Upload
lucien
View
31
Download
0
Embed Size (px)
DESCRIPTION
How to prepare the seminar?. Read through the paper several times. Think ! What’s the main question of this paper? How do the authors design the experiments? What’re the results of this paper? What’re the contributions of their work? - PowerPoint PPT Presentation
Citation preview
Read through the paper several times.Read through the paper several times.
ThinkThink !! What’s the main question of this paper?What’s the main question of this paper?
How do the authors design the experiments?How do the authors design the experiments?
What’re the results of this paper?What’re the results of this paper?
What’re the contributions of their work?What’re the contributions of their work?
Try to prepare excellent powerpoint file! Try to prepare excellent powerpoint file!
Rehearsal it with your classmates!!Rehearsal it with your classmates!!
課程名稱:專題討論(下) 學分數: 1
開課系所:生科三(甲) 上課時間: Wed, 2:40~4:30 PM
任課教師: 賴金美老師( [email protected] )上課地點: ES
406
目的 :
為培養學生閱讀及表達科學性論文之能力,本專討將由老師指定或同學自行選訂與細胞生化相關之期刋論文為題材,藉由課堂的報告與問題討論以增進同學在生化科學方面的實力。
評分:由老師與同學共同評分: 1. Teacher (70%): Presentation: 70% [Power point (20%); Paper
presentation & Discussion( 50%)]
Question: 30%
2. Classmates (30%)
週次週次 日期日期 課程內容課程內容 週次週次 日期日期 課程內容課程內容
1122 月月 2727
日日 專討課程介紹與示範專討課程介紹與示範 101044 月月 2323
日日 期中考週(停課)期中考週(停課)
2233 月月 0505
日日 專討諮詢專討諮詢 111144 月月 3030
日日 陳紋平、林鳳明
3333 月月 1212
日日 專討諮詢專討諮詢 121255 月月 0707
日日 吳奇峰、吳嘉維
4433 月月 1919
日日 專討諮詢專討諮詢 131355 月月 1414
日日 郭儆寰、林恩羽
5533 月月 2525
日日 古政宏、方欣怡 141455 月月 2121
日日
6644 月月 0202
日日 陳柏蓁、吳婉甄 151555 月月 2828
日日
7744 月月 0909
日日 陳志瑋、卓芳如 161666 月月 0404
日日
8844 月月 1616
日日 蕭婷分、歐陽偉棠 181866 月月 1111
日日 期末考週期末考週
http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/Click Click
1. Key word1. Key word
oror Ex. j biol chem [ta]Ex. j biol chem [ta] biochem j [ta]biochem j [ta]
You can choose “type of article”You can choose “type of article” “ “date”date” “ “author”author”
http://ntuml.mc.ntu.edu.tw/index.asphttp://ntuml.mc.ntu.edu.tw/index.asp
Nucleotide exchange factor GEF-H1 mediates cross-talk
between microtubules and the actin cytoskeleton
Krendel M, Zenke FT, Bokoch GM.Krendel M, Zenke FT, Bokoch GM.
Nat Cell Biol. 2002 Apr;4(4):294-301. Nat Cell Biol. 2002 Apr;4(4):294-301.
Example:
Cytoskeleton
--- composed of three well-defined filamentous structures
- Microtubules (tubulin) - Microfilaments (actin) - Intermediate filaments
Moter protein
--- is used to move cellular cargo.
- Microtubules : kinesin, dynein. - Microfilaments (actin) : myosin
is important in many physiological processes
--- including embryonic development, the inflammatory --- including embryonic development, the inflammatory immune immune response, wound repair, and tumor formation and response, wound repair, and tumor formation and metastasis.metastasis.
--- is powered by the activity of the actin cytoskeleton--- is powered by the activity of the actin cytoskeleton
actin polymerization driving leading edge protrusion actin polymerization driving leading edge protrusion
acto-myosin contractility promoting cell body advancementacto-myosin contractility promoting cell body advancement
The role of microtubules in migrating cells :The role of microtubules in migrating cells :
master regulators or obsolete ?master regulators or obsolete ?
Microtubules do not directly contribute to the generation of forces Microtubules do not directly contribute to the generation of forces drive cell migration in most cell typesdrive cell migration in most cell types
The loss of microtubules prevents directional movement of cells in culture.The loss of microtubules prevents directional movement of cells in culture.( J. Embryol. Exp. Morphol. 24: 625-40. 1970 )( J. Embryol. Exp. Morphol. 24: 625-40. 1970 )
Micriotubules may be involved in the regulation of actin-dependent Micriotubules may be involved in the regulation of actin-dependent
motility.motility.
