@article{9887, abstract = {Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.}, author = {Johnson, Alexander J and Dahhan, Dana A and Gnyliukh, Nataliia and Kaufmann, Walter and Zheden, Vanessa and Costanzo, Tommaso and Mahou, Pierre and Hrtyan, Mónika and Wang, Jie and Aguilera Servin, Juan L and van Damme, Daniël and Beaurepaire, Emmanuel and Loose, Martin and Bednarek, Sebastian Y and Friml, Jiří}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {51}, publisher = {National Academy of Sciences}, title = {{The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis}}, doi = {10.1073/pnas.2113046118}, volume = {118}, year = {2021}, } @article{8284, abstract = {Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.}, author = {Steiner, Julia and Sazanov, Leonid A}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter}}, doi = {10.7554/eLife.59407}, volume = {9}, year = {2020}, } @phdthesis{8340, abstract = {Mitochondria are sites of oxidative phosphorylation in eukaryotic cells. Oxidative phosphorylation operates by a chemiosmotic mechanism made possible by redox-driven proton pumping machines which establish a proton motive force across the inner mitochondrial membrane. This electrochemical proton gradient is used to drive ATP synthesis, which powers the majority of cellular processes such as protein synthesis, locomotion and signalling. In this thesis I investigate the structures and molecular mechanisms of two inner mitochondrial proton pumping enzymes, respiratory complex I and transhydrogenase. I present the first high-resolution structure of the full transhydrogenase from any species, and a significantly improved structure of complex I. Improving the resolution from 3.3 Å available previously to up to 2.3 Å in this thesis allowed us to model bound water molecules, crucial in the proton pumping mechanism. For both enzymes, up to five cryo-EM datasets with different substrates and inhibitors bound were solved to delineate the catalytic cycle and understand the proton pumping mechanism. In transhydrogenase, the proton channel is gated by reversible detachment of the NADP(H)-binding domain which opens the proton channel to the opposite sites of the membrane. In complex I, the proton channels are gated by reversible protonation of key glutamate and lysine residues and breaking of the water wire connecting the proton pumps with the quinone reduction site. The tight coupling between the redox and the proton pumping reactions in transhydrogenase is achieved by controlling the NADP(H) exchange which can only happen when the NADP(H)-binding domain interacts with the membrane domain. In complex I, coupling is achieved by cycling of the whole complex between the closed state, in which quinone can get reduced, and the open state, in which NADH can induce quinol ejection from the binding pocket. On the basis of these results I propose detailed mechanisms for catalytic cycles of transhydrogenase and complex I that are consistent with a large amount of previous work. In both enzymes, conformational and electrostatic mechanisms contribute to the overall catalytic process. Results presented here could be used for better understanding of the human pathologies arising from deficiencies of complex I or transhydrogenase and could be used to develop novel therapies.}, author = {Kampjut, Domen}, isbn = {978-3-99078-008-4}, issn = {2663-337X}, pages = {242}, publisher = {Institute of Science and Technology Austria}, title = {{Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes}}, doi = {10.15479/AT:ISTA:8340}, year = {2020}, } @phdthesis{8350, abstract = {Cytoplasm is a gel-like crowded environment composed of tens of thousands of macromolecules, organelles, cytoskeletal networks and cytosol. The structure of the cytoplasm is thought to be highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules is very restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the jammed nature of the cytoplasm at the microscopic scale, large-scale reorganization of cytoplasm is essential for important cellular functions, such as nuclear positioning and cell division. How such mesoscale reorganization of the cytoplasm is achieved, especially for very large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, has only begun to be understood. In this thesis, I focus on the recent advances in elucidating the molecular, cellular and biophysical principles underlying cytoplasmic organization across different scales, structures and species. First, I outline which of these principles have been identified by reductionist approaches, such as in vitro reconstitution assays, where boundary conditions and components can be modulated at ease. I then describe how the theoretical and experimental framework established in these reduced systems have been applied to their more complex in vivo counterparts, in