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（ bio2028@mails.fju.edu.tw ）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%)

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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: bio2028@mails.fju.edu.tw

TEL: 2905-3595 TEL: 2905-3595