TY - THES AB - Clathrin-Mediated Endocytosis (CME) is an aspect of cellular trafficking that is constantly regulated for mediating developmental and physiological responses. The main aim of my thesis is to decipher the basic mechanisms of CME and post-endocytic trafficking in the whole multicellular organ systems of Arabidopsis. The first chapter of my thesis describes the search for new components involved in CME. Tandem affinity purification was conducted using CLC and its interacting partners were identified. Amongst the identified proteins were the Auxilin-likes1 and 2 (Axl1/2), putative uncoating factors, for which we made a full functional analysis. Over-expression of Axl1/2 causes extreme modifications in the dynamics of the machinery proteins and inhibition of endocytosis altogether. However the loss of function of the axl1/2 did not present any cellular or physiological phenotype, meaning Auxilin-likes do not form the major uncoating machinery. The second chapter of my thesis describes the establishment/utilisation of techniques to capture the dynamicity and the complexity of CME and post-endocytic trafficking. We have studied the development of endocytic pits at the PM – specifically, the mode of membrane remodeling during pit development and the role of actin in it, given plant cells possess high turgor pressure. Utilizing the improved z-resolution of TIRF and VAEM techniques, we captured the time-lapse of the endocytic events at the plasma membrane; and using particle detection software, we quantitatively analysed all the endocytic trajectories in an unbiased way to obtain the endocytic rate of the system. This together with the direct analysis of cargo internalisation from the PM provided an estimate on the endocytic potential of the cell. We also developed a methodology for ultrastructural analysis of different populations of Clathrin-Coated Structures (CCSs) in both PM and endomembranes in unroofed protoplasts. Structural analysis, together with the intensity profile of CCSs at the PM show that the mode of CCP development at the PM follows ‘Constant curvature model’; meaning that clathrin polymerisation energy is a major contributing factor of membrane remodeling. In addition, other analyses clearly show that actin is not required for membrane remodeling during invagination or any other step of CCP development, despite the prevalent high turgor pressure. However, actin is essential in orchestrating the post-endocytic trafficking of CCVs facilitating the EE formation. We also observed that the uncoating process post-endocytosis is not immediate; an alternative mechanism of uncoating – Sequential multi-step process – functions in the cell. Finally we also looked at one of the important physiological stimuli modulating the process – hormone, auxin. auxin has been known to influence CME before. We have made a detailed study on the concentration-time based effect of auxin on the machinery proteins, CCP development, and the specificity of cargoes endocytosed. To this end, we saw no general effect of auxin on CME at earlier time points. However, very low concentration of IAA, such as 50nM, accelerates endocytosis of specifically PIN2 through CME. Such a tight regulatory control with high specificity to PIN2 could be essential in modulating its polarity. AU - Narasimhan, Madhumitha ID - 6269 SN - 2663-337X TI - Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ER - TY - GEN AU - Kim, Olena AU - Borges Merjane, Carolina AU - Jonas, Peter M ID - 11222 IS - Suppl. 1 KW - hippocampus KW - mossy fibers KW - readily releasable pool KW - electron microscopy SN - 2309-8503 T2 - Intrinsic Activity TI - Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy VL - 7 ER - TY - THES AB - Lymph nodes are es s ential organs of the immune s ys tem where adaptive immune responses originate, and consist of various leukocyte populations and a stromal backbone. Fibroblastic reticular cells (FRCs) are the main stromal cells and form a sponge-like extracellular matrix network, called conduits , which they thems elves enwrap and contract. Lymph, containing s oluble antigens , arrive in lymph nodes via afferent lymphatic vessels that connect to the s ubcaps ular s inus and conduit network. According to the current paradigm, the conduit network dis tributes afferent lymph through lymph nodes and thus provides acces s for immune cells to lymph-borne antigens. An elas tic caps ule s urrounds the organ and confines the immune cells and FRC network. Lymph nodes are completely packed with lymphocytes and lymphocyte numbers directly dictates the size of the organ. Although lymphocytes cons tantly enter and leave the lymph node, its s ize remains remarkedly s table under homeostatic conditions. It is only partly known how the cellularity and s ize of the lymph node is regulated and how the lymph node is able to swell in inflammation. The role of the FRC network in lymph node s welling and trans fer of fluids are inves tigated in this thes is. Furthermore, we s tudied what trafficking routes are us ed by cancer cells in lymph nodes to form distal metastases.We examined the role of a mechanical feedback in regulation of lymph node swelling. Using parallel plate compression and UV-las er cutting experiments we dis s ected the mechanical force dynamics of the whole lymph node, and individually for FRCs and the caps ule. Physical forces generated by packed lymphocytes directly affect the tens ion on the FRC network and capsule, which increases its resistance to swelling. This implies a feedback mechanism between tis s ue pres s ure and ability of lymphocytes to enter the organ. Following inflammation, the lymph node swells ∼10 fold in two weeks . Yet, what is the role for tens ion on the