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  1. Article ; Online: Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM.

    Begemann, Isabell / Keller, Ulrike / Nüsse, Harald / Klingauf, Jürgen / Galic, Milos

    Cells

    2020  Volume 9, Issue 6

    Abstract: Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated ... ...

    Abstract Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches.
    MeSH term(s) Animals ; Cell Membrane/ultrastructure ; Cells, Cultured ; Cytosol/metabolism ; Gold/chemistry ; Humans ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Protein Transport ; Reproducibility of Results
    Chemical Substances Gold (7440-57-5)
    Language English
    Publishing date 2020-05-26
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2661518-6
    ISSN 2073-4409 ; 2073-4409
    ISSN (online) 2073-4409
    ISSN 2073-4409
    DOI 10.3390/cells9061329
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function.

    Begemann, Isabell / Galic, Milos

    Frontiers in synaptic neuroscience

    2016  Volume 8, Page(s) 28

    Abstract: Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in ... ...

    Abstract Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications.
    Language English
    Publishing date 2016-08-23
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 2592086-8
    ISSN 1663-3563
    ISSN 1663-3563
    DOI 10.3389/fnsyn.2016.00028
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy.

    Begemann, Isabell / Viplav, Abhiyan / Rasch, Christiane / Galic, Milos

    Scientific reports

    2015  Volume 5, Page(s) 17973

    Abstract: Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to ... ...

    Abstract Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence - scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital.
    Language English
    Publishing date 2015-12-09
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/srep17973
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  4. Article ; Online: Force-control at cellular membranes.

    Galic, Milos / Begemann, Isabell / Viplav, Abhiyan / Matis, Maja

    Bioarchitecture

    2014  Volume 4, Issue 4-5, Page(s) 164–168

    Abstract: Force-regulation at cellular membranes relies on dynamic molecular platforms that integrate intra- and extracellular signals to control cell shape and function. To correctly respond to a continuously changing environment, activity of these platforms ... ...

    Abstract Force-regulation at cellular membranes relies on dynamic molecular platforms that integrate intra- and extracellular signals to control cell shape and function. To correctly respond to a continuously changing environment, activity of these platforms needs to be tightly controlled in space and time. Over the last few years, curvature-dependent mechano-chemical signal translation—a receptor-independent signaling mechanism where physical forces at the plasma membrane trigger nanoscale membrane deformations that are then translated into chemical signal transduction cascades—has emerged as a new signaling principle that cells use to regulate forces at the membrane. However, until recently, technical limitations have precluded studies of this force-induced curvature-dependent signaling at the physiological scale. Here, we comment on recent advancements that allow studying curvature-dependent signaling at membranes, and discuss processes where it may be involved in. Considering its general impact on cell function, a particular focus will be put on the curvature-dependence of feedback loops that control actin-based forces at cellular membranes.
    MeSH term(s) Cell Membrane/metabolism ; Cell Physiological Phenomena/physiology ; Humans
    Language English
    Publishing date 2014
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1949-100X
    ISSN (online) 1949-100X
    DOI 10.1080/19490992.2015.1005524
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis.

    Singh, Amrita / Saha, Tanumoy / Begemann, Isabell / Ricker, Andrea / Nüsse, Harald / Thorn-Seshold, Oliver / Klingauf, Jürgen / Galic, Milos / Matis, Maja

    Nature cell biology

    2018  Volume 20, Issue 10, Page(s) 1126–1133

    Abstract: Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue ... ...

    Abstract Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis
    MeSH term(s) Animals ; Animals, Genetically Modified ; Cell Polarity ; Cytoskeleton/metabolism ; Drosophila Proteins/genetics ; Drosophila Proteins/metabolism ; Drosophila melanogaster/genetics ; Drosophila melanogaster/metabolism ; Epithelial Cells/cytology ; Epithelial Cells/metabolism ; Epithelial Cells/ultrastructure ; Epithelium/growth & development ; Epithelium/metabolism ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Microtubules/metabolism ; Morphogenesis ; Pupa/cytology ; Pupa/growth & development ; Pupa/metabolism ; Time-Lapse Imaging/methods ; Wings, Animal/growth & development ; Wings, Animal/metabolism
    Chemical Substances Drosophila Proteins ; Luminescent Proteins
    Language English
    Publishing date 2018-09-10
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1474722-4
    ISSN 1476-4679 ; 1465-7392
    ISSN (online) 1476-4679
    ISSN 1465-7392
    DOI 10.1038/s41556-018-0193-1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 5169: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)
    Zs.MG 12: Show issues

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  6. Article ; Online: Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking.

    Saha, Tanumoy / Rathmann, Isabel / Viplav, Abhiyan / Panzade, Sadhana / Begemann, Isabell / Rasch, Christiane / Klingauf, Jürgen / Matis, Maja / Galic, Milos

    Molecular biology of the cell

    2016  Volume 27, Issue 22, Page(s) 3616–3626

    Abstract: Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins ... ...

    Abstract Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension-retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.
    MeSH term(s) Actins/metabolism ; Algorithms ; Cell Shape/physiology ; Computer Simulation ; Image Processing, Computer-Assisted/methods ; Pseudopodia/metabolism ; Pseudopodia/physiology ; Software ; Spatio-Temporal Analysis ; Statistics as Topic/methods
    Chemical Substances Actins
    Language English
    Publishing date 2016-08-17
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1098979-1
    ISSN 1939-4586 ; 1059-1524
    ISSN (online) 1939-4586
    ISSN 1059-1524
    DOI 10.1091/mbc.E16-06-0406
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 2853: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)
    Zs.MO 410: Show issues

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  7. Article ; Online: Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations.

    Goudarzi, Mohammad / Tarbashevich, Katsiaryna / Mildner, Karina / Begemann, Isabell / Garcia, Jamie / Paksa, Azadeh / Reichman-Fried, Michal / Mahabaleshwar, Harsha / Blaser, Heiko / Hartwig, Johannes / Zeuschner, Dagmar / Galic, Milos / Bagnat, Michel / Betz, Timo / Raz, Erez

    Developmental cell

    2017  Volume 43, Issue 5, Page(s) 577–587.e5

    Abstract: Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. ... ...

    Abstract Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target.
    MeSH term(s) Actins/metabolism ; Animals ; Cell Membrane/metabolism ; Cell Membrane Structures/metabolism ; Cell Movement/physiology ; Cell Shape/physiology ; Cell Surface Extensions/metabolism ; Germ Cells/cytology ; Germ Cells/metabolism ; Zebrafish/metabolism
    Chemical Substances Actins
    Language English
    Publishing date 2017-11-22
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2054967-2
    ISSN 1878-1551 ; 1534-5807
    ISSN (online) 1878-1551
    ISSN 1534-5807
    DOI 10.1016/j.devcel.2017.10.030
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 5742: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)
    Zs.MG 76: Show issues

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  8. Article ; Online: Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

    Beckmann, Denise / Römer-Hillmann, Anja / Krause, Annika / Hansen, Uwe / Wehmeyer, Corinna / Intemann, Johanna / de Gorter, David J J / Dankbar, Berno / Hillen, Jan / Heitzmann, Marianne / Begemann, Isabell / Galic, Milos / Weinhage, Toni / Foell, Dirk / Ai, Rizi / Kremerskothen, Joachim / Kiener, Hans P / Müller, Sylvia / Kamradt, Thomas /
    Schröder, Christopher / Leitão, Elsa / Horsthemke, Bernhard / Rosenstiel, Philip / Nordström, Karl / Gasparoni, Gilles / Gasparoni, Nina / Walter, Jörn / Li, Na / Yang, Xinyi / Chung, Ho-Ryun / Pavenstädt, Hermann / Lindemann, Nico / Schnittler, Hans J / Wang, Wei / Firestein, Gary S / Pap, Thomas / Korb-Pap, Adelheid

    Nature communications

    2021  Volume 12, Issue 1, Page(s) 3624

    Abstract: The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the ... ...

    Abstract The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.
    MeSH term(s) Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Adherens Junctions/metabolism ; Animals ; Arthritis/metabolism ; Arthritis/pathology ; Arthritis, Rheumatoid/metabolism ; Arthritis, Rheumatoid/pathology ; Cadherins/metabolism ; Cell Transformation, Neoplastic/metabolism ; Cytoskeletal Proteins/genetics ; Cytoskeletal Proteins/metabolism ; Female ; Fibroblasts/metabolism ; Homeodomain Proteins ; LIM Domain Proteins/genetics ; LIM Domain Proteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Osteoblasts ; beta Catenin/metabolism
    Chemical Substances Adaptor Proteins, Signal Transducing ; CTNNB1 protein, human ; Cadherins ; Cytoskeletal Proteins ; Homeodomain Proteins ; LASP1 protein, human ; LIM Domain Proteins ; Lasp1 protein, mouse ; beta Catenin ; osteoblast cadherin (156621-71-5)
    Language English
    Publishing date 2021-06-15
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-021-23706-8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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