Table 1.
The primers of kif3a used in this study.
Table 2.
The primers of kif3b used in this study.
Figure 1.
Full-length cDNA of the kif3a in E. sinensis.
The amino acid sequence is deduced from the nucleotide sequence. This figure shows that the full-length cDNA of kif3a consists of a 54 bp 5′ untranslated region, a 187 bp 3′ untranslated region and a 2061 bp open reading frame. The open reading frame encodes 687 amino acids.
Figure 2.
Full-length cDNA of the kif3b in E. sinensis.
The amino acid sequence can be deduced from the nucleotide sequence. This figure shows that the full-length cDNA of kif3b consists of a 159 bp 5′untranslated region, a 428 bp 3′ untranslated region and a 1761 bp open reading frame. The open reading frame encodes 587 amino acids.
Figure 3.
Comparison of the KIF3A protein in E. sinensis with homologues of other species.
This figure shows the amino acid alignment of KIF3A with its homologues using Vector NTI10 (Invitrogen, California, USA). The AYGXTGXGKX, SSRSH, and LAGSE sequences (red frame) are the putative ATP-binding domain, while the YXXXXXDLL sequence (blue frame) is the putative microtubule-binding motif. KIF3A in E. sinensis shows a 64.1, 64.1, 64.5, 64.1, and 44.2% identity with the homologues in Pan troglodytes, Cynops orientalis, Gallus gallus, Bos taurus, and Danio rerio, respectively.
Figure 4.
Comparison of the KIF3B protein in E. sinensis with homologues of other species.
This figure shows the amino acid alignment of KIF3B with its homologues using Vector NTI10 (Invitrogen, California, USA). The VVVRCRP, NGTIFA, GQTGTGKT, and DGENHIRVGKLNLVDLAGSERQ sequences (red frame) are the putative ATP-binding domain, while the HIPYRDSKLTRLL sequence (blue frame) is the putative microtubule-binding motif. KIF3B in E. sinensis shows a 40.3, 47.1, 49.5 and 48.6% identity with the homologues in Aureococcus anophagefferens, Octopus tankahkeei, Xenopus laevis, and Danio rerio, respectively. The identity between KIF3A and KIF3B is about 41.6% in E. sinensis.
Figure 5.
The phylogenetic tree of KIF3A protein and its homologues.
This figure shows the phylogenetic tree of KIF3A and its homologues in other species that were constructed through the neighbor-joining method in Mega 5 (version 5.0) software. We examined the KIF3A from E. sinensis, Oncorhynchus mykiss, Bos taurus, Cynops orientalis, Danio rerio, Pan troglodytes, Homo sapiens, Gallus gallus, Glytapanteles indiensis, Culex quinquefasciatus, and Loa loa. The putative protein of E. sinensis is most closely related to Loa loa.
Figure 6.
The phylogenetic tree of KIF3B protein and its homologues.
This figure shows the phylogenetic tree of KIF3B and its homologues in other species that were constructed through the neighbor-joining method in Mega 5 (version 5.0) software. We examined the KIF3B from E. sinensis, Mus musculus, Danio rerio, Aureococcus anophagefferens, Columba livia, Drosophila melanogaster, Gallus gallus, Octopus tankahkeei, and Xenopus laevis. The putative protein of E. sinensis is most closely related to Octopus tankahkeei.
Figure 7.
The major structural features of KIF3A and KIF3B in E. sinensis.
This figure shows the three structural domains in KIF3A and KIF3B. They all have three domains consisting of the head domain, the stalk domain, and the tail domain. As for KIF3A, the N-terminal (1–420 aa) contains the conserved head (yellow bar) that can move along the microtubules, the stalk domain (421–570 aa) can form an extended coiled-coil region (blue bar), and the C-terminal (571–687 aa) may contain a divergent tail (green bar) that carries a series of cargoes. As for KIF3B, the N-terminal (1–360 aa) contains the conserved head (yellow bar) that can move along the microtubules, the stalk domain (361–500 aa) can form an extended coiled-coil region (blue bar), and the C-terminal (501–587 aa) contains divergent tail (green bar) that carries a series of cargoes. (B) The figure shows the putative 3-D structure of KIF3A and KIF3B. They all contain three domains: the head domain, the stalk domain, and the tail domain. They are all marked in different colors. (C) This figure shows the model pattern of the heterodimer containing KIF3A and KIF3B.
Figure 8.
Semi-quantitative RT-PCR analysis of kif3a gene in different tissues.
(A) This figure shows the expression of kif3a in different tissues of E. sinensis (upper panel). β-actin was used as a positive control (lower panel). The expression of kif3a is high in testis of E. sinensis. (B) This figure shows the quantitative analysis of the expression of kif3a in different tissues. Kif3a is highly expressed in the hepatopancreas and gill. The expression of kif3a in testis is the lowest of these tissues. H: heart, M: muscle, HE: hepatopancreas, G: gill, T: testis.
Figure 9.
Semi-quantitative RT-PCR analysis of kif3b gene in different tissues.
(A) This figure shows the expression of kif3b in different tissues of E. sinensis (upper panel). β-actin was used as a positive control (lower panel). The expression of kif3b is relatively low in testis of E. sinensis. (B) This figure shows the quantitative analysis of the expression of kif3b in different tissues. Kif3b is highly expressed in the hepatopancreas, gill, and heart. The expression of kif3b in testis is the lowest of these tissues. H: heart, M: muscle, HE: hepatopancreas, G: gill, T: testis.
Figure 10.
In situ hydridization of kif3a mRNA during spermiogenesis of E. sinensis.
(A, B, I) Early stage of spermiogenesis. These figures show that kif3a mRNA signals (arrows; blue signal; purple dots) are weakly distributed in the cytoplasm of the round spermatids. (C, D, J) Middle stage of spermiogenesis. These figures show that kif3a mRNA signals (arrows; blue signal; purple dots) are not only distributed in the cytoplasm, but also concentrated on some parts of the nucleus. The expression of kif3a mRNA in the middle stage is larger than that in the early stage. (E, F, K) Late stage of spermiogenesis. These figures show that kif3a mRNA signals (arrows; blue signal; purple dots) are strongly distributed in the nucleus, the cytoplasm complex, the acrosomal tubule (AT) and the apical cap (AC). (G, H, L) Mature sperm. These figures show that kif3a mRNA signals (arrows; blue signal; purple dots) are mostly distributed in the acrosomal tubule (AT), the apical cap (AC), the cytoplasm complex and the nucleus. The three layers (fibrous layer FL, middle layer ML and lamellar structures LS) also have some weak signals of kif3a mRNA in these figures. The expression of kif3a was not decreased in this stage. (M) Control without mRNA. C: centriole.
Figure 11.
In situ hydridization of kif3b mRNA during spermiogenesis of E. sinensis.
(A, B, I) Early stage of spermiogenesis. These figures show that kif3b mRNA signals (arrows; blue signal; purple dots) are weakly distributed in the cytoplasm of the round spermatids. (C, D, J) Middle stage of spermiogenesis. These figures show that kif3b mRNA signals (arrows; blue signal; purple dots) are distributed in the cytoplasm and some parts of the nucleus. The expression of kif3b mRNA in the middle stage is much higher than that in the early stage. (E, F, K) Late stage of spermiogenesis. These figures show that kif3b mRNA signals (arrows; blue signal; purple dots) are strongly distributed in the nucleus, the cytoplasm complex, the acrosomal tubule (AT) and the apical cap (AC). The signals of kif3b expression dramatically increased compared with the middle stage. (G, H, L) Mature sperm. These figures show that kif3b mRNA signals (arrows; blue signal; purple dots) are distributed in the acrosomal tubule (AT), the apical cap (AC), the cytoplasm complex, the nucleus, and the three layers (fibrous layer FL, middle layer ML and lamellar structures LS). The expression of kif3b was not decreased in this stage. (M) Control without mRNA. C: centriole.
Figure 12.
Western blot analysis of KIF3A in E. sinensis.
The extracts of E. sinensis were probed with anti-KIF3A polyclonal antibody (upper panel). Anti-β-actin polyclonal antibody (lower panel) were also used to probe the tissue extracts). This figure shows that the expression of KIF3A protein is higher in the heart (H) and muscle (M) than it is in the testis (T). The molecular weight of β-actin is 42 KD and the molecular weight of KIF3A is about 75 KD.
Figure 13.
Immunofluorescent localization of KIF3A and tubulin in the early stage during spermiogenesis in E. sinensis.
(A) DAPI nuclear staining (blue staining). (B) KIF3A staining (red staining). (C) Tubulin staining (green staining). This figure shows tubulin is localized mostly in the cytoplasm and a small part near the nuclear membrane. (D) Merged Immunofluorescent image. This figure shows that KIF3A and tubulin was co-localized in the cytoplasm in the early stage during spermiogenesis in E. sinensis. Nucleus (blue staining).
Figure 14.
Immunofluorescent localization of KIF3A and tubulin in the middle stage during spermiogenesis in E. sinensis.
(A) DAPI nuclear staining (blue staining). (B) KIF3A staining (red staining). (C) Tubulin staining (green staining). This figure shows that tubulin is localized mostly in the cytoplasm and a small part near the nuclear membrane. (D) Merged Immunofluorescent image. This figure shows KIF3A and tubulin was co-localized in the cytoplasm in the middle stage during spermiogenesis in E. sinensis. Nucleus (blue staining).
Figure 15.
Immunofluorescent localization of KIF3A and tubulin in the mature sperm of E. sinensis.
(A) DAPI nuclear staining (blue staining). (B) KIF3A staining (red staining). (C) Tubulin staining (green staining). This figure shows tubulin is localized in the cytoplasm complex, the acrosomal tubule and near the nuclear membrane. (D) Merged Immunofluorescent image. Tubulin (green staining; arrows) and KIF3A (red staining; arrows) localization is shown. Nucleus (blue staining).