RhoRacCdc42

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Rho/Rac/Cdc42

• Dev Bio 231B / Bio Sci D154 (Winter 2008)
• Yi Lecture 3

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System Overview (Arrow Diagram)
FRONT
BACK
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Outline (Yi Lecture 3)

• Ras superfamily and Rho family
• Regulation of actin cytoskeleton and lipid
  signaling by Rho family
• RhoA protein and the uropod
• Readings
• Xu et al. paper (Figs. 1, 2 and 3)
• Background reading (optional)
• Hall Review on Rho family

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Small G-Protein (Ras) Superfamily(Monomeric)

• Ras
• Signal transduction (e.g. Ras activates Raf
  kinase which activates MAPK signaling)
• Rho
• Regulation of cytoskeleton (e.g. Cdc42)
• Rab
• Intracellular vesicular transport (i.e. protein
  trafficking)
• Arf
• Vesicle coating
• Ran
• Nucleocytoplasmic transport

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Small G-Protein Cycle
Active
GEF
GAP
Inactive
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More Detailed View of G-Protein Cycle
Guanine Dissociation Inhibitor (stabilizes GDP
form)
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Rho Family



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Rho Family Small G-Proteins Regulate Actin
Organization
Stress fibers contractile bundles of actin and
mysoin II
Lamellipodia 2D sheet-like projections
Filopodia 1D projections
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Rho/Rac/Cdc42 Stimulate Actin Nucleation
Branched
Cables
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Rho/Rac/Cdc42 Regulate Lipid Signaling

• Lipids

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Coordination between Actin Cytoskeleton and Lipids

• Rac/Cdc42 promote formation of PIPs as well as
  activating WAVE/WASP which activates Arp2/3
• Many proteins (e.g. profilin) bind both PIPs and
  actin
• Increases in PIPs leads to enhanced actin
  polymerization
• Actin stabilizes distribution of PIPs
• Actin and PIPs contribute to the localization of
  Rac/Cdc42

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Positive Feedback at Leading Edge
Actin
PIPs
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Rho GEFs
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Rho Effectors

• Rho kinase (ROCK) is a principal effector of Rho
• Rho induces the assembly of contractile
  actin-myosin filaments via ROCK
• ROCK phosphorylates and inactivates myosin light
  chain (MLC) phosphatase which leads to increased
  phosphorylation of MLC promoting actin
  cross-linking by myosin II
• Rho inhibits Rac via ROCK
• ROCK phosphorylates and activates FilGAP (a Rac
  GAP)
• Rac inhibits Rho by activation of p190RhoGAP

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Tools for Studying Rho Function

• RhoA-GTP pulldown using GST-RBD (Rho Binding
  Domain)
• RhoA-Q63L constituitively active mutant
• RhoA-T19N dominant negative mutant
• Y-27632 ROCK inhibitor
• PBD-GFP fluorescent probe for Rac-GTP and
  Cdc42-GTP

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Figs 1B 1D
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Uniform Distribution Versus Gradient

• How do neutrophils respond when fMLP is applied
  in a spatially uniform manner?
• Do they move?
• They polarize and move in random directions

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Figure 2
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Figure 3
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Lecture 3 Question to Ponder
2. Neutrophil HL-60 cells are transfected with
the PH-Akt-GFP reporter. One of these cells is
positioned between 2 micropipettes containing
equal concentrations of fMLP. The two
micropipettes are simultaneously turned on
emitting fMLP. DRAW (i.e fill-in) in the cell
where you expect to see the PH-Akt-GFP signal
under the following conditions (1 point
each)   A) No treatment control (fMLP)
B) Treatment with the Rho
kinase inhibitor Y-27632
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Summary (Yi Lecture 3)

• Rho family of monomeric G-proteins
• Rho, Rac, Cdc42
• Actin polymerization and lipid signaling at
  leading edge
• Regulated by Rac and Cdc42
• Rho activation inhibits the pseudopod and
  promotes the uropod