Grabon, A. Abstract: Phosphoinositide metabolism defines the foundation of a major signaling pathway that is conserved throughout the eukaryotic kingdom. The 4-OH phosphorylated phosphoinositides such as phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate are particularly important molecules as these execute intrinsically essential activities required for the viability of all eukaryotic cells studied thus far. Using intracellular tachyzoites of the apicomplexan parasite Toxoplasma gondii as model for assessing primordial roles for PtdIns4P signaling, we demonstrate the presence of PtdIns4P pools in Golgi/trans-Golgi (TGN) system and in post-TGN compartments of the parasite. Moreover, we show that deficits in PtdIns4P signaling result in structural perturbation of compartments that house dense granule cargo with accompanying deficits in dense granule exocytosis. Taken together, the data report a direct role for PtdIns4P in dense granule biogenesis and exocytosis. The data further indicate that the biogenic pathway for secretion-competent dense granule formation in T. gondii is more complex than simple budding of fully matured dense granules from the TGN. 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/2022.11.24.517842v1?rss=1 Authors: Ralhan, I., Chang, J., Moulton, M. J., Goodman, L. D., Lee, N. Y., Plummer, G., Pasolli, H. A., Matthies, D., Bellen, H. J., Ioannou, M. S. Abstract: During oxidative stress neurons release lipids that are internalized by glia. Defects in this coordinated process play an important role in several neurodegenerative diseases. Yet, the mechanisms of lipid release and its consequences on neuronal health are unclear. Here, we demonstrate that lipid-protein particle release by autolysosome exocytosis protects neurons from ferroptosis, a form of cell death driven by lipid peroxidation. We show that during oxidative stress, peroxidated lipids and iron are released from neurons by autolysosomal exocytosis which requires the exocytic machinery; VAMP7 and syntaxin 4. We observe membrane-bound lipid-protein particles by TEM and demonstrate that these particles are released from neurons using cryoEM. Failure to release these lipid-protein particles causes lipid hydroperoxide and iron accumulation and sensitizes neurons to ferroptosis. Our results reveal how neurons protect themselves from peroxidated lipids. Given the number of brain pathologies that involve ferroptosis, defects in this pathway likely play a key role in the pathophysiology of neurodegenerative disease. 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/2022.11.02.514845v1?rss=1 Authors: Raj, N., Greune, L., Kahms, M., Mildner, K., Franzkoch, R., Psathaki, O. E., Zobel, T., Zeuschner, D., Klingauf, J., Gerke, V. Abstract: The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of membrane required for wound repair are poorly understood. Here, we show that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, we demonstrate that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal SNARE VAMP2, which is crucial for efficient membrane repair. Thus, the here identified Ca2+-evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
My AP Biology Thoughts Unit 2 Cell Structure and FunctionWelcome to My AP Biology Thoughts podcast, my name is Morgan Bernstein and I am your host for episode #59 Unit 2: Active Transport: Endocytosis, Exocytosis, and Protein Pumps. Segment 1: Introduction to Active TransportFirst, we have to know that within any cell, things are always moving. Proteins need to get places, waste has to be excreted, and food is consumed. Two umbrella terms of movement- Passive Transport and Active Transport Passive Transport=no energy required, almost like a habit Active transport = within a vesicle, does require energy Active transport is what we will be discussing in this episode, but be sure to check out episode 58 to learn about passive transport as well! Why does active transport require energy? Goes against the concentration gradient Things are moving from low concentration to high concentration (disrupts equilibrium and requires extra energy) Can happen across a cell membrane or within the cell itself Segment 2: Examples of Active Transport: Endo/Exocytosis and PumpsThe first type of active transport is one that does cross a cell-membrane barrier, and it is known as the sodium-potassium pump. Two potassium ions into the cell and takes three sodium ions out Works because of the protein pump in the plasma membrane. Three sodium ions bind to the carrier protein pump inside the cell, and are transported out using the energy available from ATP. Protein then changes shape to allow for the potassium ions to bind to it as well, and pumps those inside of the cell membrane where they are transported for use in the cell before the process repeats. Higher concentration of potassium ions inside the cell than outside, and a higher concentration of sodium ions outside the cell, so this sodium-potassium pump is going against the concentration gradient and is therefore a form of ACTIVE transport Requires energy. Another form of active transport comes in endocytosis and exocytosis First, cytosis means cell, which is present in all three terms Endo = enter, + cytosis = cell, so endocytosis = into the cell Exo = exit, + cytosis = cell, so exocytosis = exiting the cell. Endocytosis Things brought into the cell across the membrane, but not through a pump Requires energy Happens inside a vesicle (small cellular bubble that holds and transports other molecules and ions) Molecules or ions outside of the cell are enclosed by a part of the plasma membrane, forming the vesicle, and vesicle brings the contents through the membrane into the cell for transport Exocytosis export proteins or excrete waste products Requires energy Necessary protein or waste products inside of a transport vesicle, vesicle connects with plasma membrane and contents released into outside environment. Segment 3: Connection to the CourseActive transport has many connections to our Unit 2 about Cells and to biology in general. Goes against the rules used for any other cellular movement ex. osmosis or diffusion. Usually moving from high concentrated areas to low concentrated areas, active transport is OPPOSITE and requires energy. These processes are all due to the selective permeability of the plasma membrane of cells. Membrane structure of phospholipids and proteins (w/0 = everything or nothing would be able to enter and exit a cell) No active transport without transport vesicles and protein pumps Potassium is charged- could not enter phospholipid bilayer Important to remember the characteristics and functions of the cell membrane and organelles when studying types of cellular movement such as active transport. We can also connect active transport to the most basic of everyday activities; eating and drinking phagocytosis and pinocytosis- cellular eating and cellular drinking Without this, cells would be...
Welcome all to IS PHARMACOLOGY DIFFICULT Podcast! I am Dr Radhika Vijay.In today's episode, a little brushing before I take a deep dive in true and exact process of Pharmacokinetics, let the oven preheat before true baking starts!!The heads covered are Vesicular transport and Filtration. As I solve the puzzle of tangled and knotty wool yarn of the day's talk, the beginnings of conversation are marked by definitions and explanations of Pinocytosis and Phagocytosis, two types of Endocytosis. Its own description is as simple as simplicity.After covering details along with examples for this heads mentioned, i will shift the tides of my talk towards Exocytosis, another good simple talk unfolds in words and this is followed by something too simple equalling to 2+2 addition in mathematics, which never turns greater as 5 , or less as 3, but exactly as equal to 4. Decorated with classical antique examples, I will pull down the curtains for today's talk while slowly curbing the pace and volume of my verbal proceedings, a quick tip to revise and learn better!Just grab it hard, and make a difference in your performance, nothing left untold.......... For all the updates and latest episodes of my podcast, please visit www.ispharmacologydifficult.com where you can also sign up for a free monthly newsletter of mine. It actually contains lot of updates about the medical sciences, drug information and my podcast updates also. You can follow me on different social media handles like twitter, insta, facebook and linkedin. They all are with same name "IS PHARMACOLOGY DIFFICULT". If you are listening for the first time, do follow me here, whatever platform you are consuming this episode, stay tuned, do rate and review on ITunes, Apple podcasts, stay safe, stay happy, stay enlightened, Thank you!!
Welcome all to IS PHARMACOLOGY DIFFICULT Podcast! I am Dr Radhika Vijay.In today's episode, a little brushing before I take a deep dive in true and exact process of Pharmacokinetics, let the oven preheat before true baking starts!!The heads covered are Vesicular transport and Filtration. As I solve the puzzle of tangled and knotty wool yarn of the day's talk, the beginnings of conversation are marked by definitions and explanations of Pinocytosis and Phagocytosis, two types of Endocytosis. Its own description is as simple as simplicity.After covering details along with examples for this heads mentioned, i will shift the tides of my talk towards Exocytosis, another good simple talk unfolds in words and this is followed by something too simple equalling to 2+2 addition in mathematics, which never turns greater as 5 , or less as 3, but exactly as equal to 4. Decorated with classical antique examples, I will pull down the curtains for today's talk while slowly curbing the pace and volume of my verbal proceedings, a quick tip to revise and learn better!Just grab it hard, and make a difference in your performance, nothing left untold.......... For all the updates and latest episodes of my podcast, please visit www.ispharmacologydifficult.com where you can also sign up for a free monthly newsletter of mine. It actually contains lot of updates about the medical sciences, drug information and my podcast updates also. You can follow me on different social media handles like twitter, insta, facebook and linkedin. They all are with same name "IS PHARMACOLOGY DIFFICULT". If you are listening for the first time, do follow me here, whatever platform you are consuming this episode, stay tuned, do rate and review on ITunes, Apple podcasts, stay safe, stay happy, stay enlightened, Thank you!!
