Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.

We recently showed that a nuclear location signal (NLS)‐containing karyophile forms a stable complex with cytoplasmic components for nuclear pore‐targeting The complex, termed nuclear pore‐targeting complex (PTAC), contained two essential proteins of 54 and 90 kDa, respectively, as estimated by electrophoresis. In this study, we found that the 54 kDa component of PTAC is the mouse homologue of Xenopus importin (m‐importin). Cytoplasmic injection of the antibodies raised against recombinant m‐importin showed an inhibitory effect on nuclear import of a karyophile in living mammalian cells. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope. Moreover, the injected antibodies co‐precipitated the karyophile, in an NLS‐dependent manner, with endogenous m‐importin in the cytoplasm. These results provide in vivo evidence that m‐importin is involved in nuclear protein import through association with a NLS in the cytoplasm before nuclear pore binding.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1␤). We found in this study that Ahr contains both nuclear localization and export signals in the NH 2 -terminal region. A fusion protein composed of ␤-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a liganddependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligandand signal-dependent nucleocytoplasmic shuttling protein.The aryl hydrocarbon receptor (Ahr) 1 binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons and certain halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Before binding ligands, Ahr is located in the cytoplasm as one component of a complex that has a molecular mass of about 280 kDa (1). This complex is composed of Ahr, two molecules of the 90-kDa heat shock protein, and possibly a 43-kDa protein (2). After ligand binding, Ahr dissociates from the complex and translocates to the nucleus (3). The heterodimer of Ahr and Ahr nuclear translocator (ARNT) constitutes a transcription factor and binds specific DNA sequences called XREs (xenobioticresponsive elements) in the enhancer regions of the CYP1A1 and several other proteins involved in xenobiotic metabolism (4). Because these enzymes are involved in the metabolism of polycyclic aromatic hydrocarbons to active genotoxic metabolites, Ahr plays an important role in carcinogenesis caused by these compounds (5-7).Because ARNT was first cloned as a factor required for ligand-dependent nuclear translocation of Ahr from the cytoplasm to the nucleus (8), the subcellular localization of ARNT was believed to be cytoplasmic. In fact, most ARNT was recovered in the cytosolic fraction by cell fractionation. However, immunohistochemical analysis has shown that ARNT is localized predominantly in the nucleus, regardless of the presence or absence of ligands (9, 10). This controversial subject was clarified by our recent study in which a nuclear localization signal (NLS) of the amino acid residues between 39 and 61 of human ARNT was found to be a novel bipartite type recognized by th...

The nuclear accumulation of ␤-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of ␤-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, ␤-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, ␤-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent ␤-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of ␤-catenin is insensitive to nuclear Ran-GTP depletion. These results show that ␤-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin ␤ in a Ran-unassisted manner. We further showed that ␤-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of ␤-catenin involves both nuclear import and export of this molecule. INTRODUCTIONThe trafficking of macromolecules across the nuclear envelope plays a key role in the coordination of cytoplasmic and nuclear events. The exchange of macromolecules occurs at the nuclear pore complex (NPC), which spans the double lipid bilayer of the nuclear envelope. The NPC is a large proteinaceous structure of ϳ125 MDa in size and mediates bidirectional transport via several different mechanisms (for reviews, see Davis, 1995;Fabre and Hurt, 1997). Small molecules, such as ions, low-molecular-weight metabolites, and proteins smaller than 20 -40 kDa cross 10-nm-diameter aqueous channels of the NPC by passive diffusion, whereas larger molecules are generally transported through the gated channels of the NPC via an active, receptor-mediated mechanism.A number of recent discoveries have led to the development of a model for receptor-mediated active nuclear import and export (for reviews, see Corbett and Silver, 1997;Gö rlich, 1997;Nakielny et al., 1997;Nigg, 1997;Ullman et al., 1997;Imamoto et al., 1998;Mattaj and Englmeier, 1998;Ohno et al., 1998). The model involves two essential elements, which are required for both the import and export pathways: 1) soluble transport factors, which recognize respective signals present in each protein, which is either imported into or exported out of the nucleus; and 2) a small GTPase Ran that affects the affinity between the transport factors and signals by binding directly to the transport factors. Import substrates form a complex with import factors in the cytoplasm, are transported through the NPC, and are then released from the import factors on the nucleoplasmic side of NPC when the GTP-bound form of Ran binds to the import factors. Export substrates form a complex with export factors and Ran-GTP inside the nucleus, are transported through the NPC, and are then released from the export factors w...

Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis.