@misc{18296, abstract = {It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the murine hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.}, author = {Kim, Olena}, keywords = {Hippocampal mossy fiber synapses, short-term potentiation, long-term potentiation, presynaptic plasticity, electron microscopy, freeze-fracture replica labeling, paired recordings, forskolin, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), neuromodulation, synaptic vesicle pools, presynaptic Ca2+ channels, Munc13, docking, priming, active zone}, publisher = {Institute of Science and Technology Austria}, title = {{Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons}}, doi = {10.15479/AT:ISTA:18296}, year = {2024}, } @phdthesis{14510, abstract = {Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment control of protein regulators for vesicle maturation and release. During the early stages of endocytosis, an area of flat membrane is remodelled by proteins to create a spherical vesicle against intracellular forces. After the Clathrin-coated vesicle (CCV) is fully formed, scission machinery releases it from the plasma membrane, and cargo proceeds for recycling or degradation through early endosomes / Trans Golgi network. Protein machineries that mediate membrane bending and vesicle release in plants are unknown. However, studies show, that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against highturgor pressure compared to other model systems. First, by using biochemical and advanced live microscopy approaches we investigate the TPLATE complex, a plant-specific endocytosis protein complex. We found that TPLATE is peripherally associated with clathrin-coated vesicles and localises at the rim of endocytosis events. Next, our study of plant Dynamin-related protein 1C (DRP1C), which was hypothesised previously to play a role in vesicle release, shows the recruitment of the protein already at the early stages of endocytosis. Moreover, DRP1C assembles into organised ring-like structures and is able to induce membrane deformation and tubulation, suggesting its role also in membrane bending during early CME. Based on the data from mammalian and yeast systems, plant DynaminRelated Proteins 2 and SH3P2 protein are strong candidates to be part of the plant vesicle scission machinery; however, their precise role in plant CME has not been yet elucidated. Here, we characterised DRP2s and SH3P2 roles in CME by combining high-resolution imaging of endocytic events in vivo and protein characterisation. Although DRP2s and SH3P2 arrive together during late CME and physically interact, genetic analysis using ∆sh3p1,2,3 mutant and complementation with non-DRP2-interacting SH3P2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. Summarising our research, these observations provide new important insights into the mechanism of plant CME and show that, despite plants posses many homologues of mammalian and yeast CME components, they do not necessarily act in the same manner. }, author = {Gnyliukh, Nataliia}, isbn = {978-3-99078-037-4}, issn = {2663-337X}, keywords = {Clathrin-Mediated Endocytosis, vesicle scission, Dynamin-Related Protein 2, SH3P2, TPLATE complex, Total internal reflection fluorescence microscopy, Arabidopsis thaliana}, pages = {180}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanism of clathrin-coated vesicle formation during endocytosis in plants}}, doi = {10.15479/at:ista:14510}, year = {2023}, } @article{10406, abstract = {Multicellular organisms develop complex shapes from much simpler, single-celled zygotes through a process commonly called morphogenesis. Morphogenesis involves an interplay between several factors, ranging from the gene regulatory networks determining cell fate and differentiation to the mechanical processes underlying cell and tissue shape changes. Thus, the study of morphogenesis has historically been based on multidisciplinary approaches at the interface of biology with physics and mathematics. Recent technological advances have further improved our ability to study morphogenesis by bridging the gap between the genetic and biophysical factors through the development of new tools for visualizing, analyzing, and perturbing these factors and their biochemical intermediaries. Here, we review how a combination of genetic, microscopic, biophysical, and biochemical approaches has aided our attempts to understand morphogenesis and discuss potential approaches that may be beneficial to such an inquiry in the future.}, author = {Mishra, Nikhil and Heisenberg, Carl-Philipp J}, issn = {1545-2948}, journal = {Annual Review of Genetics}, keywords = {morphogenesis, forward genetics, high-resolution microscopy, biophysics, biochemistry, patterning}, pages = {209--233}, publisher = {Annual Reviews}, title = {{Dissecting organismal morphogenesis by bridging genetics and biophysics}}, doi = {10.1146/annurev-genet-071819-103748}, volume = {55}, year = {2021}, } @inbook{9756, abstract = {High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms.