When rat bone marrow macrophages were incubated with acetyl lignin (EP3) in the presence of a 10% solution of fetal bovine serum, the macrophages secreted tumor necrosis factor (TNF-α) in a dose-dependent manner. This was followed by macrophage multinucleation. EP3 was found to have a significant effect on TNF-α secretion at a minimum dose of 5 μ/ml and produced no significant further increase at levels above 50 μg/ml, while multinucleation was most active at 10 μg/ml. However, multinucleation did not occur at higher concentrations of EP3 (50 μg/ml and 100 μg/ml). Secretion of TNF-α was significantly reduced in the absence of fetal bovine serum, whereas multinucleation was very active, starting after 6 h of incubation. At concentrations of 100 μg/ml, sulfonyl lignin (LS) and dextran sulfate (DS) only induced low levels of TNF-α secretion from macrophages, but induced active multinucleation. The multinucleation induced by addition of LS or DS was inhibited by further addition of EP3. Thus, macrophage multinucleation was most active when a low level of TNF-α was secreted from the macrophages.

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Axonal microtubules have two essential roles: providing the track for organelle transport and forming the cytoskeletal framework to maintain axonal morphology. Microtubule-associated proteins (MAPs) are essential for the formation of cytoskeletal architecture. However, they may have additional roles on the regulation of organelle transport by their interaction with motor proteins on the microtubules.
We first examined the effects of axonal MAPs on the organelle movement along microtubules in a heterologous system using COS fibroblasts, which express no axonal MAPs, such as tau or MAP2C. Transfection of tau or MAP2C gene suppressed organelle movement almost completely in this cell type, hence interaction of axonal MAPs with microtubules interferes with organelle transports.
It is known that the phosphorylation of MAPs reduces their interaction with microtubules. In this sense, phosphorylation of MAPs can be a good candidate for the molecular switch to regulate the organelle transport. As a second set of experiments, we investigated the effects of modulating cAMP dependent protein kinase pathway on organelle transports in primary sensory neurons, where high-molecular-weight tau protein is the major MAP. We found that the application of dibutyryl cAMP enhanced transports of large organelles in the axon. Furthermore, this drug treatment phosphorylated endogenous tau protein and thus reduced the affinity of tau to microtubules.
These results indicate that axonal MAPs can work as a phosphorylation-dependent regulator of organelle transport. Local activation of protein kinase pathways in the axon might play an important role on the segregation of microtubules serving for either organelle transport or cytoskeletal architecture.

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Computer aided three-dimensional (3-D) reconstructions of Bodo sp., a free-living kinetoplastid, were made from ultrathin (100 nm) and semithin (200 nm) serial sections, which were observed by transmission electron microscopy. Organelles and membrane systems were digitized into a computer and three-dimensional images were generated using the SYNU software package. It was observed that the internal disposition of structures like the contractile vacuole, the Golgi complex, the mitochondrion, and the nucleus maintain a constant relationship relative to each other and to the cytostome and flagellar pocket. The Golgi complex and the contractile vacuole elements are not apparently connected. The contractile vacuole fills a significant volume in the cell. Volume alterations occurring during systole/diastole cycles of the contractile vacuole are compensated by cytoskeletal adaptations. There is a subpellicular microtubule-free area adjacent to the contractile vacuole. Morphological evidence indicates that the flagellar pocket may not be the only site of vacuolar content elimination. A better view of the cytoskeleton was obtained in detergent-extracted cells, where a set of curved microtubules was observed separating the flagellar pocket from the cytostome and surrounding the cytopharynx.

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We investigated effect of docosahexaenoic acid on lipoprotein synthesis and secretion by cultured eel hepatocytes. When eel hepatocytes were incubated with 1 mM docosahexaenoic acid (DHA) at 28°C for 24 h, triacylglycerol (TG), free cholesterol (FC), and cholesteryl ester (CE) syntheses from ¹⁴C-acetate increased 9.7-, 1.6-, and 8.5-fold, respectively, and the specific activity of TG was twofold that of control. These results indicate the stimulative effect of DHA on lipid synthesis. However, the radioactivity of the lipoprotein secreted by DHA-treated cells incubated with ¹⁴C-acetate and 3H-leucine reduced to 55 and 60%, respectively, compared to that by control cells, but DHA did not affect the incorporation of ³H-leucine into other secreted protein. Furthermore, the amount of the secreted lipoprotein per 24 h by DHA-treated cells also reduced to 17%. These results show that DHA inhibits the secretion of lipoprotein. Intracellular lipids such as TG, PL, and TC increased by 4.8, 1.6, and 1.8 times, respectively, in DHA-treated cells. The remarkable increase in TG in DHA-treated cells seems to be due to the stimulative effect of DHA on TG synthesis and an inhibitory effect of DHA on lipoprotein secretion, since about 70% of the secreted lipoprotein consists of TG. The chemical composition and apoprotein profile of the lipoprotein secreted by DHA-treated and control cells were almost the same. These results suggest that DHA inhibits the assembly or secretion of the lipoprotein in eel hepatocytes.

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To obtain insight into the molecular dynamics and involvement of microtubules and the related signal molecules in the regulation of cell locomotion, we studied the influence of microtubule disruption on actin stress fibers and focal adhesion assembly in addition to cell morphology. We found that all microtubule-disrupting drugs including colcemid and vinblastine rapidly and reversibly induce the formation of actin stress fibers and focal adhesions containing vinculin, accompanied by activated cell motility in serum-starved Balb/c 3T3 cells. In contrast, taxol, a microtubule-stabilizing drug, completely inhibited these effects of the microtubule-disrupting drugs. A microinjection of C3 ADP-ribosyltransferase, a specific inhibitor of rho GTPase, blocked the stress fiber and focal adhesion assembly induced by the microtubule disruption. These results suggested that microtubules contain signal molecules that regulate the formation of stress fibers and focal adhesions by activating the rho signal cascade. We postulate that microtubule-releasing and stress fiber-inducing factors link the intrinsically variable and irregular actin filament dynamics to coordinated and directional locomotion in the process of cell movement.

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Dyneins are high molecular weight ATPases that function as microtubule-based molecular motors. The axonemal dyneins, discovered 30 years ago, are responsible for the beating movement of cilia and sperm flagella, which they generate by producing sliding between adjacent microtubules. Cytoplasmic dynein, more recently discovered, is involved in diverse activities, including intracellular transport, nuclear migration, and the orientation of the cell spindle at mitosis.

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The Acanthamoeba myosin-IA and myosin-IB molecular motors bind to membranes, so they may produce the force to move organdies and membranes along actin filaments. We have determined the rate constants for the actin-activated myosin-I ATPase by pre-steady state kinetic analysis. ATP binds rapidly to myosin-I and dissociates the enzyme from actin filaments at a rate >500 s^-1. Myosin-I hydrolyzes ATP to ADP and inorganic phosphate (Pi) at 20-50 s^-1. Phosphate dissociation is the rate limiting step in the ATPase cycle, 0.01 s^-1 for myosin-I alone and at 10 s^-1 when myosin-I is bound to actin filaments. ADP dissociation is rapid. Phosphorylation controls the ATPase cycle by increasing the rate of phosphate release from myosin-I bound to actin. At steady state the major species are myosin-ATP and myosin-ADP-P_i, which rapidly bind to and dissociate from actin filaments. During the ATPase cycle myosin-I binds so weakly to actin filaments that it cannot support processive movement like kinesin, unless several motors cluster together on a membrane or actin filament. These properties of the enzyme emphasize the importance of characterizing mechanisms that promote the self-association of myosin-I isoforms at specific binding sites in cells.

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In the cells various kinds of organelles are transported and distributed to their proper destinations in the cell. Organelle transports are very important for cellular morphogenesis and functions, with the conveying and targeting of essential materials to their correct destination being conducted, often at considerable velocities. Recently we have identified at least 10 new microtubule-associated motor proteins named as KIFs (kinesin superfamily proteins). Their characterization reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Molecular cell biological approaches combining multidiciplinary methods such as new electron microscopy, biochemistry, immunocytochemistry, biophysics, molecular biology and molecular genetics have revealed precise mechanisms of organelle transports in the cells by KIFs.

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Cellular polarization depends upon the asymmetric transport of components within the cell, often along microtubule paths. Kinesin and cytoplasmic dynein are the most abundant microtubule motors powering the transport of membranous vesicles. Many additional proteins are needed to control motility and coordinate movements with other cellular activities. In this review we will summarize our understanding of the motors, motor associated proteins and their function within cells. We suggest a cyclic model for much of membrane trafficking in which the membrane anchor for the motors, kinectin, plays a central role. In such a model up-regulation of transport results in a faster cycling of kinectin between kinesin and cytoplasmic dynein-dependent transport.

