Presentation on theme: "Read through the paper several times. Think ! What’s the main question of this paper? How do the authors design the experiments? How do the authors design."— Presentation transcript:
Read through the paper several times. Think ! 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! Rehearsal it with your classmates!!
Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton Krendel M, Zenke FT, Bokoch GM. 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 immune response, wound repair, and tumor formation and metastasis. response, wound repair, and tumor formation and metastasis. --- is powered by the activity of the actin cytoskeleton actin polymerization driving leading edge protrusion acto-myosin contractility promoting cell body advancement
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 drive cell migration in most cell types drive cell migration in most cell types The loss of microtubules prevents directional movement of cells in culture. ( J. Embryol. Exp. Morphol. 24: 625-40. 1970 ) Micriotubules may be involved in the regulation of actin-dependent motility. The rate of lamellipodial protrusion in these cells is decreased Microtubules are necessary to support normal rates of leading edge Microtubules 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 GTP GDP GTP Inactive G protein Active G 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 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 ) PBD or ( 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 assay HA-tagged GEF-H1 were expressed in Cos-1 cells. IP + [ 35 S]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-H1 GEF-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.
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