Research interests

Cell polarization is a hallmark of chemotaxis, wound healing and other types of directed migration. In response to a graded stimulus in the environment the cytoskeleton is polarized as to allow migration towards the stimulus. The axonal projection of outgrowing neurons is an extreme example of cell polarization. Localized protrusion at the axonal growth cone allows the axon to extend up to 1000 cell body diameters. Axonal guidance towards target sites is mediated by a set of highly conserved extracellular guidance molecules (e.g. Slit, Netrin). Receptor engagement triggers a downstream signaling machinery which converges on the cytoskeleton. Many of the the signaling components have been identified (e.g. receptor kinases, Rho-GTPases. GAP and GEFs), but the temporal-spatial interplay that controls protrusive activity versus retraction are not well understood. Addressing these questions requires high resolution imaging approaches to study events at the level of single microtubules (see figure) and actin bundles. We have used spinning-disk confocal microscopy and total internal reflection fluorescence (TIRF) microscopy to visualize dynamics of actin, microtubule and microtubule-associated proteins (MAPs) in live neurons and other cells (1-3). Our focus is CLASP, a MAP involved in axon guidance (3) and a target of Abelson kinase phosphorylation. We make use of the above-mentioned techniques to study the effect of specific inhibitors and expression constructs on cytoskeletal dynamics.

 

Methods applied

In the Nikon Imaging Center of the University of Heidelberg we have light microscopes at our disposition which allow for high resolution dynamic imaging of GFP-constructs in live neurons. These are Spinning disk confocal and Total Internal Reflection fluorescence (TIRF) microscopy. We use image processing, deconvolution and microtubule tracking analysis algorithms for automated image analysis. We also use FRAP (Fluorescence Recovery after Photobleaching) and are in the process of implementing FLIM to study protein-protein interaction by FRET. Our main focus is axon guidance of primary neurons, but we also use cell lines and Dictyostelium amoebae to study cytoskeletal dynamics.