ANAT365A Metabolism_Lecture 2 2023.pdf

Roles for Cellular Trafficking in Metabolic Diseases Obesity Dr. Jennifer Estall Diabetes @dnachicken IRCM, Montréal [email protected] Outline of Lecture Lecture 2: • Exosomes • Nanotubes and Nanotubules • Mitochondrial associated membranes (MAMs) • What they are and why are they impo...

Roles for Cellular Trafficking in Metabolic Diseases Obesity Dr. Jennifer Estall Diabetes @dnachicken IRCM, Montréal [email protected] Outline of Lecture Lecture 2: • Exosomes • Nanotubes and Nanotubules • Mitochondrial associated membranes (MAMs) • What they are and why are they important to metabolism and disease • Cell – cell communication beyond secreted proteins • What does this mean for the field of metabolism? Exosomes -tiny secretory vesicles, don’t quite understand how budding happens • • • • • Extracellular vesicles (EVs) of 50 – 150 nm is size (larger ones have other names) Believed to be generated from the MVB (multivesicular body) RNAs and miRNAs appear to be selectively sorted into exosomes Cargo is protected from degradation while in the circulation Exosomes are rapidly taken up by other tissues (e.g. liver, lung and spleen) mRNA signals travels to other cells to give them signals, these are usually inhibitory signals for shutting down translation in other cells internalization Cell-surface receptor Guay et al. Exosomes as new players in metabolic organ cross-talk. DOM. 2017. fusion Exosomes: An alternative way for cells to communicate • Cargo: miRNAs, lncRNA, circRNA (nucleic acids) – genomic ‘signals’ Proteins – cytosolic, intracellular signals that are not traditionally secreted Membrane-bound proteins (e.g. receptors or channels) membrane is lipophilic, so you can transport lipid-soluble material Exosomes are a “new” way that tissues can communicate • Many signals can cause release of exosomes, which travel in blood to other tissues. ie. metabolic disease, diet can influence release, excersize/fasting, etc. the whole process relies on these exosomes to form on the cell surface • Another way for tissues to communicate (in addition to hormones). • The lncRNA and miRNA cargo allow distant cells to influence gene transcription in other cell types. • Inhibiting vesicle formation blocks communication. Whitham et al. EVs provide a means for tissue crosstalk during exercise. Cell Met. 2018. paper was written to show how are important in the heart during stress • When glucose is low, cardiomyocytes of the heart secrete exosomes that contain glucose transporters (GLUTs) along with the enzymes that metabolize glucose. • The exosomes are taken up by endothelial cells (lining of blood vessels), promoting the uptake of glucose from the blood, metabolism of this glucose, and production of pyruvate (fuel for heart cells). heart cells are saying we need more glucose, so the blood vessels will take up more glucose and the enzymes needed will convert that to pyruvate Roles for Exosomes in the Pathogenesis of Metabolic Disease Risk factors for Metabolic disease: Obesity, low/no exercise, diet, inflammation • Physical activity and nutrient type (e.g. high lipids) can increase or decrease release of exosomes from tissues • Diet can influence the cargo released from the muscle cells • Inflammatory signals have a big influence on what is packaged into exosomes • A huge new area of biology we don’t yet understand. Dini et al. Microvesicles and exosomes in metabolic disease and inflammation. Cytokine and Growth Factor Reviews. 51:27-39, 2020. Nanotubes and Nanotubules: Transferring “metabolism” from one cell (or organelle) to another neurons are highly metabolic cells - they need a lot of energy to transmit their cells. When mitochondria get sick and die, this is where the neurological diseases start. Mirochondira can be transported directly through tunneling nanotubules. Mitochondria can travel directly from cell to cell upon a signal. Whern there is damaged mitochondria, you can swap healthy mitochorndra to the cells that need it Chang et al. Translational Neurodegeneration 8:17, 2019. “Mitochondrial Therapy”: a) Endocytosis of mitochondrial-containing vesicles; b) Transportation via actin-based tunnels; c) absorption of exogenous mitochondria. Inter-organelle communication: How do “stationary” organelles talk to each other? many organelles have physical contacts between eachother, they are all really densely packed together Cells are not just bags of floating organelles Mitochondrial Nanotunnels two different bacteria can connect through little nanotunnels • Based on evidence that bacterial cells form bridges to communicate • Exchange of mitochondrial content (ions, metabolites, proteins, nucleic acids) Adapted from Vincent et al. Trends in Cell Biology, 2017 • Aid communication when mitochondrial movement is limited (e.g. fusion/fission is