1. GFP aggregation and mound formation
Time-lapse video movie of aggregation and mound formation of wild-type cells. 5% of the cells are expressing GFP to enable visualization by fluoresence microscopy. R. Meili, Firtel lab.
2. cAMP waves during aggregation
Time-lapse video movie of cAMP waves during aggregation. Cells are plated on a monolayer on thin agar and viewed by phase contrast microscopy (4X objective). The movie shows cells from approximately 2 hours to 7 hours after plating. S. Funamoto, Firtel lab.
3. Chemotaxis of a population of Dictyostelium cells to cAMP
Chemotaxis of Dictyostelium cells to a micropipette emitting the chemoattractant cAMP. The movie is taken by time-lapse video microscopy using DIC optics and a 20X objective. The time frame of the movie is approximately 20 minutes. Images were taken every 6 seconds. S. Lee, Firtel lab.
4. Chemotaxis of a single cell to the chemoattractant cAMP
Time-lapse video microscopy (DIC optics, 60X objective) of a single cell moving toward a micropipette containing the chemoattractant cAMP. Note that the cell changes direction in response to movement of the micropipette by extending a new pseudopod in the direction of the pipette tip. S. Lee, Firtel lab.
5. Low magnification view of aggregation
6. High magnification view of an aggregation stream
7. Translocation of the PH domain from Akt/PKB to the plasma membrane in response to uniform stimulation
Analysis of the translocation of the PH domain from the serine/threonine kinase Akt/PKB fused to GFP to the plasma membrane in response to uniformly stimulating the cells with the chemoattractant cAMP. Images were taken every 2.6 seconds. Maximal membrane localization occurs approximately 6 seconds after stimulation. The protein is rapidly lost from the membrane and is no longer detected on the membrane by 12 seconds. Akt/PKB is also activated in response to chemoattractant stimulation. Maximal enzyme activity is approximately 10-15 seconds after stimulation. R. Meili, Firtel lab.
8. Localization of the Akt/PKB PH domain to the leading edge of chemotaxing cells
Time-lapse video microscopy of the PH domain from the serine/threonine kinase Akt/PKB fused to GFP in cells chemotaxing to cAMP. The position of the micropipette is in the upper left corner of the frame (not visible). Note localization of the PH domain to the leading edge of many of the cells. R. Meili and C. Ellsworth, Firtel lab.
9. Localization of a PH domain-containing protein (phdA) to the plasma membrane in response to changes in the direction of the cAMP source
Time-lapse video movie of a GFP fusion of a novel PH domain-containing protein (phdA), which is required for proper chemotaxis, to the plasma membrane in response to directional changes in the cAMP source. The position of the micropipette is indicated by a star. Note that as the position of the micropipette is moved, the GFP fusion localizes to a position in the plasma membrane closest to the cAMP source and is lost from the membrane at its previous site. In some cases, a new pseudopod extends shortly after membrane localization of the protein and retracts as the micropipette containing cAMP is moved. S. Funamoto, Firtel lab.
10. Translocation of wild-type and mutant phdA to the plasma membrane in response to uniform stimulation
Time-lapse video movie of a GFP fusion of a novel PH domain-containing protein (phdA) to the plasma membrane in response to uniformly stimulating the cells with the chemoattractant cAMP. The wild-type protein is shown on the right. The kinetics of membrane localization are similar to those of the Akt/PKB PH domain. The right-hand images show a mutant phdA protein in which an Arg (R) residue proposed to be required for PH domain binding to PI(3,4,5)P3 is mutated to a Cys (C). Note that this protein does not translocate to the plasma membrane. The time of cAMP stimulation is marked by +cAMP. S. Funamoto, Firtel lab.
11. Localization of F-actin in ARK1 null cells chemotaxing towards cAMP visualized using ABP-GFP
Time-lapse video microscopy of ARK1 null cells expressing a GFP fusion of the F-actin binding domain from ABP120 (a gift from David Knecht, Univ. of Conn.). ARK1, an ankyrin-repeat containing kinase is required for proper chemotaxis. The GFP fusion of the actin binding domain from ABP120 is used to visualize F-actin in vivo. The movie shows the localization of the F-actin in these cells. Note the very broad leading edge and multiple pseudopodia in many of the ARK1 null cells that are not usually seen in wild-type cells. B. Sun, Firtel lab.
12. Chemotaxis of PAKa null cells to cAMP
Time-lapse video microscopy (DIC optics) of PAKa null cells chemotaxing to a micropipette containing cAMP. C. Chung, Firtel lab.
13. Translocation of the PhdA PH-domain-containing protein
Translocation of the PhdA PH-domain-containing protein to the leading edge in response to a directional signal. PhdA is required for proper chemotaxis in Dictyostelium. The movie demonstrates that PhdA rapidly translocates to the leading edge of chemotaxing amoebae in response to a micropipette containing the chemoattractant cAMP. When the micropipette is moved to a new position relative to the cell, the PhdA-GFP is lost from the old site and the old pseudopod rapidly retracts. PhdA now localizes to a position proximal to the new cAMP source and forms a new pseudopod. S. Funamoto, Firtel lab.
14. Co-localized activation of Ras and PI3K at the leading edge.
Co-localized activation of Ras and PI3K at the leading edge. The movie shows the spatial and temporal co-localization of activated Ras (Ras-GTP) and PI3K at the leading edge of wild-type chemotaxing cells. Cells are co-expressing a reporter for activated Ras (GFP-RBD, Ras binding domain) and one (RFP-for PI(3,4,5)P3, the product of PI3K. Green- GFP-RBD; Red, RFP-PHcrac; Yellow, merge of the two images. Contemporaneous image collection was achieved using a beam splitter.
15. Animated model for the role of PI(3) kinase in controlling directionality and polarity during chemotaxis
The animation shows the working model for the role of PI(3) kinase in establishing cell polarity and a leading edge based on work in Dictyostelium and neutrophils. Cells placed in a chemoattractant gradient show a preferential localization of PH domain-containing proteins to the leading edge, including CRAC (Parent et al., Cell 95, 81-91, 1998), Akt/PKB (Meili et al., EMBO J. 18:2092-2105, 1989; Servant et al., Science 287, 1037-1040, 2000), and PhdA (Funamoto et al., J. Cell Biol. 153, 705-809, 2001). This translocation is mediated by the binding of the PH domains to the lipid products of PI(3) kinase [PI(3,4,5)P3 and PI(3,4)P2], which we suggest is preferentially activated at the leading edge. This results in the establishment of cell polarity, F-actin assembly, and myosin contraction, leading to movement up the chemoattractant gradient. We have demonstrated that Akt/PKB and PhdA play specific roles in this process, including the regulation of F-actin assembly and myosin contraction (Meili et al, 1999; Funamoto et al, 2001; Chung et al, Molecular Cell 7, 937-947, 2001). The translocation of the PH domain-containing proteins is absent in PI(3) kinase null cells or in cells treated with the PI(3) kinase inhibitor LY294002. PI(3) kinase null cells or wild-type cells treated with LY294002 exhibit phenotypes similar to those observed in Akt/PKB and PhdA null strains.
Movies of PH domain translocations are available below.
Pdf files of Meili et al., Funamoto et al., and Chung et al., can be found under Firtel lab publications.