Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
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Dynamin II Expression and Morphological Comparison of NIH3T3 and NIH3T3 (ras) Cells

NIH3T3와 NIH3T3(ras) 세포에서 Dynamin II 발현 및 형태적 비교

  • Lee, Chul-Woo (Department of Oral & Maxillofacial Surgery, College of Dentistry, Chosun University) ;
  • Kim, Su-Gwan (Department of Oral & Maxillofacial Surgery, College of Dentistry, Chosun University) ;
  • Choi, Jeong-Yun (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Choi, Baik-Dong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Bae, Chun-Sik (College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University) ;
  • Jeong, Soon-Jeong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Jeong, Moon-Jin (Department of Oral Histology, College of Dentistry, Chosun University)
  • 이철우 (조선대학교 치과대학 구강외과학교실) ;
  • 김수관 (조선대학교 치과대학 구강외과학교실) ;
  • 최정윤 (조선대학교 치과대학 구강조직학교실) ;
  • 최백동 (조선대학교 치과대학 구강조직학교실) ;
  • 배춘식 (전남대학교 수의과대학 및 생물공학연구소) ;
  • 정순정 (조선대학교 치과대학 구강조직학교실) ;
  • 정문진 (조선대학교 치과대학 구강조직학교실)
  • Published : 2005.09.01
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Abstract

It has been known that ras signaling transduction leads to cell proliferation and migration including various adaptor molecules. Dynamin protein has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. Dynamin was classified into three isoforms: dynamin I is only expressed in neuronal tissue, dynamin II is expressed ubiquitously in all tissue but that of dynamin III is confined to testis. We have reported in previous study that Grb2, binding to ras, was associated with dynamin II in NIH3T3 cells. Therefore we have tried to identify the relative expression of dynamin II according to overexpressed ras protein in ras oncogene transfected cells (NIH3T3 (ras)). For the detection of differential expression of dynamin II, we have used immunofluorescent staining and western blot methods in NIH3T3 and NIH3T3 (ras) cells. Next we have described the morphological differences between NIH3T3 and NIH3T3 (ras) cells using SEM and TEM. From these experiments dynamin II was highly expressed in NIH3T3 (ras) cells. NIH3T3 cells was transformed to more spindle shape with many cell process by transfection of ras oncogene. Moreover dynamin II was more concentrated in endocytotic membrane of the NIH3T3 (ras) cells compared to that of NIH3T3 cells. The present results suggested that dynamin II may involve the intermediate messenger in Ras signaling transduction pathway.

Ras 신호전달체계는 세포내 다양한 결합 분자들과 더불어 세포의 분열과 세포의 이동에 관여한다. Dynamin 단백질은 endocytosis와 분비과정에서 vesicle를 분리하는데 관여하는 것으로 알려져 있으며, 3가지 아형으로 구분된다. Dynamin I은 신경조직에서 만 발현되고, dynamin II는 모든 조직에서 발현되지만 dynamin III는 정소를 포함한 생식기계에서만 발현된다. 선행된 연구에서 NIH3T3 세포를 이용하여 ras과발현 세포주를 만들었으며, dynamin II와 ras의 신호전달체계에 있는 Grb2가 결합한다는 것을 보고하였다. 따라서, 본 연구는 ras 단백질이 과발현되는 세포 (NIH3T3 (ras))와 대조세포인 NIH3T3의 형태학적인 차이점을 분석하고, 이 두 세포들에서 dynamin II 단백질의 발현의 차이를 비교하고자 하였다. Dynamin II의 발현차이를 분석하기 위해 형광염색을 하여 공초점 레이저현미경으로 세포내 분석을 하였으며, western blot을 시행하여 생화학적인 발현차이를 보았다. 또한, 두 세포의 미세구조적인 분석을 위하여 SEM과 TEM을 사용하였다. Dynamin II는 NIH3T3 (ras) 세포에서 발현이 증가 하였으며, NIH3T3 세포에 비하여 좀더 방추형이 었으며, 작은 세포질 돌기가 세포막을 따라 다수 신장되어있음이 관찰되었다. 또한, NIH3T3 (ras) 세포의 endocytotic vesicle이 형성되는 부위에서 dynamin II의 발현이 증가하였다. 이러한 결과로 dynamin II는 ras신호전달체계의한 신호전달분자로서 작용을 할 것으로 사료된다.

Keywords

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