Three Dimensional Reconstruction of Cellular Structure in Drosophila Retina Using High Voltage Electron Microscopy

초고압전자현미경을 이용한 초파리 망막 세포의 3차원 구조

  • Mun, Ji-Young (School of Life Sciences and Biotechnology, Korea University) ;
  • Lee, Kyung-Eun (School of Life Sciences and Biotechnology, Korea University) ;
  • Han, Sung-Sik (School of Life Sciences and Biotechnology, Korea University)
  • 문지영 (고려대학교 생명과학대학 생명공학원 세포공학 및 생체3차구조 연구실) ;
  • 이경은 (고려대학교 생명과학대학 생명공학원 세포공학 및 생체3차구조 연구실) ;
  • 한성식 (고려대학교 생명과학대학 생명공학원 세포공학 및 생체3차구조 연구실)
  • Received : 2009.05.12
  • Accepted : 2009.06.19
  • Published : 2009.06.30

Abstract

Studies about the structure of Drosophila melanogaster retinal cell using electron microscopy have been carried out in details since 1960s. However, these results can have limitations in functional research because of two-dimensional structure. In this study, the adult retina of Drosophila melanogaster was investigated by employing high pressure freezing method, serial sections, high voltage electron microscopy, and 3-dimensional reconstruction method. From there results, mitochondria, microtubules, and nuclei were reconstructed as 3-dimensional structure using IMOD program. The 3D structure of these organelles showed that mitochondira mainly located in distal region near lens, and microtubule mainly located in distal and basal region. The 3D reconstruction of these organelles can be used for a critical evaluation in the dynamic change of cellular organelles caused by functional abnormality like retinal degeneration.

초파리 망막 세포의 미세구조연구는 1960년대부터 수행되어 왔으나, 이 연구들은 2차원 구조연구에 국한되어 있었다. 본 논문은 초고압 동결법, 연속절편법, 초고압 전자현미경, 그리고 3차원 구조 구현법을 이용하여 초파리 성체의 망막 세포에 존재하는 미토콘드리아, 미세소관, 그리고 핵의 3차원 분포를 연구한 결과이다. 이를 통하여 미토콘드리아는 주로 세포막 가까이에 위치하며, 그 수는 렌즈와 가까운 말단부분에 많은 것을 알 수 있었고, 미세소관은 렌즈와 가까운 망막 말단부분과 시신경과 연결되는 기부에 특히 많은 수가 존재하는 것을 알 수 있었다. 이들은 망막 변성의 원인이 되는 세포 소기관이므로, 이들의 세포 내 3차원 분포는 병의 원인을 연구하는 데 있어 중요한 자료가 될 것으로 사료된다.

Keywords

References

  1. Bravo-Nuevo A, Williams N, Geller S, Stone J: Mitochondrial deletions in normal and degenerating rat retina. Adv Exp Med Biol 533 : 241-248, 2003
  2. Eckmiller MS: Defective cone photoreceptor cytoskeleton, alignment, feedback, and energetics can lead to energy depletion in macular degeneration. Prog Retin Eye Res 23(5) : 495-522, 2004 https://doi.org/10.1016/j.preteyeres.2004.04.005
  3. Farquhar MG, Palade GE: Junctional complexes in various epithelia. J Cell Biol 17 : 375-412, 1963 https://doi.org/10.1083/jcb.17.2.375
  4. Gorska-Andrzejak J, Stowers RS, Borycz J, Kostyleva R, Schwarz TL, Meinertzhagen IA: Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesinassociated protein. J Comp Neurol 463(4) : 372-388, 2003 https://doi.org/10.1002/cne.10750
  5. Grayson C, Bartolini F, Chapple JP, Willison KR, Bhamidipati A, Lewis SA, Luthert PJ, Hardcastle AJ, Cowan NJ, Cheetham ME: Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3. Hum Mol Genet 11(24) : 3065-3074, 2002 https://doi.org/10.1093/hmg/11.24.3065
  6. Hama K, Arii T, Kosaka T: Three-dimensional organization of neuronal and glial processes: high voltage electron microscopy. Microsc Res Tech 29(5) : 357-367, 1994 https://doi.org/10.1002/jemt.1070290506
  7. Hess MW, Pfaller K, Hampolz B, Longato S, Teis D, Florl A, Gutleben K, Huber LA: Microscopy of the Drosophila facet eye: vademecum for standardized fixation, embedding, and sectioning. Microsc Res Tech 69(2) : 93-98, 2006 https://doi.org/10.1002/jemt.20268
  8. Knabe W, Kuhn HJ: The role of microtubules and microtubuleorganising centres during the migration of mitochondria. J Anat 189(Pt 2) : 383-391, 1996
  9. Monaghan P, Perusinghe N, Muller M: High-pressure freezing for immunocytochemistry. J Microsc 192(Pt 3) : 248-258, 1998 https://doi.org/10.1046/j.1365-2818.1998.00387.x
  10. Muller-Reichert T, Hohenberg H, O'Toole ET, McDonald K: Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J Microsc 212(Pt 1) : 71-80, 2003 https://doi.org/10.1046/j.1365-2818.2003.01250.x
  11. Perkins GA, Ellisman MH, Fox DA: Three-dimensional analysis of mouse rod and cone mitochondrial cristae architecture: bioenergetic and functional implications. Mol Vis 9 : 60-73, 2003
  12. Perkins GA, Ellisman MH, Fox DA: The structure-function correlates of mammalian rod and cone photoreceptor mitochondria: observations and unanswered questions. Mitochondrion 4(5-6) : 695-703, 2004 https://doi.org/10.1016/j.mito.2004.07.020
  13. Perkins GA, Frey TG: Recent structural insight into mitochon-dria gained by microscopy. Micron 31(1) : 97-111, 2000 https://doi.org/10.1016/S0968-4328(99)00065-7
  14. Perry MM: Further studies on the development of the eye of Drosophila melanogaster. I. The ommatidia. J Morphol 124 (2) : 227-248, 1968 https://doi.org/10.1002/jmor.1051240208
  15. Sandberg K: Methods for image segmentation in cellular tomography. Methods Cell Biol 79 : 769-798, 2007 https://doi.org/10.1016/S0091-679X(06)79030-6
  16. Studer D, Graber W, Al-Amoudi A, Eggli P: A new approach for cryofixation by high-pressure freezing. J Microsc 203 (Pt 3) : 285-294, 2001 https://doi.org/10.1046/j.1365-2818.2001.00919.x