Applied Microscopy

  • 제35권3호
  • /
  • Pages.121-128
  • /
  • 2005
  • /
  • 2287-5123(pISSN)
  • /
  • 2287-4445(eISSN)
한국현미경학회 (Korean Society of Microscopy)
권호 표지

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

Dynamin II Expression and Morphological Comparison of NIH3T3 and NIH3T3 (ras) Cells

  • 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)
  • 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)
  • 발행 : 2005.09.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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.

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.

키워드

참고문헌

  1. Ando A, Yonezawa K, Gout I, Nakata T, Ueda H, Hara K, Kitamura Y, Noda Y, Takenawa T, Hirokawa N, Waterfield MD, Kasuga M: A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment. EMBO J 13 : 3033-3038, 1994
  2. Artalejo CR, Lemmon MA, Schlessinger J, Palfrey HC: Specific role of the PH domain of dynamin-1 in the regulation of rapid endocytosis in adrenal chromaffin cells. EMBO J 16 : 1565-1574, 1997 https://doi.org/10.1093/emboj/16.7.1565
  3. Cook TA, Urrutia R, McNiven MA: Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues. Proc Natl Acad Sci 91 : 644-648, 1994
  4. Crespo P, Mischak H, Gutkind JS: Overexpression of mammalian protein kinase C-zeta does not affect the growth characteristics of NIH3T3 cells. Biochem Biophys Res Commun 213(1) : 266-272, 1995 https://doi.org/10.1006/bbrc.1995.2125
  5. Downing AK, Driscoll PC, Gout I, Salim K, Zvelebil MJ, Waterfield MD: Three-dimensional solution structure of the pleckstrin homology domain from dynamin. Curr Biol 4 : 884-891, 1994 https://doi.org/10.1016/S0960-9822(00)00197-4
  6. Gibson TJ, Hyvonen M, Musacchio A, Saraste M, Birney E: PH domain: the first anniversary. Trends Biochem Sci 19(9) : 349-353, 1994 https://doi.org/10.1016/0968-0004(94)90108-2
  7. Gold ES, Underhill DM, Morrissette NS, Guo JB, McNiven MA, Aderem A: Dynamin 2 is required for phagocytosis in macrophages. J Exp Med 190 : 1849-1856, 1999 https://doi.org/10.1084/jem.190.12.1849
  8. Grabs D, Slepnev VI, Songyang Z, David C, Lynch M, Cantley LC, De Camilli P: The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence. J Biol Chem 272 : 13419-13425, 1997 https://doi.org/10.1074/jbc.272.20.13419
  9. Jeong MJ, Yoo J, Lee SS, Lee KI, Cho A, Kwon BM, Moon MJ, Park YM, Han MY: Increase GTP-binding to dynamin II does not stimulate receptor-mediated endocytosis. Biochm Biophys Res Commun 283 : 136-142, 2001 https://doi.org/10.1006/bbrc.2001.4681
  10. Jones SM, Howell KE, Henley JR, Cao H, McNiven MA: Role of dynamin in the formation of transport vesicles from the trans-Golgi network. Science 279 : 573-577, 1998 https://doi.org/10.1126/science.279.5350.573
  11. Herskovits JS, Shpetner HS, Burgess CC, Vallee RB: Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc Natl Acad Sci 90 : 11468-11472, 1993
  12. Huff SY, Quilliam LA, Cox AD, Der CJ: R-Ras is regulated by activators and effectors distinct from these control ras function. Oncogene 14 : 133-143, 1997 https://doi.org/10.1038/sj.onc.1200815
  13. Kessels MM, Qualmann B: The syndapin protein family: linking membrane trafficking with the cytoskeleton. J Cell Sci 117 : 3077-3086, 2004 https://doi.org/10.1242/jcs.01290
