Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43

The frontotemporal dementia mutation R406W blocks tau’s interaction with the membrane in an annexin A2–dependent manner

The R406W mutation prevents tau from functioning as a linker between the membrane and the microtubule cytoskeleton

Live cell imaging of cytoskeletal dynamics using fluorescence photoactivation

Neurodegeneration in selected brain areas, associated with abnormal behavior of cytoskeletal proteins or altered organization of the cytoskeletal filament network, exhibits a characteristic feature of many neurodegenerative diseases. Therefore, focusing on analyzing the dynamics of cytoskeletal proteins under disease-relevant conditions using live cell imaging approaches could provide a better understanding of the cellular mechanisms underlying neurodegeneration. Fluorescence photoactivation (FPA) provides a novel tool to label and track living cells, organelles, or even single molecules in living systems in a spatio-temporal manner with high sensitivity. Fusion of photoactivatable fluorescence proteins to cytoskeletal proteins allows analyzing cytoskeletal dynamics in neurons in real-time and provides the unique opportunity to determine the effect of disease-relevant conditions on cytoskeletal dynamics in living neurons. Aim of the thesis was to study the motion of different cytoskeletal proteins: the microtubule associated protein tau and the growth associated actin-binding protein GAP-43. Expression of both proteins is developmentally regulated and may play an important role in neuronal polarization. Furthermore, both proteins show enrichment at the distal part of the neurite, the growth cone. The mechanisms, how distal trapping of tau and trafficking and enrichment of GAP-43 at the tip are regulated, are unclear. To scrutinize the dissipation of both proteins in living neurons, we constructed a panel of PAGFP-tagged fusion constructs and expressed them in differentiated PC12 cells as a neuronal model system. Using FPA in combination with computer-assisted image processing, we could identify the dissipation and trapping mechanisms of both proteins. The data indicate that FPA provides a useful and versatile approach to determine protein distribution in living cells during development and disease-like conditions

Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression

Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor

Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics

GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-beta A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor