Keiko Satoh

CAST: A novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and Munc13-1

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Identification of activity‐regulated proteins in the postsynaptic density fraction

Background: The postsynaptic density (PSD) at synapses is a specialized submembranous structure where neurotransmitter receptors are linked to cytoskeleton and signalling molecules. Activity‐dependent dynamic change in the components of the PSD is a mechanism of synaptic plasticity. Identification of the PSD proteins and examination of their modulations dependent on synaptic activity will be valuable for an understanding of the molecular basis of learning and memory.

Frabin, a Novel FGD1-related Actin Filament-binding Protein Capable of Changing Cell Shape and Activating c-Jun N-terminal Kinase

We purified from rat brain a novel F-actin-binding protein with a M r of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated M r of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.

Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord

Postmitotic neural precursors are generated in the ventricular zone (VZ) of the developing neural tube and immediately migrate to the mantle layer (ML) where they differentiate into mature neurons. Although the regulation of neuronal differentiation and migration has extensively been studied, the behavior of the early postmitotic precursors migrating toward the ML is largely unknown. In this study, we have identified Neph3 as a specific marker for early postmitotic neural precursors in the VZ of the developing spinal cord. Analysis of Neph3 localization by immunofluorescence and immunoelectron microscopy revealed that early neural precursors in the VZ possessed not only pia-connected processes but also ones that reached the ventricle. This apical extension of processes was confirmed by analyzing another early postmitotic marker, Dll1 mRNA, which was actively transported toward the ventricle and accumulated at the termini of the processes. Furthermore, adherens junctions (AJs) were formed around the apical end of processes extending from Neph3- and Dll1 mRNA-positive postmitotic precursors. Taken together, these observations suggest that migrating early postmitotic neural precursors in the VZ of the developing spinal cord form a neuroepithelial cell-like bipolar morphology and communicate with their neighboring cells through AJs.