difficult in skeletal muscle cells) Mitochondrial Nanotunnels – how are they important to cell function? • Possible “Sensing Probes” for the intracellular environment —> there may be sensors to see if htere are harmful or safe signals in the area • Possibly mitochondria reaching out for help. Respiratory deficient mitochondria could be calling out to: • Improve metabolism or make more efficient mitochondria (substrates needed or excess metabolites to share)? • Increase ratio of good to bad mtDNA (per mitochondria) with cells that don’t divide often, they accumulate mitochondrial damage. when you accumulate mutations in mtDNA, you will gather many more mutations. Some people are able to live with that fine, because they can swap healthy mitochondria. So as long as the ratio of helathy:unhelathy mitochondria is fine, then you can remain healthy. WHen that ratio is off, then you will develip disease. Adapted from Vincent et al. Trends in Cell Biology, 2017 “Each mitochondrion is estimated to contain 2-10 mtDNA copies” - BBRC 1992 Wiesner et al. 183 (2):553-9 Can we talk? Communication between different organelles Filadi et al. PNAS, 112(17), 2015. Red: mitochondria Green: Endoplasmic reticulum Discovery of ER-mitochondrial interactions in fish glands (1959) in fish, they identified interactions between mitochondria in fish membranes, Miochondria Associated Membranes - they showed that there is a dirrect tunnel between mitoch. and ER ER mitochondria Hayashi T1, Su TP. Cell. 2007 Nov 2;131(3):596-610. ER Mito Copeland and Dalton, J Biophys Biochem Cytol (JCB), 1959 Ribosomes Why does this happen? ER Csordás G et al. J Cell Biol 2006 25;174(7):915-21 MAM: Mitochondria-Associated (endoplasmic reticulum) Membrane How are MAMs defined? • MAMs are characterized by: • Membranes in direct contact with mitochondria • Have a higher concentration of lipid than ER or mitochondrial membrane • Have a unique lipid profile at connection point not just random lipids • Have a protein profile facilitating unique signaling, lipid synthesis, protein folding, and tethering. signalling that happens on ER and Mitochondria is one thing, but the signalling in that tube is another • 10% - 100% of the surface of the mitochondria is involved you can have a lot of surface or none covered by these tubes, and its an active, dynamic process, where these tubes can break and reform • The are heterogeneous, formation of each MAM may be celltype- and stimulus dependent. • Formation of connections is probably an active process (ER/mito protrusions are seen “reaching” toward each other) Reference material: New Insights into the role of mitochondria-associated ER membrane. Int Rev Cell Mol Biol. 2011;292:73-117. How to study MAMs: First need to isolate them from other membranes one of the ways to isolate them is to use a sucrose gradient, put liquid that has different conc. of sucrose, which makes different layers have different densities. The MAMS are the soft white layer. This old technology was used to isolate these MAMS. Full Protocol at: Isolation of mitochondrial-associated membranes and mitochondria from animal tissues and cells. Wieckowski, M et al. Nature Protocols. Doi:10.1038/nprot.2009.151 MAMs contain a unique signature of lipids that are different from other sites on ER membrane • MAMs are enriched with cholesterol and ceramides. PtEt – phosphatidylethanol, SM – Sphingomyelins, GlcCer – made MAMS be lipophillic, which allows them to be isolated glucosylceramides -this was found by disrupting formation of MAMS by removing cholesterol P3 – microsomal fraction -MAMS are detergent resistent, but when you remove cholesterol, you can • Depletion of these lipids: (MβC and FB1 deplete lipids) • Causes MAM-associated proteins to redistribute to the bulk ER (non-DRM: detergent-resistant membrane) • Reorganization of the mitochondrial network and dissociation of MAMs fully dissolve the MAMS without problems “rescue” Depletes cholesterol from PM Depletes chol. from whole cell ê cholesterol and ceramide Inhibits ceramide synthesis Inhibitor of sphingolipid biosynthesis (accumulate precursors, (é) ceramide) Sig–1R: protein found in MAMs Hayashi and Fujimoto, Mol Pharm. 