The rate of lamellipodial protrusion in these cells is decreasedThe rate of lamellipodial protrusion in these cells is decreased
Microtubules are necessary to support normal rates of leading edgeMicrotubules are necessary to support normal rates of leading edge
protrusion.protrusion.
Micriotubules
(Journal of Cell Science 114, 3795-3803. 2001)
Regulation of the actin cytoskeleton by microtubules relies on the activity of Rho family GTPase.
Microtubule disassembly results in the activation of Rho,
which enhances myosin contractility and stress fibre formation.
What’s the molecular mechanisms through which microtubules
modulate the activity of Rho GTPase ?
Rho family: small GTP-binding protein
GEF GEFGTP
GDPGDP GTP
InactiveG protein
ActiveG protein
To address that GEF is responsible for regulating Rho
activity in response to microtubule depolymerization.
modulate actin filament organization and
myosin sontractility.
Intracellular localization of GEF-H1
---- localize to microtubules
Cytosolic localization of GEF-H1
Human cDNA clone (GeneBank accession number: AB014551)
The encoded protein is different from GER-H1 only in its N-termius, which does not contain a zinc finger motif.
Transfect EGFP-GEF-H1
HeLa
-tubulin
Coiled microtubule bondles
To determine whether the nucleotide exchange activity of GEF-H1 To determine whether the nucleotide exchange activity of GEF-H1
was required for its effects on cell morphology.was required for its effects on cell morphology.
Using site-directed mutagenesis to generate Y393A GEF-H1.( in the conserved QRITKY sequence in the Dbl homology domain )
The substitutiion completely abolished nucleotide
exchange activity in vitro. (data not shown)
Y393A
This observation suggests that the morphological effects
of GEF-H1 were mediated by activation of the Rho family
GTPases.
To identify specific Rho GTPase responsible for the cytoskeletal
effects of GEF-H1 expression.
Cotransfected GEF-H1 & p21-binding domains (PBD) of
Rhotekin ( and effector of Rho ) RBD
Pak ( and effector of Rac and Cdc42 ) PBDor
( The Rhotekin RBD and Pak PBD can bind to
active Rho GTPase and specifically inhibit
Rho or Rac/Cdc42-dependent pathway )
The effects of truncated GEF-H1 constructs on cell morphology rely on the activation of a Rho-dependent pathway.
In vitro nuceotide exchange assayIn vitro nuceotide exchange assay
HA-tagged GEF-H1 were expressed in Cos-1 cells.
IP
+ [35S]GTP-S
Rac1, Cdc42. RhoA
To address the apparent discrepancy between the fact that all GEF-H1
Constructs have similar activity in vitro and that only non-microtubule-bound
GEF-H1 constructs induced a Rho-dependent change in cell morphology.
To analyse the ability of various GEF-H1 versions to
activate Rho GTPase in vivo.
RBD/PBD pull-down assays
HeLa cells
contransfect GEF-H1 and Myc-RhoA
GTP-boune RhoA was precipitated with GST-RBD
Western -myc
To perform more precise measurements of Rho GTPase activation
Using a reporter gene assay that relies on the ability of Rho to activate the transcription of reporter genes fused to the SRE promoter element.
The ability of mutant GER-H1 constructs to induce morphological changes is functionlly connected to the higher exchange activity of these proteins, demonstrated by SRE reporter gene activation and the RBD pull-down assay in vivo.
Full length GEF-H1GEF-H1 (1-894)
Mutant version of GEF-H1
Guanine nucleotide exchange activity Less active highly active
Microtubule localization
Yes lack
Microtubule association has an inhibitory effect on GEF-H1 activity ?
Disruption of microtubules
Induce activation of GEF-H1 ??
GEF-H1 localized to microtubules.
non-microtubule-bound GEF-H1 constructs
--- with highly active guanine nucleotide exchante activity
--- induced a Rho-dependent change in cell morphology.
A model for the regulation of GEF-H1 activity by microtubules.
If you still have any questions, If you still have any questions,
please do not hesitate to contact please do not hesitate to contact
me!! me!!
Lab: LS303Lab: LS303
Email: Email: [email protected]
TEL: 2905-3595 TEL: 2905-3595