Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy
碩士 === 國立陽明大學 === 生醫光電工程研究所 === 97 === The Ca2+ influx is essential for initiating messange to regulate several vital processes. The store-operated Ca2+ channels (SOCs) constructed by Orai1 with ER membrane peotein STIM1 were recently found to be a unique Ca2+ entry pathway which is the major Ca2+ s...
Main Authors: | , |
---|---|
Other Authors: | |
Format: | Others |
Language: | zh-TW |
Published: |
2009
|
Online Access: | http://ndltd.ncl.edu.tw/handle/8vmdqv |
id |
ndltd-TW-097YM005114013 |
---|---|
record_format |
oai_dc |
spelling |
ndltd-TW-097YM0051140132019-05-15T20:07:01Z http://ndltd.ncl.edu.tw/handle/8vmdqv Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy 以螢光生命週期顯微術觀測鈣庫調節鈣離子通之道調控蛋白質間的動態交互作用 Ping-Chun Huang 黃柄鈞 碩士 國立陽明大學 生醫光電工程研究所 97 The Ca2+ influx is essential for initiating messange to regulate several vital processes. The store-operated Ca2+ channels (SOCs) constructed by Orai1 with ER membrane peotein STIM1 were recently found to be a unique Ca2+ entry pathway which is the major Ca2+ signaling cascade for non-excitable cells. The SOCs also functional exist in various types of cells such as excitable neuroendocrines like rat pheochromocytoma PC12 cells where voltage-gated Ca2+ channels are the major entry route. Althogh Orai1 together with STIM1 are thought to be interact with each other thorugh biochemical evidence, it is still unrevealed to define the detail mechanism about their dynamic interactions during store-operated conditions in situ. To solve this problem in this thesis, the green fluorescence protein (EGFP) targeted Orai1 (to either N- or C-terminal; wild type or double deletion type or Orai1, as donor) and mOrange flagged STIM1 (as acceptor) were used as a fluorescence resonance energy transfer (FRET) pair within living PC12 cells under a new constructed one-photon excitation fluorescence lifetime imaging microscopy (FLIM) adapting the strategy of time-correlated single photon counting (TCSPC). The activity of SOCs, namely the store-operated Ca2+ entry (SOCE) was measured using fura-2 imaging method. The ER Ca2+ ATPase inhibitor thapsigargin (TG) was used to activate the SOCE. The lifetime map and histogram/distribution of each single cells were acquired under FLIM. Both the color coded liftime map (plasma membrane) and the distribution (100 ps left shift) of EGFP tagged Orai1 changed signficantly after 20-minute administering of TG. The efficiency xi of FRET from each experimental sets was also calculatecd and compared. In summary, we further show the dynamic interactions between the essential SOC protein Orai1 and STIm1 with the novel FLIM-FRET technique within living PC12 cells. De Ming Yang 楊德明 2009 學位論文 ; thesis 101 zh-TW |
collection |
NDLTD |
language |
zh-TW |
format |
Others
|
sources |
NDLTD |
description |
碩士 === 國立陽明大學 === 生醫光電工程研究所 === 97 === The Ca2+ influx is essential for initiating messange to regulate several
vital processes. The store-operated Ca2+ channels (SOCs) constructed by
Orai1 with ER membrane peotein STIM1 were recently found to be a
unique Ca2+ entry pathway which is the major Ca2+ signaling cascade for
non-excitable cells. The SOCs also functional exist in various types of
cells such as excitable neuroendocrines like rat pheochromocytoma PC12
cells where voltage-gated Ca2+ channels are the major entry route.
Althogh Orai1 together with STIM1 are thought to be interact with each
other thorugh biochemical evidence, it is still unrevealed to define the
detail mechanism about their dynamic interactions during store-operated
conditions in situ. To solve this problem in this thesis, the green
fluorescence protein (EGFP) targeted Orai1 (to either N- or C-terminal;
wild type or double deletion type or Orai1, as donor) and mOrange
flagged STIM1 (as acceptor) were used as a fluorescence resonance
energy transfer (FRET) pair within living PC12 cells under a new
constructed one-photon excitation fluorescence lifetime imaging
microscopy (FLIM) adapting the strategy of time-correlated single photon
counting (TCSPC). The activity of SOCs, namely the store-operated Ca2+
entry (SOCE) was measured using fura-2 imaging method. The ER Ca2+
ATPase inhibitor thapsigargin (TG) was used to activate the SOCE. The
lifetime map and histogram/distribution of each single cells were
acquired under FLIM. Both the color coded liftime map (plasma
membrane) and the distribution (100 ps left shift) of EGFP tagged Orai1
changed signficantly after 20-minute administering of TG. The efficiency
xi
of FRET from each experimental sets was also calculatecd and compared.
In summary, we further show the dynamic interactions between the
essential SOC protein Orai1 and STIm1 with the novel FLIM-FRET
technique within living PC12 cells.
|
author2 |
De Ming Yang |
author_facet |
De Ming Yang Ping-Chun Huang 黃柄鈞 |
author |
Ping-Chun Huang 黃柄鈞 |
spellingShingle |
Ping-Chun Huang 黃柄鈞 Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
author_sort |
Ping-Chun Huang |
title |
Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
title_short |
Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
title_full |
Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
title_fullStr |
Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
title_full_unstemmed |
Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
title_sort |
observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy |
publishDate |
2009 |
url |
http://ndltd.ncl.edu.tw/handle/8vmdqv |
work_keys_str_mv |
AT pingchunhuang observingdynamicinteractionsofstoreoperatedcalciumchannelproteinsbyfluorescencelifetimeimagingmicroscopy AT huángbǐngjūn observingdynamicinteractionsofstoreoperatedcalciumchannelproteinsbyfluorescencelifetimeimagingmicroscopy AT pingchunhuang yǐyíngguāngshēngmìngzhōuqīxiǎnwēishùguāncègàikùdiàojiégàilízitōngzhīdàodiàokòngdànbáizhìjiāndedòngtàijiāohùzuòyòng AT huángbǐngjūn yǐyíngguāngshēngmìngzhōuqīxiǎnwēishùguāncègàikùdiàojiégàilízitōngzhīdàodiàokòngdànbáizhìjiāndedòngtàijiāohùzuòyòng |
_version_ |
1719097222758400000 |