17 research outputs found
Cortical actomyosin network organization in epithelial cells
Epithelzellen, die Modell-Zelllinien dieser Dissertation, sind fĂŒr die Aufteilung und Abtrennung verschiedener Kompartimente eines Organismus zustĂ€ndig, indem sie sich zu GrenzflĂ€chen zusammenschliessen, welche hĂ€ufig hohen physikalischen Spannungen und KrĂ€ften
ausgesetzt sind. Um diese physikalischen KrÀfte zu verarbeiten oder sie selbst zu produzieren,
verwenden Epithelzellen, wie alle anderen Zelltypen auch, das Zytoskelett, das sich im Allgemeinen aus den Komponenten Mikrotubuli, IntermediÀr-Filamenten und Aktin sowie den
damit korrespondierenden Motorproteinen Dynein, Kinesin sowie Myosin zusammensetzt.
In dieser Dissertation wird das Zusammenspiel von Aktin und Myosin auf der apikalen Seite
von Epithelzellen untersucht. Im Falle von konfluenten Zellen mit vollstĂ€ndig ausgebildeten Zell-Zell-Kontakten sind auf der apikalen Seite der Zellen Mikrovilli zu finden, kleine, mit Aktin-BĂŒndeln gefĂŒllte AusstĂŒlpungen aus der ZelloberflĂ€che, welche fĂŒr die optimierte Nahrungsaufnahme sowie als Antennen fĂŒr Signalverarbeitung zustĂ€ndig sind. Im Zuge der
Arbeit konnten wir feststellen, dass sich der Aktin-Myosin-Aufbau auf der apikalen Seite
von Einzelzellen ohne Zell-Zell-Kontakte, sogenannten nicht-konfluenten Zellen, grundsĂ€tzlich Ă€ndert. Mittels Fluoreszenz-Mikroskopie und anderen experimentellen Methoden zeigen wir, dass zwar Ă€hnliche AusstĂŒlpungen auf der apikalen OberflĂ€che von Einzelzellen zu finden, diese jedoch hĂ€ufig verlĂ€ngert, gebogen, hoch-dynamisch und oft parallel zur Zellmembran
orientiert sind. Wir zeigen mittels molekularbiologischer Methoden, dass ein zusĂ€tzliches, innerhalb der apikalen Zellmembran liegendes isotropes Akto-Myosin-Netzwerk fĂŒr die dynamische Reorganisation der Mikrovilli-AusstĂŒlpungen verantwortlich ist.
Der Identifzierung des isotropen Akto-Myosin-Netzwerkes, welches eine der Hauptaussagen
dieser Dissertation ist, wird eine detaillierte Analyse der dynamischen Netzwerkreorganisation
angefĂŒgt, die mittels temporaler und örtlicher Bild-Korrelationsanalysen charakteristische
Zeiten und LĂ€ngen der Dynamik definiert. Des Weiteren entwickeln wir mehrere Bild-
Analyseverfahren, allen voran die Methode der iterativen temporalen Bildkorellation sowie des
optischen Flusses, wodurch wir eine Oszillation der Netzwerk-Reorganisationsgeschwindigkeit
identifizieren und parametrisieren können. Verschiedene, auf Fluoreszenzmikroskopie und automatisierter optischer Fluss-Bildanalyse basierende Experimente geben Hinweise auf zwei
mögliche ErklĂ€rungen fĂŒr die identifizierten Oszillationen. Sowohl Myosin aktivitĂ€tsregulierende Proteine als auch spontan auftretende Spannungsfluktuationen im unter Zugspannung liegenden Netzwerk können mögliche Ursachen fĂŒr die identifizierten Netzwerkoszillationen sein. Obwohl eine eindeutige zellulĂ€re Funktion des apikalen Akto-Myosin-Netzwerkes im Rahmen dieser Doktorarbeit noch nicht identifiziert werden konnte, so können wir aufgrund von verschiedenen Resultaten dennoch postulieren, dass das hier identifizierte Netzwerk eine entscheidende Rolle bei der Zellmigration und Signaltransduktion einnimmt. UnabhĂ€ngig davon reprĂ€sentiert das hier gefundene Netzwerk die faszinierende Möglichkeit, ein aktives, zweidimensionales Akto-Myosin-Netzwerk nicht nur in vitro, sondern in seiner natĂŒrlichen Umgebung studieren und biophysikalische Eigenschaften analysieren zu können.The cytoskeleton plays a central role in cellular morphogenesis by generating, sensing and
transmitting physical forces. Actin filaments are key cytoskeletal elements that are mostly
located close to the cell cortex. They can generate protrusive or contractile forces in combination with myosin motor proteins. We have identified a novel, highly dynamic actin structure at the apical side of non-polarized epithelial cells that is driven by an underlying non muscle myosin II network. By using various image analysis techniques, such as maximum intensity tracking, optical flow and correlation analysis, we observe contractile actomyosin activity within subregions of the cell cortex. The resulting spatially restricted mechanical forces differ in directionality which leads to shear stress and friction within the apical cell cortex. Additionally, we identified a global oscillatory behavior using autocorrelation analysis methods. The actomyosin network oscillates between states of low and high activity, as confirmed by iterative temporal image correlation (ITIC), a newly developed method for global feature extraction from image sequences, and highest intensity tracking. These remarkable features of subcellular cortex regulation give important insights into how mechanical force generation and propagation control cell shape and migration in non-polarized epithelial cells
Quantitative nanoscale imaging using transmission He ion channelling contrast: Proof-of-concept and application to study isolated crystalline defects
A newly developed microscope prototype, namely npSCOPE, consisting of a Gas Field Ion Source (GFIS) column and a position sensitive Delay-line Detector (DLD)
was used to perform Scanning Transmission Ion Microscopy (STIM) using keV He+ ions. One experiment used 25 keV ions and a second experiment used 30 keV ions.
