Atsuko Honda

Identification of functional marker proteins in the mammalian growth cone

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.

Direct, Ca2+-dependent Interaction between Tubulin and Synaptotagmin I A POSSIBLE MECHANISM FOR ATTACHING SYNAPTIC VESICLES TO MICROTUBULES

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca2+, and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 μm tubulin. The Ca2+ dependence mainly resulted from Ca2+ binding to the Ca2+ ligands of synaptotagmin I. The C-terminal region of β-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca2+. These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca2+ concentrations.

Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals. SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Rufy3 is an adapter protein for small GTPases that activates a Rac guanine nucleotide exchange factor to control neuronal polarity

RUN and FYVE domain–containing 3 (Rufy3) is an adapter protein for small GTPase proteins and is bound to activated Rap2, a Ras family protein in the developing neuron. Previously, we reported the presence of a rapid cell polarity determination mechanism involving Rufy3, which is likely required for in vivo neuronal development. However, the molecular details of this mechanism are unclear. To this end, here we produced Rufy3 knock-out (Rufy3-KO) mice to study the role of Rufy3 in more detail. Examining Rufy3-KO neurons, we found that Rufy3 is recruited via glycoprotein M6A to detergent-resistant membrane domains, which are biochemically similar to lipid rafts. We also clarified that Rufy3, as a component of a ternary complex, induces the assembly of Rap2 in the axonal growth cone, whereas in the absence of Rufy3, the accumulation of a Rac guanine nucleotide exchange factor, T-cell lymphoma invasion and metastasis 2 (Tiam2/STEF), is inhibited downstream of Rap2. We also found that Rufy3 regulates the cellular localization of Rap2 and Tiam2/STEF. Taken together, we conclude that Rufy3 is a physiological adapter for Rap2 and activates Tiam2/STEF in glycoprotein M6A-regulated neuronal polarity and axon growth.

Direct binding of synaptotagmin to tubulin

Synaptotagmin I (tagmm) is a synaptic vesicle protein thought to be a major Caz+ sensor in transmitter release \~a its interaction with syntaxin and SNAP-25 Moreover, it binds other proteins including neurexins and the clathrrn .AP-2. suggesting that the protein is also involved in other presynapticfunctions We have found tagmm binds to the cytoskelrtal protein tubulin When the fusion protem of glutathione ,S-transferase (GST)-the cytoplasmic portion of tagmin immobilized on glutathione-Sepharose was Incubated with the soluble fraction of rat brain. tubulins were only maJor proteins in the bound material Purified tubulins lacking MAPS and tau proteins also bound to GST-tagmin The binding was dependent on CalTo determine which of the two tubulin subunits bmds tagmin, purified tubulins blotted on to nitrocellulosefilters were incubated with a Triton X-100 extract of brain membranes. l3-Tubulin but not rr-tubulin showed significant tagmin binding. Furthermore, tagmin and tubulins were coimmunoprecipitated from theTriton X-100 extract with a monoclonal antibody against tubulin These results indicate that tagmm directly binds to l.-tubulin