particular oocytes and embryonic syncytial structures, and discuss how such complex biological systems can initiate symmetry breaking and establish patterning. Specifically, I examine an example of large-scale reorganizations taking place in zebrafish embryos, where extensive cytoplasmic streaming leads to the segregation of cytoplasm from yolk granules along the animal-vegetal axis of the embryo. Using biophysical experimentation and theory, I investigate the forces underlying this process, to show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the embryo. This wave functions in segregation by both pulling cytoplasm animally and pushing yolk granules vegetally. Cytoplasm pulling is mediated by bulk actin network flows exerting friction forces on the cytoplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. This study defines a novel role of bulk actin polymerization waves in embryo polarization via cytoplasmic segregation. Lastly, I describe the cytoplasmic reorganizations taking place during zebrafish oocyte maturation, where the initial segregation of the cytoplasm and yolk granules occurs. Here, I demonstrate a previously uncharacterized wave of microtubule aster formation, traveling the oocyte along the animal-vegetal axis. Further research is required to determine the role of such microtubule structures in cytoplasmic reorganizations therein. Collectively, these studies provide further evidence for the coupling between cell cytoskeleton and cell cycle machinery, which can underlie a core self-organizing mechanism for orchestrating large-scale reorganizations in a cell-cycle-tunable manner, where the modulations of the force-generating machinery and cytoplasmic mechanics can be harbored to fulfill cellular functions.}, author = {Shamipour, Shayan}, issn = {2663-337X}, pages = {107}, publisher = {Institute of Science and Technology Austria}, title = {{Bulk actin dynamics drive phase segregation in zebrafish oocytes }}, doi = {10.15479/AT:ISTA:8350}, year = {2020}, } @phdthesis{8353, abstract = {Mrp (Multi resistance and pH adaptation) are broadly distributed secondary active antiporters that catalyze the transport of monovalent ions such as sodium and potassium outside of the cell coupled to the inward translocation of protons. Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG) encoded in a single operon, whereas other antiporters catalyzing the same reaction are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline environments and for reduction of the intracellular concentration of toxic cations. Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp subunits have primary sequence similarity to essential redox-driven proton pumps, such as respiratory complex I and membrane-bound hydrogenases. This similarity reinforces the hypothesis that these present day redox-driven proton pumps are descended from the Mrp antiporter. The Mrp structure serves as a model to understand the yet obscure coupling mechanism between ion or electron transfer and proton translocation in this large group of proteins. In the thesis, I am presenting the purification, biochemical analysis, cryo-EM analysis and molecular structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. Numerous conditions were screened to purify Mrp to high homogeneity and to obtain an appropriate distribution of single particles on cryo-EM grids covered with a continuous layer of ultrathin carbon. A preferred particle orientation problem was solved by performing a tilted data collection. The activity assays showed the specific pH-dependent profile of secondary active antiporters. The molecular structure shows that Mrp is a dimer of seven-subunit protomers with 50 trans-membrane helices each. The dimer interface is built by many short and tilted transmembrane helices, probably causing a thinning of the bacterial membrane. The surface charge distribution shows an extraordinary asymmetry within each monomer, revealing presumable proton and sodium translocation pathways. The two largest and homologous Mrp subunits MrpA and MrpD probably translocate one proton each into the cell. The sodium ion is likely being translocated in the opposite direction within the small subunits along a ladder of charged and conserved residues. Based on the structure, we propose a mechanism were the antiport activity is accomplished via electrostatic interactions between the charged cations and key charged residues. The flexible key TM helices coordinate these electrostatic interactions, while the membrane thinning between the monomers enables the translocation of sodium across the charged membrane. The entire family of redox-driven proton pumps is likely to perform their mechanism in a likewise manner.}, author = {Steiner, Julia}, issn = {2663-337X}, pages = {191}, publisher = {Institute of Science and Technology Austria}, title = {{Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I}}, doi = {10.15479/AT:ISTA:8353}, year = {2020}, } @article{8581, abstract = {The majority of adenosine triphosphate (ATP) powering cellular processes in eukaryotes is produced by the mitochondrial F1Fo ATP synthase. Here, we present the atomic models of the membrane Fo domain and the entire mammalian (ovine) F1Fo, determined by cryo-electron microscopy. Subunits in the membrane domain are arranged in the ‘proton translocation cluster’ attached to the c-ring and a more distant ‘hook apparatus’ holding subunit e. Unexpectedly, this subunit is anchored to a lipid ‘plug’ capping the c-ring. We present a detailed proton translocation pathway in mammalian Fo and key inter-monomer contacts in F1Fo multimers. Cryo-EM maps of F1Fo exposed to calcium reveal a retracted subunit e and a disassembled c-ring, suggesting permeability transition pore opening. We propose a model for the permeability transition pore opening, whereby subunit e pulls the lipid plug out of the c-ring. Our structure will allow the design of drugs for many emerging applications in medicine.}, author = {Pinke, Gergely and Zhou, Long and Sazanov, Leonid A}, issn = {1545-9985}, journal = {Nature Structural and Molecular Biology}, number = {11}, pages = {1077--1085}, publisher = {Springer Nature}, title = {{Cryo-EM structure of the entire mammalian F-type ATP synthase}}, doi = {10.1038/s41594-020-0503-8}, volume = {27}, year = {2020}, } @article{8586, abstract = {Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.}, author = {Fäßler, Florian and Zens, Bettina and Hauschild, Robert and Schur, Florian KM}, issn = {1047-8477}, journal = {Journal of Structural Biology}, keywords = {electron microscopy, cryo-EM, EM sample preparation, 3D printing, cell culture}, number = {3}, publisher = {Elsevier}, title = {{3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy}}, doi = {10.1016/j.jsb.2020.107633}, volume = {212}, year = {2020}, } @article{8737, abstract = {Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo-electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.}, author = {Kampjut, Domen and Sazanov, Leonid A}, issn = {1095-9203}, journal = {Science}, number = {6516}, publisher = {American Association for the Advancement of Science}, title = {{The coupling mechanism of mammalian respiratory complex I}}, doi = {10.1126/science.abc4209}, volume = {370}, year = {2020}, } @article{8971, abstract = {The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.}, author = {Fäßler, Florian and Dimchev, Georgi A and Hodirnau, Victor-Valentin and Wan, William and Schur, Florian KM}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology, General Physics and Astronomy, General Chemistry}, publisher = {Springer Nature}, title = {{Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction}}, doi = {10.1038/s41467-020-20286-x}, volume = {11}, year = {2020}, } @article{7490, abstract = {In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.}, author = {Narasimhan, Madhumitha and Johnson, Alexander J and Prizak, Roshan and Kaufmann, Walter and Tan, Shutang and Casillas Perez, Barbara E and Friml, Jiří}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants}}, doi = {10.7554/eLife.52067}, volume = {9}, year = {2020}, } @phdthesis{7525, abstract = {The medial habenula (MHb) is an evolutionary conserved epithalamic structure important for the modulation of emotional memory. It is involved in regulation of anxiety, compulsive behavior, addiction (nicotinic and opioid), sexual and feeding behavior. MHb receives inputs from septal regions and projects exclusively to the interpeduncular nucleus (IPN). Distinct sub-regions of the septum project to different subnuclei of MHb: the bed nucleus of anterior commissure projects to dorsal MHb and the triangular septum projects to ventral MHb. Furthermore, the dorsal and ventral MHb project to the lateral and rostral/central IPN, respectively. Importantly, these projections have unique features of prominent co-release of different neurotransmitters and requirement of a peculiar type of calcium channel for release. In general, synaptic neurotransmission requires an activity-dependent influx of Ca2+ into the presynaptic terminal through voltage-gated calcium channels. The calcium channel family most commonly involved in neurotransmitter release comprises three members, P/Q-, N- and R-type with Cav2.1, Cav2.2 and Cav2.3 subunits, respectively. In contrast to most CNS synapses that mainly express Cav2.1 and/or Cav2.2, MHb terminals in the IPN exclusively express Cav2.3. In other parts of the brain, such as the hippocampus, Cav2.3 is mostly located to postsynaptic elements. This unusual presynaptic location of Cav2.3 in the MHb-IPN pathway implies unique mechanisms of glutamate release in this pathway. One potential example of such