STIM imaging of a 50 nm thick free-standing gold membrane exhibited excellent contrast due to ion channelling and revealed rich microstructural features including
isolated nanoscale twin bands which matched well with the contrast in the conventional ion-induced Secondary Electron (SE) imaging mode. Transmission Kikuchi
Diffraction (TKD) and Backscattered Electron (BSE) imaging were performed on the same areas to correlate and confirm the microstructural features observed in
STIM. Monte Carlo simulations of the ion and electron trajectories were performed with parameters similar to the experimental conditions to derive insights related
to beam broadening and its effect in the degradation of transmission image resolution. For the experimental conditions used, STIM imaging showed a lateral resolution close to30 nm. Dark twin bands in bright grains as well as bright twin bands in dark grains were observed in STIM. Some of the twin bands were invisible in
STIM. For the specific experimental conditions used, the ion transmission efficiency across a particular twin band was found to decrease by a factor of 2.8. Surprisingly, some grains showed contrast reversal when the Field of View (FOV) was changed indicating the sensitivity of the channelling contrast to even small
changes in illumination conditions. These observations are discussed using ion channelling conditions and crystallographic orientations of the grains and twin bands.
This study demonstrates for the first time the potential of STIM imaging using keV He+ ions to quantitatively investigate channelling in nanoscale structures including
isolated crystalline defects
Calcium-Mediated Actin Reset (Caar) Mediates Acute Cell Adaptations
Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress
Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells
Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of MadinâDarby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization
Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells
Deficient spontaneous in vitro apoptosis and increased tmTNF reverse signaling-induced apoptosis of monocytes predict suboptimal therapeutic response of rheumatoid arthritis to TNF inhibition
Impact of the coronavirus disease 2019 pandemic on stroke teleconsultations in Germany in the first half of 2020
Background and purpose
The effects of the coronavirus disease 2019 (COVID-19) pandemic on telemedical care have not been described on a national level. Thus, we investigated the medical stroke treatment situation before, during, and after the first lockdown in Germany.
Methods
In this nationwide, multicenter study, data from 14 telemedical networks including 31 network centers and 155 spoke hospitals covering large parts of Germany were analyzed regarding patients' characteristics, stroke type/severity, and acute stroke treatment. A survey focusing on potential shortcomings of in-hospital and (telemedical) stroke care during the pandemic was conducted.
Results
Between January 2018 and June 2020, 67,033 telemedical consultations and 38,895 telemedical stroke consultations were conducted. A significant decline of telemedical (p < 0.001) and telemedical stroke consultations (p < 0.001) during the lockdown in March/April 2020 and a reciprocal increase after relaxation of COVID-19 measures in May/June 2020 were observed. Compared to 2018â2019, neither stroke patients' age (p = 0.38), gender (p = 0.44), nor severity of ischemic stroke (p = 0.32) differed in March/April 2020. Whereas the proportion of ischemic stroke patients for whom endovascular treatment (14.3% vs. 14.6%; p = 0.85) was recommended remained stable, there was a nonsignificant trend toward a lower proportion of recommendation of intravenous thrombolysis during the lockdown (19.0% vs. 22.1%; p = 0.052). Despite the majority of participating network centers treating patients with COVID-19, there were no relevant shortcomings reported regarding in-hospital stroke treatment or telemedical stroke care.
Conclusions
Telemedical stroke care in Germany was able to provide full service despite the COVID-19 pandemic, but telemedical consultations declined abruptly during the lockdown period and normalized after relaxation of COVID-19 measures in Germany