1. 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.
2.
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.
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3.
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.
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4.
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.
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5.
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.
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6.
Low magnification view of aggregation.
Firtel lab.
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7.
High magnification view of an aggregation stream.
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8.
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.
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9.
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.
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10.
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.
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11.
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.
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12.
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.
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13.
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.
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14.
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.
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