Welcome all to IS PHARMACOLOGY DIFFICULT Podcast! I am Dr Radhika Vijay. In today's episode, a little brushing before I take a deep dive in true and exact process of Pharmacokinetics, let the oven preheat before true baking starts!! The heads covered are Vesicular transport and Filtration. As I solve the puzzle of tangled and knotty wool yarn of the day's talk, the beginnings of conversation are marked by definitions and explanations of Pinocytosis and Phagocytosis, two types of Endocytosis. Its own description is as simple as simplicity.After covering details along with examples for this heads mentioned, i will shift the tides of my talk towards Exocytosis, another good simple talk unfolds in words and this is followed by something too simple equalling to 2+2 addition in mathematics, which never turns greater as 5 , or less as 3, but exactly as equal to 4. Decorated with classical antique examples, I will pull down the curtains for today's talk while slowly curbing the pace and volume of my verbal proceedings, a quick tip to revise and learn better!Just grab it hard, and make a difference in your performance, nothing left untold.......... For all the updates and latest episodes of my podcast, please visit www.ispharmacologydifficult.com where you can also sign up for a free monthly newsletter of mine. It actually contains lot of updates about the medical sciences, drug information and my podcast updates also. You can follow me on different social media handles like twitter, insta, facebook and linkedin. They all are with same name "IS PHARMACOLOGY DIFFICULT". If you are listening for the first time, do follow me here, whatever platform you are consuming this episode, stay tuned, do rate and review on ITunes, Apple podcasts, stay safe, stay happy, stay enlightened, Thank you!!
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.21.289314v1?rss=1 Authors: Park, C., Chen, X., Tian, C.-L., Park, G. N., Chenouard, N., Lee, H., Yeo, X. Y., Jung, S., Bi, G., Tsien, R. W., Park, H. Abstract: The balance between excitation and inhibition is essential for maintaining proper brain function in the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Inhibitory synaptic transmission faces greater kinetic demands than excitatory synaptic transmission, yet remains less well understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated due to both technical and practical limitations. Using quantum dots (QDs)-conjugated antibodies against the luminal domain of the vesicular GABA transporter (VGAT) to selectively label single GABAergic inhibitory vesicles and dual-focus imaging optics, we tracked single inhibitory vesicles up to the moment of exocytosis (i.e., fusion) in three dimensions in real time. Using three-dimensional trajectories, we found that the total travel length before fusion of inhibitory synaptic vesicles was smaller than that of synaptotagmin-1 (Syt1)-labeled vesicles. Fusion times of inhibitory vesicles were shorter compared with those of Syt1-labeled vesicles. We also found a close relationship between release probability to the proximity to fusion sites and total travel length of inhibitory synaptic vesicles. Furthermore, inhibitory synaptic vesicles exhibited a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. Thus, our results showed that inhibitory synaptic vesicles have the unique dynamics and fusion properties that facilitate their ability to support fast synaptic inhibition. Copy rights belong to original authors. Visit the link for more info
I know we can’t travel right now but we discuss how vesicles can travel inside the cell and outside the cell and catch flight not feelings and what vesicular transport can teach us about breast cancer, and some insight on what metastasizing cancer cells look for when they travel to new niche environments and what your iPhone4 v iPhone11 have in common with a super resolution microscope. We also commiserate over wild questions in a candidacy exam. https://www.scientistswholift.com
Active Transport, Endocytosis, Phagocytosis, Pinocytosis, and Exocytosis are discussed during this segment.
