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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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Folate chemotaxis is inefficient in axenic cells. Cells were grown under different conditions, then examined responding to linear attractant gradients in Insall chambers. Numbers are the means ± SEM of at least four independent experiments of at least 20 cells each. Tracks of cells from the same experiment have the same color. (A) Axenically cultivated AX2 cells responding to folate (left) and 4 h–starved cells responding to cAMP (right). (B) Bacterially grown NC4 cells (the parent of AX2) migrating toward folate. (C) Bacterially grown AX2 cells migrating toward folate.
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fig1: Folate chemotaxis is inefficient in axenic cells. Cells were grown under different conditions, then examined responding to linear attractant gradients in Insall chambers. Numbers are the means ± SEM of at least four independent experiments of at least 20 cells each. Tracks of cells from the same experiment have the same color. (A) Axenically cultivated AX2 cells responding to folate (left) and 4 h–starved cells responding to cAMP (right). (B) Bacterially grown NC4 cells (the parent of AX2) migrating toward folate. (C) Bacterially grown AX2 cells migrating toward folate.

Mentions: The main reason for studying chemotaxis in model organisms like D. discoideum is to find simple but generalizable results. It is therefore desirable to study multiple attractants to separate global from agonist-specific mechanisms. We have therefore studied chemotaxis toward folate. Compared with the well-studied cAMP system, folate uses different receptors and G protein α subunits (Srinivasan et al., 2013), which are expressed in growing cells that are not responsive to cAMP. However it has been difficult to measure folate chemotaxis with most assays (though under-agar and some micropipette assays succeed). To discover why, we exposed AX2 cells to folate gradients in a standard chemotaxis chamber (Muinonen-Martin et al., 2010). Under these conditions, starved cells chemotax efficiently toward cAMP. However, growing cells consistently failed to migrate up the folate gradient (Fig. 1 A). This was surprising, as folate is thought to be a potent attractant. To analyze the problem, we switched to using wild-type NC4, the parent strain of AX2, which does not have axenic mutations and must thus be grown on bacteria. In contrast to axenic cells, wild-type cells robustly migrated up the folate gradient (Fig. 1 B). AX2 cells grown on bacteria also show a little chemotaxis (Fig. 1 C), but still far less than NC4, which indicates that the loss of folate chemotaxis is caused by genetic differences between the wild type and the axenic AX2 strain.


PIP₃-dependent macropinocytosis is incompatible with chemotaxis.

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

Folate chemotaxis is inefficient in axenic cells. Cells were grown under different conditions, then examined responding to linear attractant gradients in Insall chambers. Numbers are the means ± SEM of at least four independent experiments of at least 20 cells each. Tracks of cells from the same experiment have the same color. (A) Axenically cultivated AX2 cells responding to folate (left) and 4 h–starved cells responding to cAMP (right). (B) Bacterially grown NC4 cells (the parent of AX2) migrating toward folate. (C) Bacterially grown AX2 cells migrating toward folate.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Folate chemotaxis is inefficient in axenic cells. Cells were grown under different conditions, then examined responding to linear attractant gradients in Insall chambers. Numbers are the means ± SEM of at least four independent experiments of at least 20 cells each. Tracks of cells from the same experiment have the same color. (A) Axenically cultivated AX2 cells responding to folate (left) and 4 h–starved cells responding to cAMP (right). (B) Bacterially grown NC4 cells (the parent of AX2) migrating toward folate. (C) Bacterially grown AX2 cells migrating toward folate.
Mentions: The main reason for studying chemotaxis in model organisms like D. discoideum is to find simple but generalizable results. It is therefore desirable to study multiple attractants to separate global from agonist-specific mechanisms. We have therefore studied chemotaxis toward folate. Compared with the well-studied cAMP system, folate uses different receptors and G protein α subunits (Srinivasan et al., 2013), which are expressed in growing cells that are not responsive to cAMP. However it has been difficult to measure folate chemotaxis with most assays (though under-agar and some micropipette assays succeed). To discover why, we exposed AX2 cells to folate gradients in a standard chemotaxis chamber (Muinonen-Martin et al., 2010). Under these conditions, starved cells chemotax efficiently toward cAMP. However, growing cells consistently failed to migrate up the folate gradient (Fig. 1 A). This was surprising, as folate is thought to be a potent attractant. To analyze the problem, we switched to using wild-type NC4, the parent strain of AX2, which does not have axenic mutations and must thus be grown on bacteria. In contrast to axenic cells, wild-type cells robustly migrated up the folate gradient (Fig. 1 B). AX2 cells grown on bacteria also show a little chemotaxis (Fig. 1 C), but still far less than NC4, which indicates that the loss of folate chemotaxis is caused by genetic differences between the wild type and the axenic AX2 strain.

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