Annexins A2 and A6 interact with the extreme N terminus of tau and thereby contribute to tau's axonal localization.

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca2+-regulated plasma membrane-binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca2+-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau-annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau-annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.

Cluster Recognition by Delaunay Triangulation of Synaptic Proteins in 3D.

The advent of super-resolution microscopy opens up the opportunity to study biological structures in unprecedented detail. However, revealing quantitative information about the spatial organization of a set of labeled proteins requires sophisticated analysis. This study introduces a novel robust cluster recognition algorithm based on Delaunay triangulation (CRADT), which can handle complex datasets generated by 3D super-resolution microscopy. This algorithm allows determining volume and shape of protein clusters in 3D. The study demonstrates its performance by applying this algorithm on dual-color 3D super-resolved measurements of mouse hippocampal synapses, stained against the presynaptic active zone marker protein Bassoon and the opposing postsynaptic density protein Homer as well as the exo- and endocytosis machinery proteins Synaptobrevin and Clathrin.

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-ß 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.

Restoring synaptic vesicles during compensatory endocytosis.

In the CNS (central nervous system), nerve cells communicate by transmitting signals from one to the next across chemical synapses. Electrical signals trigger controlled secretion of neurotransmitter by exocytosis of SV (synaptic vesicles) at the presynaptic site. Neurotransmitters diffuse across the synaptic cleft, activate receptor channels in the receiving neuron at the postsynaptic site, and thereby elicit a new electrical signal. Repetitive stimulation should result in fast depletion of fusion-competent SVs, given their limited number in the presynaptic bouton. Therefore, to support repeated rounds of release, a fast trafficking cycle is required that couples exocytosis and compensatory endocytosis. During this exo-endocytic cycle, a defined stoichiometry of SV proteins has to be preserved, that is, membrane proteins have to be sorted precisely. However, how this sorting is accomplished on a molecular level is poorly understood. In the present chapter we review recent findings regarding the molecular composition of SVs and the mechanisms that sort SV proteins during compensatory endocytosis. We identify self-assembly of SV components and individual cargo recognition by sorting adaptors as major mechanisms for maintenance of the SV protein complement.

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.

Monitoring cytoskeletal dynamics in living neurons using fluorescence photoactivation.

Neurons exhibit high temporal and spatial dynamics of their cytoskeletal organization, which is critical for the development and maintenance of axons and dendrites. Live cell imaging of fluorescence labeled proteins provides a powerful approach to scrutinize the dynamics of cytoskeletal components in living neuronal cells. Here, we describe a method to monitor and quantitatively analyze the dissipation of populations of cytoskeletal proteins in neurites of living cells using fluorescence photoactivation of fusion constructs with photoactivatable GFP (PAGFP). We present considerations on the design of the constructs, methods of gene transfer in neural cell lines and primary neurons, and implementation of photoactivation experiments using standard confocal laser scanning microscopy. In addition, we introduce general methods for data presentation and analysis using paradigmatic experiments of imaging PAGFP-neurofilament, -tubulin, and -tau in neuronally differentiated PC12 cells and primary cortical cultures. Methods include the generation of color-coded plots of 2D space-time intensity function, determination of immobile fractions, intensity shift analyses, and modeling to determine effective diffusion constants.

The frontotemporal dementia mutation R406W blocks tau's interaction with the membrane in an annexin A2-dependent manner.

Changes of the microtubule-associated protein tau are central in Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). However, the functional consequence of the FTDP-17 tau mutation R406W, which causes a tauopathy clinically resembling AD, is not well understood. We find that the R406W mutation does not affect microtubule interaction but abolishes tau's membrane binding. Loss of binding is associated with decreased trapping at the tip of neurites and increased length fluctuations during process growth. Tandem affinity purification tag purification and mass spectrometry identify the calcium-regulated plasma membrane-binding protein annexin A2 (AnxA2) as a potential interaction partner of tau. Consistently, wild-type tau but not R406W tau interacts with AnxA2 in a heterologous yeast expression system. Sequestration of Ca(2+) or knockdown of AnxA2 abolishes the differential trapping of wild-type and R406W tau. We suggest that the pathological effect of the R406W mutation is caused by impaired membrane binding, which involves a functional interaction with AnxA2 as a membrane-cytoskeleton linker.