uniqueness is the facilitation of release by GABAB receptor (GBR) activation. Presynaptic GBRs usually inhibit the release of neurotransmitters by inhibiting presynaptic calcium channels. MHb shows the highest expression levels of GBR in the brain. GBRs comprise two subunits, GABAB1 (GB1) and GABAB2 (GB2), and are associated with auxiliary subunits, called potassium channel tetramerization domain containing proteins (KCTD) 8, 12, 12b and 16. Among these four subunits, KCTD12b is exclusively expressed in ventral MHb, and KCTD8 shows the strongest expression in the whole MHb among other brain regions, indicating that KCTD8 and KCTD12b may be involved in the unique mechanisms of neurotransmitter release mediated by Cav2.3 and regulated by GBRs in this pathway. In the present study, we first verified that neurotransmission in both dorsal and ventral MHb-IPN pathways is mainly mediated by Cav2.3 using a selective blocker of R-type channels, SNX-482. We next found that baclofen, a GBR agonist, has facilitatory effects on release from ventral MHb terminal in rostral IPN, whereas it has inhibitory effects on release from dorsal MHb terminals in lateral IPN, indicating that KCTD12b expressed exclusively in ventral MHb may have a role in the facilitatory effects of GBR activation. In a heterologous expression system using HEK cells, we found that KCTD8 and KCTD12b but not KCTD12 directly bind with Cav2.3. Pre-embedding immunogold electron microscopy data show that Cav2.3 and KCTD12b are distributed most densely in presynaptic active zone in IPN with KCTD12b being present only in rostral/central but not lateral IPN, whereas GABAB, KCTD8 and KCTD12 are distributed most densely in perisynaptic sites with KCTD12 present more frequently in postsynaptic elements and only in rostral/central IPN. In freeze-fracture replica labelling, Cav2.3, KCTD8 and KCTD12b are co-localized with each other in the same active zone indicating that they may form complexes regulating vesicle release in rostral IPN. On electrophysiological studies of wild type (WT) mice, we found that paired-pulse ratio in rostral IPN of KCTD12b knock-out (KO) mice is lower than those of WT and KCTD8 KO mice. Consistent with this finding, in mean variance analysis, release probability in rostral IPN of KCTD12b KO mice is higher than that of WT and KCTD8 KO mice. Although paired-pulse ratios are not different between WT and KCTD8 KO mice, the mean variance analysis revealed significantly lower release probability in rostral IPN of KCTD8 KO than WT mice. These results demonstrate bidirectional regulation of Cav2.3-mediated release by KCTD8 and KCTD12b without GBR activation in rostral IPN. Finally, we examined the baclofen effects in rostral IPN of KCTD8 and KCTD12b KO mice, and found the facilitation of release remained in both KO mice, indicating that the peculiar effects of the GBR activation in this pathway do not depend on the selective expression of these KCTD subunits in ventral MHb. However, we found that presynaptic potentiation of evoked EPSC amplitude by baclofen falls to baseline after washout faster in KCTD12b KO mice than WT, KCTD8 KO and KCTD8/12b double KO mice. This result indicates that KCTD12b is involved in sustained potentiation of vesicle release by GBR activation, whereas KCTD8 is involved in its termination in the absence of KCTD12b. Consistent with these functional findings, replica labelling revealed an increase in density of KCTD8, but not Cav2.3 or GBR at active zone in rostral IPN of KCTD12b KO mice compared with that of WT mice, suggesting that increased association of KCTD8 with Cav2.3 facilitates the release probability and termination of the GBR effect in the absence of KCTD12b. In summary, our study provided new insights into the physiological roles of presynaptic Cav2.3, GBRs and their auxiliary subunits KCTDs at an evolutionary conserved neuronal circuit. Future studies will be required to identify the exact molecular mechanism underlying the GBR-mediated presynaptic potentiation on ventral MHb terminals. It remains to be determined whether the prominent presence of presynaptic KCTDs at active zone could exert similar neuromodulatory functions in different pathways of the brain. }, author = {Bhandari, Pradeep}, issn = {2663-337X}, keywords = {Cav2.3, medial habenula (MHb), interpeduncular nucleus (IPN)}, pages = {79}, publisher = {Institute of Science and Technology Austria}, title = {{Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway}}, doi = {10.15479/AT:ISTA:7525}, year = {2020}, } @article{7814, abstract = {Scientific research is to date largely restricted to wealthy laboratories in developed nations due to the necessity of complex and expensive equipment. This inequality limits the capacity of science to be used as a diplomatic channel. Maker movements use open-source technologies including additive manufacturing (3D printing) and laser cutting, together with low-cost computers for developing novel products. This movement is setting the groundwork for a revolution, allowing scientific equipment to be sourced at a fraction of the cost and has the potential to increase the availability of equipment for scientists around the world. Science education is increasingly recognized as another channel for science diplomacy. In this perspective, we introduce the idea that the Maker movement and open-source technologies have the potential to revolutionize science, technology, engineering and mathematics (STEM) education worldwide. We present an open-source STEM didactic tool called SCOPES (Sparking Curiosity through Open-source Platforms in Education and Science). SCOPES is self-contained, independent of local resources, and cost-effective. SCOPES can be adapted to communicate complex subjects from genetics to neurobiology, perform real-world biological experiments and explore digitized scientific samples. We envision such platforms will enhance science diplomacy by providing a means for scientists to share their findings with classrooms and for educators to incorporate didactic concepts into STEM lessons. By providing students the opportunity to design, perform, and share scientific experiments, students also experience firsthand the benefits of a multinational scientific community. We provide instructions on how to build and use SCOPES on our webpage: http://scopeseducation.org.}, author = {Beattie, Robert J and Hippenmeyer, Simon and Pauler, Florian}, issn = {2504-284X}, journal = {Frontiers in Education}, publisher = {Frontiers Media}, title = {{SCOPES: Sparking curiosity through Open-Source platforms in education and science}}, doi = {10.3389/feduc.2020.00048}, volume = {5}, year = {2020}, } @article{8139, abstract = {Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.}, author = {Johnson, Alexander J and Gnyliukh, Nataliia and Kaufmann, Walter and Narasimhan, Madhumitha and Vert, G and Bednarek, SY and Friml, Jiří}, issn = {1477-9137}, journal = {Journal of Cell Science}, number = {15}, publisher = {The Company of Biologists}, title = {{Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis}}, doi = {10.1242/jcs.248062}, volume = {133}, year = {2020}, } @unpublished{9750, abstract = {Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow1,2. Here we show in zebrafish primary germ layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase, and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. Once tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension stabilizing E-cadherin-actin complexes at the contact.}, author = {Slovakova, Jana and Sikora, Mateusz K and Caballero Mancebo, Silvia and Krens, Gabriel and Kaufmann, Walter and Huljev, Karla and Heisenberg, Carl-Philipp J}, booktitle = {bioRxiv}, pages = {41}, publisher = {Cold Spring Harbor Laboratory}, title = {{Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion}}, doi = {10.1101/2020.11.20.391284}, year = {2020}, } @phdthesis{7186, abstract = {Tissue morphogenesis in developmental or physiological processes is regulated by molecular and mechanical signals. While the molecular signaling cascades are increasingly well described, the mechanical signals affecting tissue shape changes have only recently been studied in greater detail. To gain more insight into the mechanochemical and biophysical basis of an epithelial spreading process (epiboly) in early zebrafish development, we studied cell-cell junction formation and actomyosin network dynamics at the boundary between surface layer epithelial cells (EVL) and the yolk syncytial layer (YSL). During zebrafish epiboly, the cell mass sitting on top of the yolk cell spreads to engulf the yolk cell by the end of gastrulation. It has been previously shown that an actomyosin ring residing within the YSL pulls on the EVL tissue through a cable-constriction and a flow-friction motor, thereby dragging the tissue vegetal wards. Pulling forces are likely transmitted from the YSL actomyosin ring to EVL cells; however, the nature and formation of the junctional structure mediating this process has not been well described so far. Therefore, our main aim was to determine the nature, dynamics and potential function of the EVL-YSL junction during this epithelial tissue spreading. Specifically, we show that the EVL-YSL junction is a mechanosensitive structure, predominantly made of tight junction (TJ) proteins. The process of TJ mechanosensation depends on the retrograde flow of non-junctional, phase-separated Zonula Occludens-1 (ZO-1) protein clusters towards the EVL-YSL boundary. Interestingly, we could demonstrate that ZO-1 is present in a non-junctional pool on the surface of the yolk cell, and ZO-1 undergoes a phase separation process that likely renders