  14. Kharbanda S, Saleem A, Yuan Z, Emoto Y, Prasad KV, Kufe D: Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2 mediated association of the focal adhesion kinase pp125FAK and dynamin. Proc Natl Acad Sci USA 92 : 6132-6136, 1995
  15. Khosravi-Far R, White MA, Westwick JK, Solski PA, Chrzanowska-Wodnicka M, Van Aelst L, Wigler MH, Der CJ: Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol Cell Biol 16(7) : 3923-3933, 1996
  16. Li G, D'Souza-Schorey C, Barboeri MA, Cooper JA, Stahl PD: Uncoupling of membrane ruffling and pinocytosis during ras signal transduction. J Biol Chem 272 : 10337-10340, 1997 https://doi.org/10.1074/jbc.272.16.10337
  17. Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skolnik EY, Bar-Sagi D, Schlessinger J: The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70(3) : 431-442, 1992 https://doi.org/10.1016/0092-8674(92)90167-B
  18. Maier O, Knoblich M, Westermann P: Dynamin II binds to the trans-Golgi network. Biochem Biophys Res Commun 223 : 229-233, 1996 https://doi.org/10.1006/bbrc.1996.0876
  19. Noda Y, Nakata T, Hirokawa N: Localization of dynamin : widespread distribution in mature neurons and association with membranous organelles. Neuroscience 55 : 113-127, 1993 https://doi.org/10.1016/0306-4522(93)90459-S
  20. Obar RA, Collins CA, Hammarback JA, Shpetner HS, Vallee RB: Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature 347 : 256-261, 1990 https://doi.org/10.1038/347256a0
  21. Okamoto PM, Herskovits JS, Vallee RB: Role of the basic, proline-rich region of Dynamin in Src Homology 3 domain binding and endocytosis. J Biol Chem 272 : 11629-11635, 1997 https://doi.org/10.1074/jbc.272.17.11629
  22. Schafer DA: Regulating actin dynamics at membranes: a focus on dynamin. Traffic 5 : 463-469, 2004 https://doi.org/10.1111/j.1600-0854.2004.00199.x
  23. Shupliakov O, Low P, Grabs D, Gad H, Chen H, David C, Takei K, De Camilli P, Brodin L: Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions. Science 276 : 259-263, 1997 https://doi.org/10.1126/science.276.5310.259
  24. Song BD, Yarar D, Schmid SL: An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo. Mol Biol Cell 15 : 2243-2252, 2004 https://doi.org/10.1091/mbc.E04-01-0015
  25. Sontag JM, Fykse EM, Ushkaryov Y, Liu JP, Robinson PJ, Sudhof TC: Differential expression and regulation of multiple dynamins. J Biol Chem 269 : 4547-4554, 1994
  26. Vidal M, Montiel JL, Cussac D, Cornille F, Duchesne M, Parker F, Tocque B, Roques BP, Garbay C: Differential interactions of the growth factor receptor bound protein 2N-SH3 domain with son of sevenless and dynamin. J Biol Chem 273 : 5343-5348, 1998 https://doi.org/10.1074/jbc.273.9.5343
  27. Vojtek AB, Der CJ: Increasing complexity of the Ras signaling pathway. J Biol Chem 273 : 19925-19928, 1998 https://doi.org/10.1074/jbc.273.32.19925
  28. Wang Z, Moran MF: Requirement for the adapter protein GRB2 in EGF recepter endocytosis. Science 272 : 1935-1939, 1996 https://doi.org/10.1126/science.272.5270.1935
  29. Warnock DE, Terlecky LJ, Schmid SL: Dynamin GTPase is stimulated by crosslinking through the C-terminal proline-rich domain. EMBO J 14 : 1322-1328, 1995
  30. Yoon SY, Koh WS, Lee KI, Park YM, Han MY: Dynamin II associates with Grb2 SH3 domain in ras transformed NIH3T3 cells. Biochm Biophs Res Commun 234 : 539-543, 1997 https://doi.org/10.1006/bbrc.1997.6676