FRC network and caps ule, and how are lymphocytes able to enter in conditions that resist swelling remain open ques tions . We s how that tens ion on the FRC network is important to limit the swelling rate of the organ so that the FRC network can grow in a coordinated fashion. This is illustrated by interfering with FRC contractility, which leads to faster swelling rates and a dis organized FRC network in the inflamed lymph node. Growth of the FRC network in turn is expected to releas e tens ion on thes e s tructures and lowers the res is tance to swelling, thereby allowing more lymphocytes to enter the organ and drive more swelling. Halt of swelling coincides with a thickening of the caps ule, which forms a thick res is tant band around the organ and lowers tens ion on the FRC network to form a new force equilibrium.The FRC and conduit network are further believed to be a privileged s ite of s oluble information within the lymph node, although many details remain uns olved. We s how by 3D ultra-recons truction that FRCs and antigen pres enting cells cover the s urface of conduit s ys tem for more than 99% and we dis cus s the implications for s oluble information exchangeat the conduit level.Finally, there is an ongoing debate in the cancer field whether and how cancer cells in lymph nodes s eed dis tal metas tas es . We s how that cancer cells infus ed into the lymph node can utilize trafficking routes of immune cells and rapidly migrate to blood vessels. Once in the blood circulation, these cells are able to form metastases in distal tissues. AU - Assen, Frank P ID - 6947 SN - 2663-337X TI - Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking ER - TY - THES AB - Brain function is mediated by complex dynamical interactions between excitatory and inhibitory cell types. The Cholecystokinin-expressing inhibitory cells (CCK-interneurons) are one of the least studied types, despite being suspected to play important roles in cognitive processes. We studied the network effects of optogenetic silencing of CCK-interneurons in the CA1 hippocampal area during exploration and sleep states. The cell firing pattern in response to light pulses allowed us to classify the recorded neurons in 5 classes, including disinhibited and non-responsive pyramidal cell and interneurons, and the inhibited interneurons corresponding to the CCK group. The light application, which inhibited the activity of CCK interneurons triggered wider changes in the firing dynamics of cells. We observed rate changes (i.e. remapping) of pyramidal cells during the exploration session in which the light was applied relative to the previous control session that was not restricted neither in time nor space to the light delivery. Also, the disinhibited pyramidal cells had higher increase in bursting than in single spike firing rate as a result of CCK silencing. In addition, the firing activity patterns during exploratory periods were more weakly reactivated in sleep for those periods in which CCK-interneuron were silenced than in the unaffected periods. Furthermore, light pulses during sleep disrupted the reactivation of recent waking patterns. Hence, silencing CCK neurons during exploration suppressed the reactivation of waking firing patterns in sleep and CCK interneuron activity was also required during sleep for the normal reactivation of waking patterns. These findings demonstrate the involvement of CCK cells in reactivation-related memory consolidation. An important part of our analysis was to test the relationship of the identified CCKinterneurons to brain oscillations. Our findings showed that these cells exhibited different oscillatory behaviour during anaesthesia and natural waking and sleep conditions. We showed that: 1) Contrary to the past studies performed under anaesthesia, the identified CCKinterneurons fired on the descending portion of the theta phase in waking exploration. 2) CCKinterneuron preferred phases around the trough of gamma oscillations. 3) Contrary to anaesthesia conditions, the average firing rate of the CCK-interneurons increased around the peak activity of the sharp-wave ripple (SWR) events in natural sleep, which is congruent with new reports about their functional connectivity. We also found that light driven CCK-interneuron silencing altered the dynamics on the CA1 network oscillatory activity: 1) Pyramidal cells negatively shifted their preferred theta phases when the light was applied, while interneurons responses were less consistent. 2) As a population, pyramidal cells negatively shifted their preferred activity during gamma oscillations, albeit we did not find gamma modulation differences related to the light application when pyramidal cells were subdivided into the disinhibited and unaffected groups. 3) During the peak of SWR events, all but the CCK-interneurons had a reduction in their relative firing rate change during the light application as compared to the change observed at SWR initiation. Finally, regarding to the place field activity of the recorded pyramidal neurons, we showed that the disinhibited pyramidal cells had reduced place field similarity, coherence and spatial information, but only during the light application. The mechanisms behind such observed behaviours might involve eCB signalling and plastic changes in CCK-interneuron synapses. In conclusion, the observed changes related to the light-mediated silencing of CCKinterneurons have unravelled characteristics of this interneuron subpopulation that might change the understanding not only of their particular network interactions, but also of the current theories about the emergence of certain cognitive processes such as place coding needed for navigation or hippocampus-dependent memory consolidation. AU - Rangel Guerrero, Dámaris K ID - 6849 SN - 2663-337X TI - The role of CCK-interneurons in regulating hippocampal network dynamics ER - TY - JOUR AB - A process of restorative patterning in plant roots correctly replaces eliminated cells to heal local injuries despite the absence of cell migration, which underpins wound healing in animals. Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing. AU - Marhavá, Petra AU - Hörmayer, Lukas AU - Yoshida, Saiko AU - Marhavy, Peter AU - Benková, Eva AU - Friml, Jiří ID - 6351 IS - 4 JF - Cell SN - 00928674 TI - Re-activation of stem cell pathways for pattern restoration in plant wound healing VL - 177 ER - TY - JOUR AB - Plants as sessile organisms are constantly under attack by herbivores, rough environmental situations, or mechanical pressure. These challenges often lead to the induction of wounds or destruction of already specified and developed tissues. Additionally, wounding makes plants vulnerable to invasion by pathogens, which is why wound signalling often triggers specific defence responses. To stay competitive or, eventually, survive under these circumstances, plants need to regenerate efficiently, which in rigid, tissue migration-incompatible plant tissues requires post-embryonic patterning and organogenesis. Now, several studies used laser-assisted single cell ablation in the Arabidopsis root tip as a minimal wounding proxy. Here, we discuss their findings and put them into context of a broader spectrum of wound signalling, pathogen responses and tissue as well as organ regeneration. AU - Hörmayer, Lukas AU - Friml, Jiří ID - 6943 JF - Current Opinion in Plant Biology SN - 1369-5266 TI - Targeted cell ablation-based insights into wound healing and restorative patterning VL - 52 ER - TY - JOUR AB - Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. AU - Tabata, Shigekazu AU - Jevtic, Marijo AU - Kurashige, Nobutaka AU - Fuchida, Hirokazu AU - Kido, Munetsugu AU - Tani, Kazushi AU - Zenmyo, Naoki AU - Uchinomiya, Shohei AU - Harada, Harumi AU - Itakura, Makoto AU - Hamachi, Itaru AU - Shigemoto, Ryuichi AU - Ojida, Akio ID - 7391 IS - 12 JF - iScience SN - 2589-0042 TI - Electron microscopic detection of single membrane proteins by a specific chemical labeling VL - 22 ER - TY - JOUR AB - Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers7,8,9. AU - Kampjut, Domen AU - Sazanov, Leonid A ID - 6848 IS - 7773 JF - Nature SN - 0028-0836 TI - Structure and mechanism of mitochondrial proton-translocating transhydrogenase VL - 573 ER - TY - JOUR AB - Grid cells with their rigid hexagonal firing fields are thought to provide an invariant metric to the hippocampal cognitive map, yet environmental geometrical features have recently been shown to distort the grid structure. Given that the hippocampal role goes beyond space, we tested the influence of nonspatial information on the grid organization. We trained rats to daily learn three new reward locations on a cheeseboard maze while recording from the medial entorhinal cortex and the hippocampal CA1 region. Many grid fields moved toward goal location, leading to long-lasting deformations of the entorhinal map. Therefore, distortions in the grid structure contribute to goal representation during both learning and recall, which demonstrates that grid cells participate in mnemonic coding and do not merely provide a simple metric of space. AU - Boccara, Charlotte N. AU - Nardin, Michele AU - Stella, Federico AU - O'Neill, Joseph AU - Csicsvari, Jozsef L ID - 6194 IS - 6434 JF - Science SN - 0036-8075 TI - The entorhinal cognitive map is attracted to goals VL - 363 ER - TY - THES AB - A major challenge in neuroscience research is to dissect the circuits that orchestrate behavior in health and disease. Proteins from a wide range of non-mammalian species, such as microbial opsins, have been successfully transplanted to specific neuronal targets to override their natural communication patterns. The goal of our work is to manipulate synaptic communication in a manner that closely incorporates the functional intricacies of synapses by preserving temporal encoding (i.e. the firing pattern of the presynaptic neuron) and connectivity (i.e. target specific synapses rather than specific neurons). Our strategy to achieve this goal builds on the use of non-mammalian transplants to create a synthetic synapse. The mode of modulation comes from pre-synaptic uptake of a synthetic neurotransmitter (SN) into synaptic vesicles by means of a genetically targeted transporter selective for the SN. Upon natural vesicular release, exposure of the SN to the synaptic cleft will modify the post-synaptic potential through an orthogonal ligand gated ion channel. To achieve this goal we have functionally characterized a mixed cationic methionine-gated ion channel from Arabidopsis thaliana, designed a method to functionally characterize a synthetic transporter in isolated synaptic vesicles without the need for transgenic animals, identified and extracted multiple prokaryotic uptake systems that are substrate specific for methionine (Met), and established a primary/cell line co-culture system that would allow future combinatorial testing of this orthogonal transmitter-transporter-channel trifecta. Synthetic synapses will provide a unique opportunity to manipulate synaptic communication while maintaining the electrophysiological integrity of the pre-synaptic cell. In this way, information may be preserved that was generated in upstream circuits and that could be essential for concerted function and information processing. AU - Mckenzie, Catherine ID - 7132 SN - 2663-337X TI - Design and characterization of methods and biological components to realize synthetic neurotransmission ER -