2004 Fiscal Year Final Research Report Summary
Structural analysis of the molecular organization of the membrane undercoat
| Project/Area Number |
14380315
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Nagoya University |
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Principal Investigator |
USUKURA Jiro Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (30143415)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIZAWA Yuji Nagoya University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (80252229)
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| Project Period (FY) |
2002 – 2004
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| Keywords | Cell membrane / Membrane undercoat / immunocytochemistry / CRMP-2 / IQCAP-1 / Rapid freezing / Neural growth cone / freeze-etchine |
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| Research Abstract |
Cell membrane consists of domains with distinct functions and different sizes, which also plays most important roles in life. Peripheral membrane proteins forming membrane undercoat regulate function and movement of intrinsic membrane proteins within various domains. This project aimed to reveal molecular organization of such membrane undercoat. For this purpose immuno-freeze-etching replica method combined with electron microscopic tomography was employed and available for three-dimensional analysis of membrane undercoat of neural growth cone. In common, membrane undercoat consisted of actin filaments, microtubules, actin related proteins (Arp2/3) and small amount of spectrin. Those filaments formed complicated meshwork and attached closely onto the cytoplasmic surface of the plasma membrane. It is very interested what and how neuronal growth factors are associated in this membrane undercoat. In particular, we focused on CRMP-2 because it was involved in initial formation of axon. Alt
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hough CRMP-2 were localized predominantly on microtubules in axons, it was found frequently on clathrin coats being associated with other unknown particles in membrane undercoat of growth cone. This suggests that CRMP-2 is supposed to participate in endocytosis in the growth cone as well.
However, the exact function of CRMP-2 has not been unknown. If CRMP-2 controls growing axon by regulating microtubules and/or actin filaments assembly as the substrate of Rho-kinase in axon, it may have different function in growth cone such as the membrane recycling. We also have been trying to observe the membrane undercoat at living state without any decoration.
Fortunately, hydrated membrane undercoat was observed directly under the electron microscope equipped with liquid helium cooled stage using the cell unroofed by original method. Microtubles, actin filaments and clathrin coated pits were found in the similar way of freeze-etching method. This success is a very important step for forthcoming research project. Less
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