Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm.
ABSTRACT: Kinesin was previously immunolocalized to mitotic apparatuses (MAs) of early sea urchin blastomeres (Scholey, J.M., M.E. Porter, P.M. Grissom, and J.R. McIntosh. 1985. Nature [Lond.]. 318:483-486). Here we report evidence that this MA-associated motor protein is a conventional membrane-bound kinesin, rather than a kinesin-like protein. Our evidence includes the observation that the deduced amino acid sequence of this sea urchin kinesin heavy chain is characteristic of a conventional kinesin. In addition, immunolocalizations using antibodies that distinguish kinesin from kinesin-like proteins confirm that conventional kinesin is concentrated in MAs. Finally, our immunocytochemical data further suggest that conventional kinesin is associated with membranes which accumulate in MAs and interphase asters of early sea urchin embryos, and with vesicles that are distributed in the perinuclear region of coelomocytes. Thus kinesin may function as a microtubule-based vesicle motor in some MAs, as well as in the interphase cytoplasm.
Project description:Sea urchin (Strongylocentrotus intermedius) has long been a model species for developmental and evolutionary research, but only a few studies have focused on gene mapping. Here, we reported a high-density genetic map containing 4,387 polymorphism specific-length amplified fragment (SLAF) markers spanning 21 linkage groups (LG) for sea urchin. Based on this genetic map and phenotyping data for eight economic traits, 33 potentially significant QTLs were detected on ten different LGs with explanations ranging from 9.90% to 46.30%, partly including 10 QTLs for test diameter, six QTLs for body weight and eight QTLs for Aristotle's lantern weight. Moreover, we found a QTL enrichment LG, LG15, gathering QTLs for test diameter, body weight, gonad weight, light orange-yellow color difference (?E1) and light yellow color difference (?E2). Among all QTLs, we genotyped four QTLs for test diameter, Aristotle's lantern weight and body weight using High Resolution Melting (HRM) technology. Finally, we used the verified SNP marker (detected using SLAF sequencing) to explore their marker-assisted selection (MAS) breeding application potential and found that SNP-29 associated tightly with body weight and that heterozygous genotype was a dominant genotype, indicating that SNP-29 was a promising marker for MAS.
Project description:We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.
Project description:The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.
Project description:We identified cis-regulatory elements based on their dynamic chromatin accessibility during the gastrula-larva stages of sea urchin and sea star and studied their evolution in these echinoderm species Overall design: ATAC-seq assays in wild-type sea urchin embryos, at 48 hpf developmental stage
Project description:Using MethylC-seq we investigated single-base resolution methylomes of sea urchin during development. Overall design: MethylC-seq was performed on (i) 24 hpf, 48 hpf, 72 hpf embryos and adult tissue (tube feet) of purple sea urchin in biological replicates.
Project description:The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.
Project description:This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.
Project description:Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.
Project description:Using hMeDIP-seq we validated the single-base resolution hydroxymethylomes (ACE-seq) of sea urchin, lancelet and zebrafish embryos. Overall design: hMeDIP-seq was perfomred on (i) purple sea urchin 72 hpf embryos; (ii) european lancelet 36 hpf embryo; (iii) zebrafish 24 hpf embryo (in replicates). Input DNA sequencing has been performed in parallel.
Project description:Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis.The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven approximately 50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14-55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis.These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.