Andrews further explains how Ca2+-dependent exocytosis of lysosomes aids membrane repair. Her laboratory showed that after lysosomal exocytosis, an injury to the plasma membrane would also trigger a Ca2+-dependent endocytosis that is required for the repair mechanism. Andrews laboratory showed that lysosomes release the enzyme acid sphingomyelinase (ASM) which induce the endocytosis required for plasma membrane repair.
Tumor cells WASH away the extracellular matrix Tumor cells invade through extracellular matrices by forming actin-rich structures called invadopodia, which contain the transmembrane matrix metalloproteinase MT1-MMP. Monteiro et al. reveal that the Arp2/3 activating protein WASH works with the exocyst complex to deliver MT1-MMP from late endosomes to the invadopodial plasma membrane. This biosights episode presents the paper by Monteiro et al. from the December 23, 2013, issue of The Journal of Cell Biology and includes an interview with senior author Philippe Chavrier (Institut Curie, Paris, France). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu
Professor Patrik Rorsman talks about Diabetes and how beta cells within the pancreas control insulin secretion. Patrik Rorsman is professor of diabetic medicine in the Oxford Centre for Diabetes, Endocrinology and Metabolism. Professor Rorsman has been at the forefront of research on hormone-secreting cells in the pancreas for more than 20 years, work that is highly relevant to understanding the causes and treatment of type 2 diabetes. The Wellcome Trust award will allow him to study further the metabolic and hormonal regulation of hormone secretion in the pancreas.
Professor Patrik Rorsman talks about Diabetes and how beta cells within the pancreas control insulin secretion. Patrik Rorsman is professor of diabetic medicine in the Oxford Centre for Diabetes, Endocrinology and Metabolism. Professor Rorsman has been at the forefront of research on hormone-secreting cells in the pancreas for more than 20 years, work that is highly relevant to understanding the causes and treatment of type 2 diabetes. The Wellcome Trust award will allow him to study further the metabolic and hormonal regulation of hormone secretion in the pancreas.
Professor Patrik Rorsman talks about diabetes and how beta cells within the pancreas control insulin secretion. While the lifestyle causes of type-2 diabetes are now known, the molecular details of the disease remain unclear. Professor Patrik Rorsman is researching the processes that control insulin secretion and determine defects associated with clinical diabetes. Professor Rorsman has been at the forefront of research on hormone-secreting cells in the pancreas for more than 20 years, work that is highly relevant to understanding the causes and treatment of type-2 diabetes. This research may lead to new diabetes medicines, and improved beta cells for transplantation.
Activation of the calcium-sensing receptor promotes its trafficking to the cell surface and enhances signaling.
The actin cytoskeleton has been proposed to regulate exocytosis in many different ways. Nightingale et al. use a two-color, live-cell imaging assay to reveal two contrasting functions of actin in distinct stages of Weibel–Palade body secretion: actin-based anchors inhibit the fusion of these secretory granules with the plasma membrane but, post-fusion, a contractile actin ring squeezes granule content out of the cell. This biosights episode presents the paper by Nightingale et al. from the August 22, 2011, issue of The Journal of Cell Biology, and includes an interview with authors Thomas Nightingale and Daniel Cutler (MRC Laboratory of Molecular Cell Biology, London, UK). Produced by Caitlin Sedwick and Ben Short. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu
The success of acrosomal exocytosis, a complex process with a variety of inter-related steps, relies on the coordinated interaction of participating signaling molecules. Since the acrosome reaction resembles Ca(2+)-regulated exocytosis in neurons, we investigated whether cognate neuronal binding partners of the multi-PDZ domain protein MUPP1, which recruits molecules that control the initial tethering and/or docking between the acrosomal vesicle and the plasma membrane, are also expressed in spermatozoa, and whether they contribute to the regulation of acrosomal secretion. We observed that CaMKIIalpha colocalizes with MUPP1 in the acrosomal region of epididymal spermatozoa where the kinase selectively binds to a region encompassing PDZ domains 10-11 of MUPP1. Furthermore, we found that pre-treating mouse spermatozoa with a CaMKII inhibitor that directly blocks the catalytic region of the kinase, as well as a competitive displacement of CaMKIIalpha from PDZ domains 10-11, led to a significant increase in spontaneous acrosomal exocytosis. Since Ca(2+)-calmodulin releases CaMKIIalpha from the PDZ scaffolding protein, MUPP1 represents a central signaling platform to dynamically regulate the assembly and disassembly of binding partners pertinent to acrosomal secretion, thereby precisely adjusting an increase in Ca(2+) to synchronized fusion pore formation.
