Kinesin transports RNA: isolation and characterization of an RNA-transporting granule

RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.     

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.     

Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head

Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.     

Discrimination of the Cell Surface of the Coccolithophorid Pleurochrysis haptonemofera from Light Scattering and Fluorescence After Fluorescein-Isothiocyanate-Labeled Lectin Staining Measured by Flow Cytometry

We investigated the extent of calcification on the cell surface of the coccolithophorid Pleurochrysis haptonemofera using flow cytometry. Side scattering (SSC) by coccolith-bearing cells was higher than that by naked cells, suggesting the difference was due to scattering of the laser beam by the coccoliths. SSC of coccolith-bearing cells under acidic conditions corresponded well to the extracellular Ca content, although SSC could not be used to detect a delicate change in the coccolith thickness. The increase in SSC during the reproduction of coccoliths after decalcification was consistent with the increase in the number of coccoliths on the cell surface. The fluorescence after fluorescein-isothiocyanate-labeled lectin staining suggests that α-d-mannose, α-d-glucose, d-galactose, d-N-acetylgalactosamine, or derivatives of them are included in the coccoliths. Measurement of SSC and fluorescence after fluorescein-isothiocyanate-labeled lectin staining enabled rapid and quantitative determination of the status on the cell surface and isolation of desirable cells for physiological studies by cell sorting. Received May 22, 2001; accepted July 30, 2001.     

Defect in Synaptic Vesicle Precursor Transport and Neuronal Cell Death in KIF1A Motor Protein–deficient Mice

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Development of blood-cerebrospinal fluid barrier to horseradish peroxidase in the avian choroidal epithelium

Summary Four neurons in the brain of the migratory locust were immunohistologically identified with an anti-met-enkephalin antiserum. The perikarya of two of these cells are located in the center of each of the two groups of lateral protocerebral neurosecretory cells. The fibres coming from these perikarya terminate in numerous immunoreactive ramifications visible at the periphery of both tractus I to the corpora cardiaca, through which pass the neurosecretory products of the pars intercerebralis. The other two cell bodies are located at the bases of the two optic lobes; their fibres enter the posterior part of the protocerebrum and ramify around the root of the nervus corporis cardiaci II, another area through which neurosecretory products pass. The topographic distribution of these met-enkephalin arborizations suggests that these four neurons may act as neuromodulators of the acitivity of the major neurosecretory cells in the brain of this insect.     

Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units

Based on the both of results for X-ray studies of tetrahydrothiazolopyridine derivative 1c and FXV673, we synthesized a series of thiazol-5-ylpyridine derivatives containing pyridine N-oxide and 2-carbamoylthiazole units to optimize the S4 binding element. N-Oxidation of thiazol-5-ylpyridine increased the anti-fXa activity more than 10-fold independent on the position of N-oxide. The 4-pyridine N-oxide derivatives 3a and 3d excelled over the tetrahydrothiazolopyridine 1b in potency. 2-Methylpyridine N-oxide 3d exhibited 49-fold selectivity over thrombin. Our modeling study proposed a binding mode that the pyridine N-oxide ring of 3a stuck into the "cation hole" , and the oxide anion of 3a occupied in the almost same space to that of FXV673. From observations of the SAR and modeling studies, we suggested the possibilities that the formation of hydrogen bond with the oxide anion in the "cation hole" and the affinity of cationic pyridine ring to S4 subsite were responsible for increase in anti-fXa activity.     

Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.