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The sites and roles of microtubule assembly/disassembly in mitosis are reviewed, together with evidence that microtubule assembly can push, and disassembly can pull, organelles such as the kinetochore on chromosomes which remain attached to the assembling or disassembling end of the microtubule, in vivo and in vitro. Motor proteins with weakened or inhibited transport activity can bind objects to the assembling or disassembling microtubule end, and may be involved in the dynamic attachment of the kinetochore to the spindle microtubules in mitosis.

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In epithelial and endothelial cells, the tight junction (TJ) seals cells to create a primary barrier to the diffusion of solutes across the cell sheet, and it also works as a boundary between the apical and basolateral membrane domains to create their polarization. An integral membrane protein working at TJ is postulated to exist, but it has remained elusive for quite some time. Most recently, using mAbs, we identified an integral membrane protein named occludin that was exclusively localized at TJ both in epithelial and endothelial cells. Here we overview our recent studies on the structure and function of occludin.

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In germinating fatty seedlings, microbodies are functionally transformed to leaf peroxisomes from glyoxysomes during greening, and then converted to glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to glyoxysomes during senescence. The reversible transformations of microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.

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1, 3-β-glucan synthase catalyzes the synthesis of a l, 3-/9-linked glucan polymer which produces the main rigidity of the yeast cell wall. Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall. This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase. In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme. Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth. In this article, we will focus on biochemistry of 1, 3-β-glucan synthase and signaling through rho type GTPase.

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A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

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The secretory pathway of eukaryotic cells consists of a number of distinct membrane-bound compartments interconnected by vesicular traffic. Each compartment has a characteristic content of proteins and lipids, which must be maintained. This is achieved in most cases by active sorting - proteins may reach the wrong compartment but are continually retrieved. A good example is the retrieval system for lumenal ER proteins. These proteins carry a specific sorting signal, typically the tetrapeptide KDEL, which is bound by a receptor in the Golgi apparatus. The receptor-ligand complex, together with escaped ER membrane proteins, returns to the ER. Many of the components of vesicle traffic, including the coat proteins required for vesicle budding from the ER, those that form retrograde vesicles on post-ER compartments, and integral membrane proteins that target the vesicles to their correct destination, have been identified. The sorting events that occur can largely be understood in terms of specific protein-protein interactions involving these components. However, sorting of some membrane proteins, including the vesicle targeting molecules, is influenced by their transmembrane domains, and it is likely that segregation of these is dependent on the composition and biophysical properties of the lipid bilayer, which very between compartments. The secretory pathway is thus a dynamic entity, split into discrete organdies by the constant segregation and recycling of lipids and proteins, processes that are ultimately driven by the mechanics of vesicle formation and fusion.

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The fission yeast Schizosaccharomyces pombe initiates sexual development under starved conditions. Nutritional starvation decreases the level of intracellular cAMP. This decrease induces expression of the stell gene, which encodes a key transcription factor for genes required for mating and meiosis. Mutational analyses of S. pombe genes encoding components of the cAMP cascade have shown that S. pombe cells stay in the mitotic cell cycle as long as the level of cAMP-dependent protein kinase activity is high, but are committed to mating and meiosis if this activity is lowered. To initiate meiosis in S. pombe, a protein kinase encoded by pat1 (also called ran1) should be inactivated. This inactivation results from deprivation of nutrients via a cascade of expression of genes including ste11. The mei2 gene encodes a factor indispensable for the initiation of meiosis, and its expression is regulated directly by Ste11. If Pat1 kinase is intact, it blocks Mei2 function. Mei2 is required at two distinct stages of meiosis, once prior to premeiotic DNA synthesis and then prior to the first meiotic division (meiosis I). Mei2 is an RNA-binding protein, and forms a complex with a specific RNA species to promote meiosis I. This RNA species, named meiRNA, is polyadenylated but is unlikely to encode a protein product. It is essential for meiosis I, but not for either cell growth or premeiotic DNA synthesis. These observations unequivocally demonstrate that RNA plays a critical role in the control of meiosis.

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