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Neuronal communication and endocrine signaling are fundamental for integrating the function of tissues and cells in the body. Hormones released by endocrine cells are transported to the target cells through the circulation. By contrast, transmitter release from neurons occurs at specialized intercellular junctions, the synapses. Nevertheless, the mechanisms by which signal molecules are synthesized, stored, and eventually secreted by neurons and endocrine cells are very similar. Neurons and endocrine cells have in common two different types of secretory organelles, indicating the presence of two distinct secretory pathways. The synaptic vesicles of neurons contain excitatory or inhibitory neurotransmitters, whereas the secretory granules (also referred to as dense core vesicles, because of their electron dense content) are filled with neuropeptides and amines. In endocrine cells, peptide hormones and amines predominate in secretory granules. The function and content of vesicles, which share antigens with synaptic vesicles, are unknown for most endocrine cells. However, in B cells of the pancreatic islet, these vesicles contain GABA, which may be involved in intrainsular signaling.' Exocytosis of both synaptic vesicles and secretory granules is controlled by cytoplasmic calcium. However, the precise mechanisms of the subsequent steps, such as docking of vesicles and fusion of their membranes with the plasma membrane, are still incompletely understood. This contribution summarizes recent observations that elucidate components in neurons and endocrine cells involved in exocytosis. Emphasis is put on the intracellular aspects of the release of secretory granules that recently have been analyzed in detail.
Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells.
1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.
The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin. The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain.
The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.
Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release ofcatecholamines from permeabi1ized chromaffin cells [(1989) FEBS Lett. 242, 245-248; (1989) J. Neurochern., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions ofboth neurotoxins on exocytosis were presently compared using streptolysin O-permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca2 +-stimulated catecholamine release from these cells. Addition of dithiothreitollowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.
Tetanus toxin is one of the most poisonous substances known. It is first produced by Clostridium tetani as a single-chain protein [1,2]. Subsequent proteolytic processing leads to a pharmacologically more active [3], two-chain form consisting of disulfide-linked heavy (98 kDa) and light (53 kDa) chains. The individual chains alone produced by reduction are not neurotoxic [4]. Intoxication requires hours before transmitter release is blocked. This delay reflects the time required for the binding of the toxin to the plasma membrane and its subsequent internalization before the toxin can interact with its as yet unknown intracellular target (cf. [2]). Adrenal medullary chromaffin cells, closely related to neurones, lack the capacity to bind tetanus toxin [2] and therefore are insensitive to externally applied toxin [5,6]. Sensitivity may be introduced by pretreatment of such cells with a ganglioside mixture containing tri- and tetrasialogangliosides as putative receptors (Marxen, Fuhrmann and Bigalke, personal communication). When directly injected into these cells tetanus toxin inhibits Ca2+ -induced exocytosis as revealed by cell capacitance measurement [7]. Binding and internalization can be by-passed using permeabilized cell preparations. With these methods the cytoplasm becomes fully accessible to extrinsic substances including Ca2+, drugs and toxins. Pheochromocytoma (PC 12) or adrenal medullary chromaffin cells in culture, when permeabilized with pore-forming proteins (cf. [8]) still release catecholamines in response to micromolar concentrations of Ca2 + [9-13]. Cells can be made permeable to low molecular mass solutes with staphylococcal er-toxin [9-14] and to large proteins such as immunoglobulins with streptolysin 0 (SLO) [8,13,15]. We have recently looked for an interference of various forms of tetanus toxin or its chains with the Ca2+ -stimulated [3H]noradrenaline release from SLO-permeabilized chromaffin cells.
The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 μM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by α-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.
The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular 86Rb+ and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.
Tue, 1 Jan 1980 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7495/1/7495.pdf Gratzl, Manfred ddc:610, Medizin