Molecular cloning and characterization of KIFC1-like kinesin gene (ot-kifc1) from Octopus tankahkeei

Faculty of Life Science and Bioengineering, Ningbo University, Zhejiang 315211, PR China.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology (Impact Factor: 1.55). 03/2010; 156(3):174-82. DOI: 10.1016/j.cbpb.2010.03.004
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Spermiogenesis in Octopus tankahkeei involves striking cellular reorganization to generate a mature spermatozoon. This process may require spermatid-specific adaptation of cytoskeleton and associated molecular motor proteins. KIFC1 is a C-terminal kinesin motor with important roles in acrosome biogenesis and nuclear reshaping during spermiogenesis in rat. Here, we have cloned and characterized the gene encoding a homologue of rat KIFC1, termed as ot-kifc1, from the testis of O. tankahkeei. The 2229 bp complete cDNA contains a 75 bp 5'-untranslated region, a 1992 bp open reading frame and a 162 bp 3'-untranslated region. The deduced protein shares an overall identity of 40%, 41%, 39% and 41% with its counterpart from human, rat, mouse and African clawed frog, respectively. Tissue expression analysis revealed ot-kifc1 was expressed in testis, gill and hepatopancreas, but not in other tissues examined. In situ hybridization result showed the ot-kifc1 message was hardly detectable in early spermatid, concentrated at the tail region of intermediate spermatid, abundant in spermatid undergoing dramatic elongation and compression, enriched at one end in late spermatids and disappeared in mature sperm. In conclusion, the expression of ot-kifc1 at specific stages of spermiogenesis suggests a role for this motor in major cytological transformations.

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    • "Spermatid differentiation in Octopus tankahkeei can be divided into several continuous stages based on the morphological features of major organelles (Figure 1A). During O. tankahkeei spermiogenesis, a manchette-analogous structure is also temporarily present in close proximity to the external nuclear membrane (Figure 1B) and has a role in the elongation and compaction of the developing nucleus [44]–[46]. The dramatic nature of the sperm nuclear shaping process in this organism enables it to be a good model system to investigate whether a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery has evolved. "
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    ABSTRACT: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.
    PLoS ONE 12/2010; 5(12):e15616. DOI:10.1371/journal.pone.0015616 · 3.23 Impact Factor
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    • "Because of its intimacy to the primary cilium, KIF3A is also implicated in some signaling pathways such as Hedgehog and Wnt pathways (Corbit et al., 2008; Ocbina and Anderson, 2008) and might control embryonic body planning, particularly for determination of left–right asymmetry (Takeda et al., 1999) and patterning of vertebrate skeleton (Kolpakova-Hart et al., 2007). Spermiogenesis of the octopod Octopus tankahkeei is a good model system to study the involvement of kinesin in cytological changes because of the dramatic nature of these changes in this organism (Li and Dong, 2003; Zhu et al., 2005, 2006; Li et al., 2010; Wang et al., 2010; Yu et al., 2010). Preciously, we identified a gene encoding a protein homologous to rat KIFC1 (Wang et al., 2010), a C-terminal retrograde motor protein potentially responsible for vesicles transport from Golgi to acrosome and reshaping of nucleus (Yang and Sperry, 2003; Yang et al., 2006). "
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    ABSTRACT: KIF3A is a subunit of the heterotrimeric Kinesin-II motor which achieves fame for its pivotal roles in the assembly and maintenance of cilia and flagella and in intracellular transport of membrane bound organelles and protein complexes in various tissues. Its intimacy to the cell's antenna, namely the primary cilia, makes it also involved in some signaling transduction pathways. To test the idea that KIF3A functions during spermiogenesis of the octopod Octopus tankahkeei, we hereby identified a gene (designated as ot-kif3a) encoding a protein apparently homologous to a group of KIF3As, from the testis of this organism. The full-length ot-kif3a comprised a 344bp 5' untranslated region, a 2241bp open reading frame and a 147bp 3' untranslated region. The putative protein consisted of 746 amino acid residues with a calculated molecular weight of 85kDa and a predicted isoelectric point of 6.36. It shared an overall sequence identity of 69%, 69%, 69% and 67% to KIF3A from Homo sapiens, Rattus norvegicus, Mus musculus and Danio rerio, respectively. Tissue distribution profile analysis unraveled its presence in all the tissues examined. In situ hybridization of mRNA in spermionenic cells demonstrated that ot-kif3a was expressed moderately at the beginning of spermiogenesis. The abundance of transcripts increased in intermediate spermatid and peaked in drastically remodeling and final spermatids. In mature sperm, the message was still visible in the head and tail. The temporal and spatial expression dynamics of ot-kif3a during spermiogenesis supports the possibility that the putative motor protein, OtKIF3A, participates in the major cytological events during this differentiation program and is vital for the acquisition of the final cellular phenotype.
    Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 11/2010; 157(3):237-45. DOI:10.1016/j.cbpa.2010.07.004 · 1.97 Impact Factor
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    ABSTRACT: KIF3A, the subunit within the kinesin-2 superfamily, is a typically N-terminal motor protein, which is involved in membranous organelle and intraflagellar transport. During spermatogenesis, KIF3A plays a critical role in the formation of flagella and cilia. KIF3A is also related to the left-right asymmetry, the signal pathway, DNA damage and tumorigenesis. We used RT-PCR and in situ hybridization to clone the kif3a gene, and we identified its function in the testis of the Chinese fire-bellied newt Cynops orientalis (termed as co-kif3a). The full-length sequence of co-kif3a was 2193 bp, containing a 56 bp 5'UTR, 2073 bp ORF encoding a protein of 691 amino acids and a 64 bp 3'UTR. The secondary structure analysis showed that co-KIF3A had three motor domains, representing the N-terminal motor domain (1-400 aa), α-helix domain (400-600 aa) and C-terminal tail domain (600-691 aa). The amino acid sequence of co-KIF3A shared an identity of 55.9%, 90.9%, 89.9%, 91.3% and 85.7% with its counterparts in Aedes aegypti, Mus musculus, Xenopus tropicalis, Homo sapiens and Danio rerio, respectively. The calculated molecular weight of the putative co-KIF3A was 79 kDa and its estimated isoelectric point was 6.8. RT-PCR result showed that co-kif3a was expressed in several examined tissues, with a high level in the testis and low levels in liver, muscle and ovum. Kif3a was weakly expressed in the heart and spleen, and barely detected in the intestine. In situ hybridization analysis demonstrated that in early spermatid co-kif3a was expressed around the nuclear membrane. When the tail began to emerge in the middle spermatid, mRNA transcript was abundantly concentrated in the flagellum. The mRNA signal was still very strong along all the flagellum in late spermatid. In mature spermatid, the message was weak. Therefore, co-KIF3A probably plays a functional role in the spermiogenesis of C. orientalis.
    Molecular Biology Reports 07/2011; 39(4):4207-14. DOI:10.1007/s11033-011-1206-3 · 2.02 Impact Factor
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