2010 MAMs contain a unique set of proteins MAM is very different - has many different proteins, is very unique, its not like a normal piece of mitochondria H: Homogenate Mc: Crude mitochondrial fraction Mp: Pure mitochondria Er: Endoplasmic reticulum MAM: mitochondrial-associated ER membrane C: cytosol N: Nucleus Specialized MAM domains contain: ER proteins – IP3R, calretuculin, CNX Mito proteins – VDAC, p66shc Unique proteins – FACL-4, Mnf2 Protein signature and visualization of labeled proteins in cells allows construction of a 3D model you have proteins involved in fission/fusion, membrane docking, channels involved in calcium metabolism Wieckowski, M et al. Nature Protocols, 2009. Raturi and Simmen. BBA – Molecular Cell Research, 2013. MAMs are enriched in proteins necessary for ion transport and cell signaling • • • • • IP3 receptors Ryanodine receptors Ca+2/ATPase pumps (SERCA) VDAC Sigma-1 receptors Ca+2 transport • • • • don’t need Bax to know Bak Cytochrome c Bcl-2 Apoptosis • • • • • • • Akt ACAT G6Pase DGAT FACL4 PSS-2 mTOR Metabolism • • • • Mitofusin-2 VDAC1/IP3R Fis1/Bap31 PACS-2 Mitochondria dynamics and tethering of MAM (ER-mito) (especially lipid transport and synthesis) -breaking/fusing of mitochondria Proposed roles of MAMs in metabolism • Reservoir for specific lipid species (i.e. ceramides) – ceramide excess has been implicated in metabolic syndrome ceramides are really toxic to cells, so storage would be useful to avoid this • Site of lipid metabolism, phospholipid synthesis, and transport lipids themselves are broken down for energy, so cell can have access to lipids for energy • Platform for enzymes involved in controlling metabolism and cell growth lots of eznymes are concentrated in MAMS • Mediate autophagy (destruction of cell components) • Permit Ca+2 flow into mitochondria (can stimulate metabolism) calcium comes from outside of the cell, and gets concentrated in ER, which is connected to the MAMs that act as a direct door for when calcium is needed Lopez-Crisosto C. et al., BBA. 2015 Creating microdomains (“hot spots”) of second messenger signals really high conc. of Calcium inside MAMs • Calcium concentration in proximity to mitochondria can reach 20-fold higher than in cytosol (hot spot). High [Ca2+] • Postulated that the MAM surface is like a “quasisynapse” where local calcium goes primarily to the mitochondria with very little spillage. • Calcium activates the tricarboxylic acid (TCA) cycle to promote ATP production. Janikiewicz et al. Cell Death and Disease 2018 really efficient way to bring Calcium to mitochondria when its needed MAMs are dynamic and move/reorient in response to cell signaling there have to be ways to turn this system off: • Receptors for calcium-induced signaling molecules (IP3 and AMF) are colocalized at the MAM interface. when you don’t have Calcium signalling, or an active receptor, the interaction is very tight. When you DO have calcium signalling, you have a block of the MAM attachment. THis is tightly regulated • Cell signaling can rapidly promote MAM rearrangement. AMFR AMFR Goetz et al. Journal of Cell Science, 2007 What metabolic signals could benefit from these mito-ER interactions? • Skeletal muscle - muscle contraction requires tight calcium signaling, e.g. adrenergic signaling needed during exercise MAMs can form/break during skeletal muscle contraction • Heart contraction – relies very heavily on mitochondria and the rise and fall of calcium concentrations for ATP production all of these are off/on processes, so they need MAMs only when they need them and to shut them off when they don’t • Insulin secretion – local concentrations of calcium at mitochondria and ATP production are tightly linked to insulin secretion • Liver – mitochondrial dysfunction, disturbances in lipid synthesis/catabolism and calcium signaling lead to fatty liver disease liver is PACKED full of mitochondria, and in ppl who have metabloc disease, the MAMs are malformed Mito/ER interactions might help regulate metabolism in the fasted versus fed state Adapted from Theurey and Rieusset Trends in Endo and Met, 2017 when liver needs lots of energy, you have lots of ER-mito interaction transition of fasting to feeding: when fasting, you only have glucagon, but don’t have much glucose. Liver is source of energy when you arent eating. GLucagon is the signal, promotes the connection of ER to mito, promotes Calcium transfer to mitochondria what happens with insulin secretion? when you eat? once you have phosphoAKT, you will break ER-mitochondria interaction, and Calcium influx into mitochondira will stop because you don’t need the energy production anymore Altered MAM structure and function is a feature of many metabolic diseases most of time, in metabolic disease, MAM formation is DOWN Hippocampus/Cortex: Increased ER-mitochondrial targets found in fibroblasts from patients with Alzheimer's disease controversial data Lopez-Crisosto C. et al., BBA. 2015 Take home messages: • Movement between and within cells, and membrane dynamics are essential parts of efficient metabolism in cells • When we think of what causes metabolic disease, we often do not focus on cellular trafficking, but it plays a big role • It has been understudied because we lacked high resolution tools to study these processes (in vivo is exponentially harder!) • Understanding these basic processes in cells will help us work out what underlies complex disease. 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