the protein responsive to flows. These flows are directed towards the junction and mediate proper tension-dependent recruitment of ZO-1. Upon reaching the EVL-YSL junction ZO-1 gets incorporated into the junctional pool mediated through its direct actin-binding domain. When the non-junctional pool and/or ZO-1 direct actin binding is absent, TJs fail in their proper mechanosensitive responses resulting in slower tissue spreading. We could further demonstrate that depletion of ZO proteins within the YSL results in diminished actomyosin ring formation. This suggests that a mechanochemical feedback loop is at work during zebrafish epiboly: ZO proteins help in proper actomyosin ring formation and actomyosin contractility and flows positively influence ZO-1 junctional recruitment. Finally, such a mesoscale polarization process mediated through the flow of phase-separated protein clusters might have implications for other processes such as immunological synapse formation, C. elegans zygote polarization and wound healing.}, author = {Schwayer, Cornelia}, issn = {2663-337X}, pages = {107}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanosensation of tight junctions depends on ZO-1 phase separation and flow}}, doi = {10.15479/AT:ISTA:7186}, year = {2019}, } @article{7406, abstract = {Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.}, author = {Mckenzie, Catherine and Spanova, Miroslava and Johnson, Alexander J and Kainrath, Stephanie and Zheden, Vanessa and Sitte, Harald H. and Janovjak, Harald L}, issn = {0165-0270}, journal = {Journal of Neuroscience Methods}, pages = {114--121}, publisher = {Elsevier}, title = {{Isolation of synaptic vesicles from genetically engineered cultured neurons}}, doi = {10.1016/j.jneumeth.2018.11.018}, volume = {312}, year = {2019}, } @article{6087, abstract = {Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz−/− follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.}, author = {Xia, Peng and Gütl, Daniel J and Zheden, Vanessa and Heisenberg, Carl-Philipp J}, journal = {Cell}, number = {6}, pages = {1379--1392.e14}, publisher = {Elsevier}, title = {{Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity}}, doi = {10.1016/j.cell.2019.01.019}, volume = {176}, year = {2019}, } @phdthesis{6269, abstract = {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. }, author = {Narasimhan, Madhumitha}, issn = {2663-337X}, pages = {138}, publisher = {Institute of Science and Technology Austria}, title = {{Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants }}, doi = {10.15479/at:ista:th1075}, year = {2019}, } @phdthesis{6947, abstract = {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.}, author = {Assen, Frank P}, issn = {2663-337X}, pages = {142}, publisher = {Institute of Science and Technology Austria}, title = {{Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking}}, doi = {10.15479/AT:ISTA:6947}, year = {2019}, } @phdthesis{395, abstract = {Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g. autism spectrum disorder, intellectual disability, epilepsy) remains a great challenge. Recent advancements in geno mics, like whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that were discovered, the etiological variability and the heterogeneous phenotypic outcomes, the need for genotype -along with phenotype- based diagnosis of individual patients becomes a requisite. Driven by this rationale, in a previous study our group described mutations, identified via whole - exome sequencing, in the gene BCKDK – encoding for a key regulator of branched chain amin o acid (BCAA) catabolism - as a cause of ASD. Following up on the role of BCAAs, in the study described here we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized mainly at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation and severe neurolo gical abnormalities. Additionally, deletion of Slc7a5 from the neural progenitor cell population leads to microcephaly. Interestingly, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Furthermore, whole - exome sequencing of patients diagnosed with neurological dis o r ders helped us identify several patients with autistic traits, microcephaly and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. In conclusion, our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for t he BCAA s in human bra in function. Together with r ecent studies (described in chapter two) that have successfully made the transition into clinical practice, our findings on the role of B CAAs might have a crucial impact on the development of novel individualized therapeutic strategies for ASD. }, author = {Tarlungeanu, Dora-Clara}, issn = {2663-337X}, pages = {88}, publisher = {Institute of Science and Technology Austria}, title = {{The branched chain amino acids in autism spectrum disorders }}, doi = {10.15479/AT:ISTA:th_992}, year = {2018}, }