Work plan
To obtain new insights into the execution of cellular signaling as a function of extracellular cues, one major research line will be the production of adapted microenvironments that present stimuli for various signaling pathways in a defined spatial and temporal electron microscopy and routine biochemical analysis. To engineer cellular microenvironments, an approach based on the self-organizing spatial positioning of single signaling molecules attached to inorganic or polymeric supports has proven to offer the highest spatial resolution with respect to the position of single signaling molecules.

 

Figure: Regulation of the spatial organization of receptors by cell adhesion to nanoscopic interfaces (Patla et al. 2010, Nat. Cell Biol.; Selhuber- Unkel et al. 2010, Biophys. J.).  
 

 

 

 

 

This technology was partly developed and refined within CellNetworks and has since been applied to a number of initial questions. The utilization of a biomaterial in this approach allows for adjusting and fine-tuning important properties of the cellular microenvironment, such as viscoelasticity, signaling molecule composition and spatial patterning of signaling molecules in well-defined nanoscopic geometries. These and other types of interfaces can be fabricated as 2- and 3- dimensional matrices. Functional imaging, such as fluorescence cross-correlation spectroscopy imaging, structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED) and photoactivated localization microscopy (PALM) will be used for the analysis of the molecular behavior of the signaling molecules.

 

 

Collaborative projects: The Spatz, Knop & new junior group labs will use spatially and temporal constricted exposure of membrane-affecting enzymes and osmolytes to the surface of individual yeast cells to find out to what extent reaction-diffusion mechanisms control the major 4 MAP kinase signaling cascades. Microscopic wide channels in substrates will be used to apply spatially confined but temporally changing stimuli to the cell membrane and the intracellular response will be assessed.

The Frischknecht and Spatz labs will explore the ideal physical, chemical and structural design of microenvironments for optimized adhesion  and migration of malaria parasites using nanoscopically defined 2- and 3-dimensional matrices. The goal will be to finally uncover the functional details of the adhesion and migration apparatus in malaria sporozoites, which to date remains unclear.

The Pollerberg group together with the Rappold, Engel, and Tanaka labs will investigate the behavior of microtubules and microtubule-associated proteins (MAPs) in neuronal growth cones in response to the cellular microenvironment. For this, growth cones will be challenged by a local, defined contact to cells or proteins and the elicited signaling downstream of the regionally activated cell adhesion molecules will be quantitatively analyzed in terms of chemical/ functional modifications of selected MAPs. In addition, tools for automated analysis of microtubule dynamics in neurons will be developed and combined with analysis of neuronal adhesion site dynamics in response to neuronal attractants (Engel, Rohr, Pollerberg labs, new junior group).

The Wittbrodt and Gruss labs will explore how tightly controlled spindle positioning influences cell fate decisions and the formation in organogenesis of multicellular organisms. They will investigate the interaction between extracellular cues, cell-cell communication and spindle positioning. To achieve this they will use methods developed for screens and functional studies in fish embryos.

The Hufnagel and Knop labs will explore bicoid signaling in Drosophila embryos by imaging the diffusion properties of fluorescently-labeled bicoid (using their light sheet-based FCS microscope) and by imaging of a fluorescent timer fusion with bicoid. Through this they will acquire the relevant parameters to investigate the still open question of how a paradigmatic morphogen gradient is formed and maintained.

The Fackler lab in collaboration with ex66 perts in microfluidics (new junior group, Tanka and Spatz labs) will analyze how the spatial and temporal organization of HIV-1 Nef (Negative regulatory factor) alters the biophysical and biological properties of immunological synapses (IS) to adjust T cell receptor (TCR) signaling outputs and adhesion forces in IS. Together with a molecular analysis of downstream TCR signaling events and their impact on HIV-1 replication, these studies will yield new insights on how Nef optimizes virus spread in the infected host.