}, author = {Kaufmann, Walter and Kleindienst, David and Harada, Harumi and Shigemoto, Ryuichi}, booktitle = { Receptor and Ion Channel Detection in the Brain}, isbn = {9781071615218}, keywords = {Freeze-fracture replica: Deep learning, Immunogold labeling, Integral membrane protein, Electron microscopy}, pages = {267--283}, publisher = {Humana}, title = {{High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)}}, doi = {10.1007/978-1-0716-1522-5_19}, volume = {169}, year = {2021}, } @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}, } @inproceedings{11222, author = {Kim, Olena and Borges Merjane, Carolina and Jonas, Peter M}, booktitle = {Intrinsic Activity}, issn = {2309-8503}, keywords = {hippocampus, mossy fibers, readily releasable pool, electron microscopy}, location = {Innsbruck, Austria}, number = {Suppl. 1}, publisher = {Austrian Pharmacological Society}, title = {{Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy}}, doi = {10.25006/ia.7.s1-a3.27}, volume = {7}, year = {2019}, } @misc{5569, abstract = {Nela Nikolic, Tobias Bergmiller, Alexandra Vandervelde, Tanino G. Albanese, Lendert Gelens, and Isabella Moll (2018) “Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations” Nucleic Acids Research, doi: 10.15479/AT:ISTA:74; microscopy experiments by Tobias Bergmiller; image and data analysis by Nela Nikolic.}, author = {Bergmiller, Tobias and Nikolic, Nela}, keywords = {microscopy, microfluidics}, publisher = {Institute of Science and Technology Austria}, title = {{Time-lapse microscopy data}}, doi = {10.15479/AT:ISTA:74}, year = {2018}, } @phdthesis{10663, abstract = {The superconducting state of matter enables one to observe quantum effects on the macroscopic scale and hosts many fascinating phenomena. Topological defects of the superconducting order parameter, such as vortices and fluxoid states in multiply connected structures, are often the key ingredients of these phenomena. This dissertation describes a new mode of magnetic force microscopy (Φ0-MFM) for investigating vortex and fluxoid sates in mesoscopic superconducting (SC) structures. The technique relies on the magneto-mechanical coupling of a MFM cantilever to the motion of fluxons. The novelty of the technique is that a magnetic particle attached to the cantilever is used not only to sense the state of a SC structure, but also as a primary source of the inhomogeneous magnetic field which induces that state. Φ0-MFM enables us to map the transitions between tip-induced states during a scan: at the positions of the tip, where the two lowest energy states become degenerate, small oscillations of the tip drive the transitions between these states, which causes a significant shift in the resonant frequency and dissipation of the cantilever. For narrow-wall aluminum rings, the mapped fluxoid transitions form concentric contours on a scan. We show that the changes in the cantilever resonant frequency and dissipation are well-described by a stochastic resonance (SR) of cantilever-driven thermally activated phase slips (TAPS). The SR model allows us to experimentally determine the rate of TAPS and compare it to the Langer-Ambegaokar-McCumber-Halperin (LAMH) theory for TAPS in 1D superconducting structures. Further, we use the SR model to qualitatively study the effects of a locally applied magnetic field on the phase slip rate in rings containing constrictions. The states with multiple vortices or winding numbers could be useful for the development of novel superconducting devices, or the study of vortex interactions and interference effects. Using Φ0-MFM allows us to induce, probe and control fluxoid states in thin wall structures comprised of multiple loops. We show that Φ0-MFM images of the fluxoid transitions allow us to identify the underlying states and to investigate their energetics and dynamics even in complicated structures.}, author = {Polshyn, Hryhoriy}, keywords = {physics, superconductivity, magnetic force microscopy, phase slips}, pages = {103}, publisher = {University of Illinois at Urbana-Champaign}, title = {{Magnetic force microscopy studies of mesoscopic superconducting structures}}, year = {2017}, } @misc{5560, abstract = {This repository contains the data collected for the manuscript "Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity". The data is compressed into a single archive. Within the archive, different folders correspond to figures of the main text and the SI of the related publication. Data is saved as plain text, with each folder containing a separate readme file describing the format. Typically, the data is from fluorescence microscopy measurements of single cells growing in a microfluidic "mother machine" device, and consists of relevant values (primarily arbitrary unit or normalized fluorescence measurements, and division times / growth rates) after raw microscopy images have been processed, segmented, and their features extracted, as described in the methods section of the related publication.