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PIP₃-dependent macropinocytosis is incompatible with chemotaxis.

Veltman DM, Lemieux MG, Knecht DA, Insall RH - J. Cell Biol. (2014)

Bottom Line: In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear.Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate.Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases).

View Article: PubMed Central - HTML - PubMed

Affiliation: Beatson Institute for Cancer Research, Glasgow G61 1BD, Scotland, UK.

ABSTRACT
In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.

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PIP3 patches organize macropinosomes. (A and B) Confocal series showing turnover of PIP3 patches in NC4 and axenically grown AX2 cells expressing the PIP3 marker PH-CRAC-GFP. Arrowheads mark budding. Bar, 10 µm. (C) Confocal series showing protrusions in an axenically cultivated AX2 cell expressing PH-CRAC-GFP and the F-actin marker Lifeact-mRFP. Arrows indicate negative curvature. Bar, 5 µm. This corresponds with Video 1. (D) Kymograph of a line drawn through the protrusion in C.
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fig3: PIP3 patches organize macropinosomes. (A and B) Confocal series showing turnover of PIP3 patches in NC4 and axenically grown AX2 cells expressing the PIP3 marker PH-CRAC-GFP. Arrowheads mark budding. Bar, 10 µm. (C) Confocal series showing protrusions in an axenically cultivated AX2 cell expressing PH-CRAC-GFP and the F-actin marker Lifeact-mRFP. Arrows indicate negative curvature. Bar, 5 µm. This corresponds with Video 1. (D) Kymograph of a line drawn through the protrusion in C.

Mentions: In wild-type cells, macropinocytosis could readily be visualized with the PIP3 marker PH-CRAC-GFP (Fig. 3 A), despite the substantially lower rate of fluid uptake. The PIP3 is strictly confined to the nascent vesicle; levels increase during invagination and reach a maximum as the vesicle closes. The entire process is very fast, with a mean time of 8 s between appearance of PIP3 and budding of the vesicle (n = 27, SD = 4 s). Macropinosomes in wild-type cells are small and do not contribute to cell movement, and are readily distinguishable from the large, convex, and spiky F-actin pseudopods.


PIP₃-dependent macropinocytosis is incompatible with chemotaxis.

Veltman DM, Lemieux MG, Knecht DA, Insall RH - J. Cell Biol. (2014)

PIP3 patches organize macropinosomes. (A and B) Confocal series showing turnover of PIP3 patches in NC4 and axenically grown AX2 cells expressing the PIP3 marker PH-CRAC-GFP. Arrowheads mark budding. Bar, 10 µm. (C) Confocal series showing protrusions in an axenically cultivated AX2 cell expressing PH-CRAC-GFP and the F-actin marker Lifeact-mRFP. Arrows indicate negative curvature. Bar, 5 µm. This corresponds with Video 1. (D) Kymograph of a line drawn through the protrusion in C.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926956&req=5

fig3: PIP3 patches organize macropinosomes. (A and B) Confocal series showing turnover of PIP3 patches in NC4 and axenically grown AX2 cells expressing the PIP3 marker PH-CRAC-GFP. Arrowheads mark budding. Bar, 10 µm. (C) Confocal series showing protrusions in an axenically cultivated AX2 cell expressing PH-CRAC-GFP and the F-actin marker Lifeact-mRFP. Arrows indicate negative curvature. Bar, 5 µm. This corresponds with Video 1. (D) Kymograph of a line drawn through the protrusion in C.
Mentions: In wild-type cells, macropinocytosis could readily be visualized with the PIP3 marker PH-CRAC-GFP (Fig. 3 A), despite the substantially lower rate of fluid uptake. The PIP3 is strictly confined to the nascent vesicle; levels increase during invagination and reach a maximum as the vesicle closes. The entire process is very fast, with a mean time of 8 s between appearance of PIP3 and budding of the vesicle (n = 27, SD = 4 s). Macropinosomes in wild-type cells are small and do not contribute to cell movement, and are readily distinguishable from the large, convex, and spiky F-actin pseudopods.

Bottom Line: In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear.Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate.Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases).

View Article: PubMed Central - HTML - PubMed

Affiliation: Beatson Institute for Cancer Research, Glasgow G61 1BD, Scotland, UK.

ABSTRACT
In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.

Show MeSH
Related in: MedlinePlus