• Molecules and Cells
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• 제41권12호
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• pp.1045-1051
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• 2018

The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.

• Reproductive and Developmental Biology
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• 제34권2호
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• pp.73-79
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• 2010

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing $\gamma$-tubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The $\gamma$-tubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas $\gamma$-tubulin spots in the SCNT embryos were observed only during the inter-anaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.49-49
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• 2002

γ - tubulin is an essential, invariant constitutive centrosomal protein, which plays key roles in microtubule patterning and defining the microtubule intrinsic polarity. Although γ-tubulin was also present in cattle oocytes and zygotes, no details have been provided on its recruitment and localization to date. In this study, we determined γ-tubulin distribution chronologically in conjunction with microtubule dynamics during fertilization and parthenogenesis, with a view to understanding the molecular basis of zygotic centrosome reconstitution in cattle. (omitted)

• 한국가축번식학회지
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• 제19권3호
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• pp.205-216
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• 1995

Microtubules와 micrfilaments는 포유동물 난자이 주요한 세포 구조물들로, 이들은 난자의 성숙, 수정 및 배발달시 핵질의 이동과 세포질 분열에 직접 관여하는 것으로 알려져 왔다. 난자내 세포구조물의 정확한 움직임은 정상적인 배 발달을 위해 필수적이다. Microtubules는 $\alpha$, $\beta$- bubulin이 서로 연결되어 이루어져 있으며, 수정시 웅성.자성전핵 움직임과 세포분열시, 유사 및 감수분열시 그 역할을 한다. 생쥐를 제외한 대부분의 동물에서 microbubules의 역할은 수정시 정자가 centrosome을 난자내로 이전하여 sperm aster를 형성함으로써 시작된다고 보고되고 있다. 따라서 정자의 도움없이 배발달이 일어나는 단위발생시 microbubules의 형성은 연구들 사이에 흥미로운 연구대상이 되고 있다. 한편 microfilaments는 세포분열시 세포질을 분할하는 기계적인 역할을 하는 것으로 알려져 있으며, 최근 생쥐 난자에서는 정자의 난자내 융합과 웅성 및 자성 전핵의 이동에 관여한다고 보고되고 있다. 포유동물 난자의 체외성숙, 체외수정을 유도할 때 여러 가지 비정상적인 핵움직임과 세포분열이 관찰되어지고, 낮은 배발달율이 보고되고 있는데, 최근 연구자들은 세포구조물, 즉 microtubules와 microfilaments의 비정상적인 역할에서 기인한다고 보고 있다. 따라서 포유동물 난자의 성숙.수정 및 단위발생시 세포구조물의 움직임과 역할 및 상호관계에 대한 정확한 이해는 체외수정율 및 배발달 향상에 중요한 기초자료로 이용되리라고 본다.

• BMB Reports
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• 제40권2호
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• pp.218-225
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• 2007

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation exp eriments demonstrated that AtMAP65-1 bound to tubulin dimers,at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with $\alpha$-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 $\Delta$N339 (amino acids 340-587); AtMAP65-1 $\Delta$N494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 $\Delta$C495 (amino acids 1-494) and AtMAP65-1 $\Delta$C340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 $\Delta$N339, $\Delta$N494, AtMAP65-1 340-494 and $\Delta$C495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 $\Delta$N339, $\Delta$N494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 $\Delta$N339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 $\Delta$N494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 $\Delta$C495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.

• 생명과학회지
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• 제27권6호
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• pp.673-679
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• 2017

미세소관을 따라 이동하는 모터단백질들은 세포내 물질수송에 필수적인 역할을 한다. Kinesin 1은 세포내에서 미세소관을 따라 움직이는 모터단백질로서 다양한 소포, mRNA, 그리고 단백질의 세포내 수송에 관여한다. Kinesin 1은 2개의 장쇄단위체(KHCs, 또는 KIF5s)와 2개의 경쇄단위체(KLCs)로 구성되어 있다. KIF5s는 N-말단에 모터도메인을 가지고 있고 C-말단의 운반체 결합도메인을 통해 다양한 운반체와 결합한다. 본 연구에서 KIF5B와 결합하는 단백질을 분리하기 위하여 효모 two-hybrid 탐색을 수행한 결과 미세소관의 plus end 결합단백질인 cytoplasmic linker protein 170 (CLIP-170)을 분리하였다. CLIP-170의 coiled-coil 도메인은 KIF5B의 운반체 결합도메인과 결합하였다. 또한 CLIP-170은 KIF5A와 KIF5C와도 결합하였다. 그리고 glutathione S-transferase (GST) pull-down을 통해 KIF5s와 CLIP-170이 단백질수준에서 결합함을 확인하였다. 생쥐 뇌파쇄액을 KIF5B 항체로 면역침강한 결과 CLIP-170이 같이 침강함을 확인하였다. 이러한 결과들은 kinesin 1이 세포내에서 CLIP-170을 운반함을 시사한다.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.301-309
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• 1997