}, author = {Bergmiller, Tobias and Andersson, Anna M and Tomasek, Kathrin and Balleza, Enrique and Kiviet, Daniel and Hauschild, Robert and Tkacik, Gasper and Guet, Calin C}, keywords = {single cell microscopy, mother machine microfluidic device, AcrAB-TolC pump, multi-drug efflux, Escherichia coli}, publisher = {Institute of Science and Technology Austria}, title = {{Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity}}, doi = {10.15479/AT:ISTA:53}, year = {2017}, } @misc{5566, abstract = {Current minimal version of TipTracker}, author = {Hauschild, Robert}, keywords = {tool, tracking, confocal microscopy}, publisher = {Institute of Science and Technology Austria}, title = {{Live tracking of moving samples in confocal microscopy for vertically grown roots}}, doi = {10.15479/AT:ISTA:69}, year = {2017}, } @misc{5555, abstract = {This FIJI script calculates the population average of the migration speed as a function of time of all cells from wide field microscopy movies.}, author = {Hauschild, Robert}, keywords = {cell migration, wide field microscopy, FIJI}, publisher = {Institute of Science and Technology Austria}, title = {{Fiji script to determine average speed and direction of migration of cells}}, doi = {10.15479/AT:ISTA:44}, year = {2016}, } @article{21514, abstract = {This paper describes observations and metrological analyses made to compare the replication quality of polymeric replicas obtained by filling micro-cavities using both hot embossing and micro-injection moulding processes. The experiments are performed with polypropylene (PP) at a constant melt temperature and a constant mould temperature, whereas hot embossing tests are carried out with the same polymer at temperatures close to the softening one. The results concerning the micro-cavities filling provide information on the reliability about the possibilities of replication topographical surface geometries. The data obtained by scanning mechanical microscopy (SMM) are used to determine the comparative filling ratio values.}, author = {Sahli, M. and Millot, C. and Roques-Carmes, Charles and Khan Malek, C. and Barriere, T. and Gelin, J.C.}, issn = {1873-4774}, journal = {Journal of Materials Processing Technology}, keywords = {Hot embossing, Micro-injection moulding, Micro-cavities replication, Polypropylene polymer, Scanning mechanical microscopy, Roughness parameters}, number = {18-19}, pages = {5851--5861}, publisher = {Elsevier}, title = {{Quality assessment of polymer replication by hot embossing and micro-injection moulding processes using scanning mechanical microscopy}}, doi = {10.1016/j.jmatprotec.2009.06.011}, volume = {209}, year = {2009}, } @article{21518, abstract = {The present investigation confirms that initially implemented procedure to produce poly(methylidene malonate 2.1.2) (PMM 2.1.2) nanoparticles (Lescure et al. Pharm Res 1994;11:1270–77) lead to products mostly containing plasticizing oligomers which strongly lowered glass-transition temperature (Tg), dramatically reduced nanoparticle consistency and rendered them too sensitive to solubilization when diluted in an aqueous medium. From MALDI-TOF spectroscopy analysis, performed on intact colloids, emerged some structural information about these oligomeric species which could result from an intramolecular cyclization mechanism occurring soon in the course of the polymerization process. Thus, with the objective of overcoming these drawbacks, this contribution deals with the variations of manufacturing specifications such as pH and magnetic stirring speed to try and modulate molecular weight (Mw) of nanoparticle constituents and reduce oligomer concentration. Although the analyses performed on these new nanoparticles were rather encouraging, the colloid formation yield became so low that it required the developement of other methodologies, excluding a previous emulsion step, and allowing a controlled production of PMM 2.1.2-made nanoparticles having better physico-chemical characteristics while keeping good pharmaceutical capabilities.}, author = {Breton, P and Guillon, X and Roy, D and Lescure, F and Riess, G and Bru, N and Roques-Carmes, Charles}, issn = {0142-9612}, journal = {Biomaterials}, keywords = {Colloid physico-chemical analysis, Colloidal drug carriers, MALDI-TOF spectroscopy, Nanoparticles, Poly(methylidene malonate), Scanning electron microscopy}, number = {1-3}, pages = {271--281}, publisher = {Elsevier}, title = {{Physico-chemical characterization, preparation and performance of poly (methylidene malonate 2.1.2) nanoparticles}}, doi = {10.1016/s0142-9612(97)00243-3}, volume = {19}, year = {1998}, }