1. Chemotaxis of Eukaryotic Cells: 1.Dictyostelium discoideum http://dictybase.org/Multimedia/index.html http://dictybase.org/Multimedia/development/development.html

2. Lodish, Darnell and Baltimore Molecular Biology of the Cell Life Cycle of Dictyostelium discoideum

3. 1. At high density and low nutrient availability, a random cell begins to release cAMP. 2. Adjacent cells chemotaxis up the cAMP gradient and the phosphodiesterase they release degrades the cAMP. 3. After a lag, the chemotaxing cells release their own cAMP. This creates a a second, greater cAMP gradient. 4. The cycle continues as the next set of cells chemotax toward the center of mass of cells and in turn release cAMP after a lag, recruiting yet more cells

4. Phosphoinositide 3-kinase (PI3K) is an enzyme found in all multicellular eukaryotic organisms. In response to cell stimulation it is activated and places a phosphate at the 3 position of the inositol ring of phosphatidylinositol-4,5- bisphosphate (PIP 2 ) to generate phosphatidylinositol-3,4,5-trisphosphate (PI- 3,4,5-P 3 or PIP 3 ). PIP 2 is at high concentrations at the plasma membrane of all eukaryotic cells and only a small fraction of this lipid is converted to PI-3,4,5- P 3 in response to cell stimulation. PI PI-4-P PI-4,5-P 2 PI-3,4,5-P 3 PI4KPI4P5K P O O O O O OH O O OC O C O Fatty Acid 1 2 3 4 5 6 P O O O P O O O P O O O O PI-3,4,5-P 3 PI3K

5. Receptor Outside Cytosol GG G  ? PIP 2 PI3K PIP 2 PIP 3 PH Recruitment of cortical actin to drive cell polarization and motility cAMP G  GG ? PIP 3 Adenylyl Cyclase cAMP (secreted) PTEN PIP 2 ??

6. A green fluorescent protein chimera of a pleckstrin homology (PH) domain that binds to the lipid phosphatidylinositol-3,4,5- trisphosphate (PIP 3 ) reveals that this lipid is generated at the leading edge of the cell in a dynamic fashion that rapidly responds to changes in the extracellular cAMP gradient. (from Richard Firtel)

7. A mutation in the PH domain that prevents binding to PI-3,4,5-P 3 prevents recruitment to the membrane (Richard Firtel)

8. From Richard Firtel

9. Local Excitation, Global Inhibition (LEGI) Model Ma et al., 2004 Biophysical J. 87, 3764 Assumptions: PI3K LEGI 1.Receptor occupation rapidly stimulates a local, membrane imbedded (slowly diffusable) component that activates a membrane imbedded PI3K binding protein. 2.This recruits PI3K from the cytosol to the membrane and results in local production of PIP 3. 3.Receptor occupation also stimulates a cytosolic component that diffuses throughout the cell and globally inactivates the membrane imbedded PI3K binding protein. 4.The activation of the global inhibitor is slower than activation of the local activator. Thus, PI3K is concentrated near activated receptors

10. Local Excitation, Global Inhibition (LEGI) Model Ma et al., 2004 Biophysical J. 87, 3764 Assumptions: PTEN Regulation 1.Receptor occupation rapidly stimulates a local, membrane imbedded (slowly diffusable) component that inactivates a membrane imbedded PTEN binding protein. 2.This locally releases PTEN from the membrane and allows local production of PIP 3. 3.Receptor occupation also stimulates a cytosolic component that diffuses throughout the cell and globally activates the membrane imbedded PTEN binding protein. 4.The activation of the global PTEN regulator is slower than activation of the local regulator. Thus, PTEN levels are reduced near activated receptors and elevated elsewhere. Note: PTEN regulation and PI3K regulation are assumed to be uncoupled

11. BS BSA RL S L PI3K BSA IIA EEA PI3K BS BSA PI3K IA Cytosolic PI3K, I and IA are freely diffusable throughout the cytosol. Membrane imbedded components (R, S, E, BS) have more restricted movement Membrane PI3K half of the LEGI model

12. Virtual Cell Model Ma et al. (Devreotes & Iglesias) Using Lowe and Schaff, 2001, Trends Biotechnol. 19, 401; Virtual Cell http://www.nrcam.uchc.edu/applications/applications.html

13. Virtual Cell Model Ma et al. (Devreotes & Iglesias)

16. The PIP 3 gradient depends on the cAMP gradient but is relatively insensitive to the absolute amount of cAMP, allowing adaptation to higher basal cAMP Virtual Cell Model Ma et al. (Devreotes & Iglesias)