본 연구에서는 체외수정, 난자내 정자 직접주입, 난자내 정자 두부 미두 주입 후의 핵과 미세소관의 변화를 관찰하였다. 핵과 미세소관의 움직임은 형광염색을 실시한 후 공초점주사현미경을 이용하여 관찰하였다. 체외수정에서 관찰된 바와 동일하게 정자를 난자에 직접주입 한 직후 정자 중편부에서 성상체가 형성되었고, 이 성상체에 의해 자성 웅성 전핵이 융합되는 것으로 관찰되었다. 그러나 난자내 정자를 직접주입하였을 경우 웅성전핵으로 발달하는 비율이 낮았다. 이는 주입된 정자가 원형질막과 perinuclear theca에 싸인 체 난자내로 들어가 난자내의 sperm nucleus decondensing factor와 정자 핵과의 반응이 억제되기 때문으로 생각된다. 정자 두부 만을 주입하였을 경우 성상체가 형성되지 않았지만 자성 웅성 전핵 사이 또는 그 주위에서 두터운 미세소관층이 관찰이 되었다. 따라서 소에 있어서는 정자의 중편부에 위치하여 microtubule organizing center (MTOC)의 역할을 하는 중심립 또는 중심체 없이도 모계에서 유래된 미세소관이 형성되어 이것이 전핵의 융합과 세포분열에 관여하는 것으로 생각된다. 정자의 미부 만을 주입하였을 경우 성상체가 형성이 되지 않았으며, 자성핵 사이에 형성된 미세소관과 떨어져서 관찰되었다. 따라서 주입된 정자의 꼬리는 미세소관형성과 관련이 없는 것으로 생각된다. 이러한 결과는 소에 있어서, 수정 시 정자로부터 유래되는 중심립 또는 중심체가 없이도 미세소관을 형성하여 미세소관에 의해 이후의 배발달이 정상적으로 일어남을 보여주고 있다.

• Earthquakes and Structures
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• 제24권2호
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• pp.127-140
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• 2023

The vibration of microtubule in human cells is the source of electrical field around it and inside cell structure. The induction of electrical field is a direct result of the existence of dipoles on the surface of the microtubules. Measuring the electrical fields could be performed using nano-scale sensors and the data could be transformed to other computers using internet of things (IoT) technology. Processing these data is feasible by artificial intelligence-based methods. However, the first step in analyzing the vibrational behavior is to study the mechanics of microtubules. In this regard, the vibrational behavior of the microtubules is investigated in the present study. A shell model is utilized to represent the microtubules' structure. The displacement field is assumed to obey first order shear deformation theory and classical theory of elasticity for anisotropic homogenous materials is utilized. The governing equations obtained by Hamilton's principle are further solved using analytical method engaging Navier's solution procedure. The results of the analytical solution are used to train, validate and test of the deep neural network. The results of the present study are validated by comparing to other results in the literature. The results indicate that several geometrical and material factors affect the vibrational behavior of microtubules.

• 한국발생생물학회지:발생과생식
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• 제15권3호
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• pp.231-237
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• 2011

The objective of this study was to elucidate the dynamics of microtubules in post-ovulatory aging in vivo and in vitro of mouse oocytes. The fresh ovulated oocytes were obtained from oviducts of superovulated female ICR mice at 16 hours after hCG injection. The post-ovulatory aged oocytes were collected at 24 and 48 hours after hCG injection from in vivo and in vitro, respectively. Immunocytochemistry was performed on ${\beta}$-tubulin and acetylated ${\alpha}$-tubulin. The microtubules were localized in the spindle assembly, which was barrel-shaped or slightly pointed at its poles and located peripherally in the fresh ovulated oocytes. The frequency of misaligned metaphase chromosomes were significantly increased in post-ovulatory aged oocytes after 48 hours of hCG injection. The spindle length and width of post-ovulatory aged oocytes were significantly different from those of fresh ovulated oocytes, respectively. The staining intensity of acetylated ${\alpha}$-tubulin showed stronger in post-ovulatory aged oocytes than that in the fresh ovulated oocytes. In the aged oocytes, the spindles had moved towards the center of the oocytes from their original peripheral position and elongated, compared with the fresh ovulated oocytes. Microtubule organizing centers were formed and observed in the cytoplasm of the aged oocytes. On the contrary, it was not observed in the fresh ovulated oocytes. The alteration of spindle formation and chromosomes alignment substantiates the poor development and the increase of disorders from the post-ovulatory aged oocytes. It might be important to fertilize on time in ovulated oocytes for the developmental competence of embryos with normal karyotypes.