The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.

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Significant progress has been made toward our understanding of the basic principle of nucleocytoplasmic transport, and the structure of transport factors, as well as the diversity of nucleocytoplasmic transport pathways. This review outlines the current knowledge of transport, and discusses the problems that remain as to how eukaryotic cells acquire additional levels for the regulation of gene expression from a diversity of nucleocytoplasmic transport pathways.

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Nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. The simplicity and specialization of this system, combined with the availability of crystal structures of NTF2, RanGDP and their complex, has facilitated the investigation of the molecular mechanism of its trafficking. NTF2 binds to both RanGDP and FxFG repeat-containing nucleoporins. Mutants engineered on the basis of structural information together with determination of binding constants have been used to dissect the roles of these interactions in transport. Thus, NTF2 binds to RanGDP sufficiently strongly for the complex to remain intact during transport through NPCs, but the interaction between NTF2 and FxFG nucleoporins is much more transient, which would enable NTF2 to move through the NPC by hopping from one repeat to another. An analogous nucleoporin hopping mechanism may also be used by carrier molecules of the importin-β family to move through NPCs.

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In eukaryotic cells, the nuclear membrane creates a barrier between the nucleus and the cytoplasm. Whereas RNA synthesis occurs in the nucleus, they mostly function in the cytoplasm; thus export of RNA molecules from the nucleus to the cytoplasm is indispensable for normal function of the cells. The molecular mechanisms involved in each kind of cellular RNA export is gradually understood. The focus of this review will be mRNA export. mRNAs are multiformed. In order to ensure that this variety of mRNA molecules are all exported, cells are probably equipped with multiple export pathways. A number of proteins is predicted to be involved in mRNA export. Ascertaining which proteins play crucial roles in the pathways is the key point in the study of mRNA export.

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The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRα1 for GDNF and GFR α2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRα1 and GFRα2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.

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The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca²⁺/CaM-binding proteins, p85 and EF-1α, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1α are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca²⁺/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca²⁺/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl. In Tetrahymena, it is known that Ca²⁺/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1α to the cytopharynx, these results suggest that the Ca²⁺/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.

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Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.

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Previous studies have shown that the motility of flagellar and ciliary axonemes in many organisms are influenced by the concentration of both ATP and ADP. Detergent-extracted cell models of Chlamydomonas oda1, a mutant lacking flagellar outer-arm dynein, displayed slightly lower flagellar beating frequencies when reactivated with ATP in the presence of an ATP-regenerating system, composed of creatine phosphate and creatine phosphokinase, than when reactivated with ATP alone. Thus, presence of a low concentration of ADP may somehow stimulate axonemal motility. To see if this motility stimulation is due to a direct effect on dynein, we analyzed the effect of ADP on the in vitro microtubule translocation caused by isolated inner-arm dyneins in the presence of ATP. Of the seven inner-arm dyneins (species a-g) fractionated by ion-exchange chromatography, most species translocated microtubules at faster speed in the presence of 0.1 mM ATP and 0.1 mM ADP than in the presence of 0.1 mM ATP alone. Most notably, species a and e did not translocate microtubules at all in the presence of the ATP-regenerating system, indicating that a trace amount of ADP is necessary for their motility. This regulation may be effected through binding of ADP to some of the four nucleotide binding sites in each dynein heavy chain.

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We prepared a cell model of Amoeba proteus by mechanical bursting to study the interaction between actin filaments (AFs) and plasma membrane (PM). The cell model prepared in the absence of Ca^{2 +} showed remarkable contraction upon addition of ATP. When the model was prepared in the presence of Ca^{2 +}, the cytoplasmic granules formed an aggregate in the central region, having moved away from PM. Although this model showed contraction upon addition of ATP in the presence of Ca²⁺, less contraction was noted. Staining with rhodamine-phalloidin revealed association of AFs with PM in the former model, and a lesser amount of association in the latter model. The interaction between AFs and PM was also studied using the isolated PM. AFs were associated with PM isolated in the absence of Ca²⁺ , but were not when Ca²⁺ was present. These results suggest that the interaction between AFs and PM is regulated by Ca²⁺.

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