Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Distal Dendritic Enrichment of HCN1 Channels in Hippocampal CA1 Is Promoted by Estrogen, but Does Not Require Reelin.

HCN1 compartmentalization in CA1 pyramidal cells, essential for hippocampal information processing, is believed to be controlled by the extracellular matrix protein Reelin. Expression of Reelin, in turn, is stimulated by 17ß-estradiol (E2). In this study, we therefore tested whether E2 regulates the compartmentalization of HCN1 in CA1 via Reelin. In organotypic entorhino-hippocampal cultures, we found that E2 promotes HCN1 distal dendritic enrichment via the G protein-coupled estrogen receptor GPER1, but apparently independent of Reelin, because GST-RAP, known to reduce Reelin signaling, did not prevent E2-induced HCN1 enrichment in distal CA1. We therefore re-examined the role of Reelin for the regulation of HCN1 compartmentalization and could not detect effects of reduced Reelin signaling on HCN1 distribution in CA1, either in the (developmental) slice culture model or in tamoxifen-inducible conditional reelin knockout mice during adulthood. We conclude that for HCN1 channel compartmentalization in CA1 pyramidal cells, Reelin is not as essential as previously proposed, and E2 effects on HCN1 distribution in CA1 are mediated by mechanisms that do not involve Reelin. Because HCN1 localization was not altered at different phases of the estrous cycle, gonadally derived estradiol is unlikely to regulate HCN1 channel compartmentalization, while the pattern of immunoreactivity of aromatase, the final enzyme of estradiol synthesis, argues for a role of local hippocampal E2 synthesis.

Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress.

Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm(2), 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.