visualizing-adhesion-formation-cells-means-advanced-spinning-disk

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

University Medical Center Hamburg-Eppendorf

The vascular endothelium as well as subendothelium are objects of many researches as it is directly involved in a multiplicity of physiological and pathological settings. Detailed study of endothelial function became feasible with the development of techniques to culture endothelial cells (EC) in vitro. Limitations of this approach have become apparent with the realization that cell culture dedifferentiate with time and do not exhibit properties of intact tissue. Here we describe the development of a novel ex vivo tissue model to study cell-vascular wall interactions by using isolated mouse aorta patches. Validation of this model was performed by demonstrating cell attachment and changes in cell shape typical for cell spreading during adhesion. A major advantage of this model is that cell-endothelium interaction and its molecular backgrounds can now be studied more feasibly on an intact and native tissue. 

Ex vivo aorta patch model for analysis of cellular adhesion.

Reelin controls neuronal migration and layer formation. Previous studies in  mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in , but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated -expressing dentate granule cells in slice cultures from , -like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from  and -like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in  slices cocultured to wild-type dentate gyrus showed that the  neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of  slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer. Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of  by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

Antonio Virgilio Failla

UKE Microscopy Imaging Facility

University Medical Center Hamburg-Eppendorf

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

Antonio Virgilio Failla

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}UKE Microscopy Imaging Facility

University Medical Center Hamburg-Eppendorf

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

UKE Microscopy Imaging Facility