LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co-migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin-1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co-precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein-LIS1 complex on transportable MTs, which is a possibility supported by our data.     

Distinct Functions of Nuclear Distribution Proteins LIS1, Ndel1 and NudCL in Regulating Axonal Mitochondrial Transport

Neurons critically depend on the long‐distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein‐LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.      

Identification of the Tapasin gene in the chicken major histocompatibility complex

 The Tapasin molecule plays a role in the assembly of major histocompatibility complex (Mhc) class I molecules in the endoplasmic reticulum, by mediating the interaction of class I-β₂-microglobulin dimers with TAP. We report here the identification of the Tapasin gene in the chicken Mhc (B complex). This gene is located at the centromeric end of the complex, between the class II B-LBI and B-LBII genes. Like its human counterpart it comprises 8 exons, but features a significantly reduced intron size as compared to the human gene. Chicken Tapasin codes for a transmembrane protein with a probable endoplasmic reticulum retention signal. Exons IV and V, and possibly exon III, code for separate domains that are related to the immunoglobulin (Ig) superfamily (this relationship was so far unrecognized for human Tapasin domain IV which has lost its two cysteines). Two different cDNAs corresponding to the Tapasin gene were isolated, possibly related to alternative splicing events; the Ig-like domain encoded by exon IV is missing in one of the cDNAs, suggesting either that this domain is not necessary for the protein to perform its function, or that the two alternatively spliced cDNAs are translated into two functionally different forms of the protein. Received: 8 July 1998 / Revised: 5 October 1998     

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

 The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Received: 20 March 1996/Accepted: 1 July 1996     

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.     

Expression pattern of XIRG, a marker for non-neural ectoderm

XIRG (for Xenopus IRG) was cloned by screening a cDNA library of UV-ventralized stage 13 Xenopus laevis embryos for specifically ventrally expressed mRNAs. Embryonic XIRG mRNA expression is restricted to non-neural ectoderm at the gastrula and neurula stages. In adult X. laevis, XIRG mRNA can be detected in skin and kidney. Extensive searches in nucleic acid and protein databases revealed homologous sequences in mouse, human and zebrafish. Mouse IRG1 mRNA is expressed in cultured macrophages as a response to bacterial lipopolysaccharide treatment. Received: 27 April 2000 / Accepted: 13 June 2000     

Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate,synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC₅₀ value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1 mg/ml.     

The chicken NKX2.8 homeobox gene: A novel member of the NK-2 gene family

 We have isolated the chicken homeobox gene NKX2.8, which represents a novel member of the NK-2 gene family. Besides the homeodomain, the NKX2.8 protein contains two other conserved sequences, a TN and an NK2 domain. NKX2.8 is expressed in the ventral foregut, the developing heart, in the epithelial layers of the branchial arches and in the dorsal mesocardium. Thus, its expression overlaps partially, but also differs significantly from another chicken tinman orthologue, the NKX2.5 gene. It is suggestive that NKX2.8 and NKX2.5 play a cooperative role in early heart development. Received: 28 January 1997 / Accepted: 8 February 1997     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000     

Isolation and characterization of OsDMC1, the rice homologue of the yeast DMC1 gene essential for meiosis

Yeast DMC1 is a meiosis-specific gene required for homologous chromosome pairing in meiosis. Using degenerate primers designed according to amino acid motifs conserved in yeast Dmc1 and Arabidopsis AtDmc1, we obtained full-length cDNA of a rice homologue of the DMC1 gene (OsDMC1) by RT-PCR and rapid amplification of cDNA ends (RACEs). OsDmc1 exhibited 53% amino acid sequence identity to yeast Dmc1 and 81% to AtDmc1. OsDMC1 was expressed at high-levels in reproductive organs, low-levels in roots, and undetectable levels in leaves and seedlings. Southern blot analyses revealed that OsDMC1 is one of two DMC1 homologues present in rice. Received: 18 December 2000 / Accepted: 22 December 2000     

Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

Highly polymorphic microsatellites of rice consist of AT repeats, and a classification of closely related cultivars with these microsatellite loci

Microsatellites consisting of AT repeats are highly polymorphic in rice genomes and can be used to distinguish between even closely related japonica cultivars in Japan. Polymorphisms of 20 microsatellite loci were determined using 59 japonica cultivars, including both domestic and modern Japanese cultivars. Although the polymorphisms of these 20 microsatellite loci indicated that the Japanese cultivars were genetically quite similar, microsatellites consisting of AT repeats showed high gene diversity even among such closely related cultivars. Combinations of these hypervariable microsatellites can be employed to classify individual cultivars, since the microsatellites were stable within each cultivar. An identification system based on these highly polymorphic microsatellites could be used to maintain the purity of rice seeds by eliminating contamination. A parentage diagnosis using 17 polymorphic microsatellite loci clearly demonstrated that plants which carried desired chromosome regions had been selected in breeding programs. Thus, these hypervariable microsatellites consisting of AT repeats should promote the selection of plants which carry desired chromosomes from genetically similar parents. Backcrossing could also help to eliminate unnecessary chromosome regions with microsatellite polymorphisms at an early stage in breeding programs. Received: 8 July 1996 / Accepted: 12 July 1996