Limits...
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.

Show MeSH

Related in: MedlinePlus

SCAR, actin, and PIP3 in pseudopods and macropinosomes. (A) TIRF and DIC image of HSPC300-GFP during progression of a normal pseudopod of axenic AX3 cells. Cells were overlayed with 0.4% agarose to ensure contact with the glass surface. (B) Confocal series of an axenically grown AX2 cell expressing PH-CRAC-RFP and the SCAR complex marker HSPC300-GFP. Arrowheads show regions marked by HSPC300-GFP. Asterisk indicates a PIP3 patch that does not form a macropinosome. The boxed regions are shown in detail (2.2× magnification) in the bottom row. This corresponds with Video 2. (C) TIRF image of an axenically cultivated AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP. (D) Profile plot of the line indicated in C. (E) Confocal image series of an axenic AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP during macropinocytosis. Colored dots indicate the edges (red, purple) and center (green) of the PIP3 patch. (F) Confocal image of an axenic AX2 cell expressing Lifeact-mRFP and PH-CRAC-GFP with a pseudopod (top left) and a macropinosome (top right). (G) Profile plot of a line drawn along the perimeter of the developing macropinosome in F. All indicated times are in seconds. Bars: (A, B, C, and F) 10 µm; (E) 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: SCAR, actin, and PIP3 in pseudopods and macropinosomes. (A) TIRF and DIC image of HSPC300-GFP during progression of a normal pseudopod of axenic AX3 cells. Cells were overlayed with 0.4% agarose to ensure contact with the glass surface. (B) Confocal series of an axenically grown AX2 cell expressing PH-CRAC-RFP and the SCAR complex marker HSPC300-GFP. Arrowheads show regions marked by HSPC300-GFP. Asterisk indicates a PIP3 patch that does not form a macropinosome. The boxed regions are shown in detail (2.2× magnification) in the bottom row. This corresponds with Video 2. (C) TIRF image of an axenically cultivated AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP. (D) Profile plot of the line indicated in C. (E) Confocal image series of an axenic AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP during macropinocytosis. Colored dots indicate the edges (red, purple) and center (green) of the PIP3 patch. (F) Confocal image of an axenic AX2 cell expressing Lifeact-mRFP and PH-CRAC-GFP with a pseudopod (top left) and a macropinosome (top right). (G) Profile plot of a line drawn along the perimeter of the developing macropinosome in F. All indicated times are in seconds. Bars: (A, B, C, and F) 10 µm; (E) 2 µm.

Mentions: The ambiguous character of these actin structures in axenic cells complicates analysis. To more accurately distinguish the potential role of PIP3 in pseudopods and macropinosomes, we examined the behavior of the SCAR/WASP and verprolin homologue (WAVE) complex, which is the definitive marker for pseudopods. It remains associated with the leading edge throughout the progression of a pseudopod and disappears from the leading edge when the pseudopod stops advancing (Fig. 4 A; Hahne et al., 2001; Veltman et al., 2012). Interestingly, high concentrations of PIP3 do not colocalize with the SCAR complex, which is recruited to a small margin surrounding each patch (Fig. 4, B–D; and Video 2). This distribution is not only visible in the PIP3 patches that develop to form macropinosomes, but also in ones that remain planar and that cannot readily be classified as either a pseudopod or a nascent macropinosome (Video 1 and Fig. 4 B, asterisk). We were unable to find any event where a PIP3 patch was followed by recruitment of SCAR to the leading edge in the fashion that is typical for pseudopods.


PIP₃-dependent macropinocytosis is incompatible with chemotaxis.

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

SCAR, actin, and PIP3 in pseudopods and macropinosomes. (A) TIRF and DIC image of HSPC300-GFP during progression of a normal pseudopod of axenic AX3 cells. Cells were overlayed with 0.4% agarose to ensure contact with the glass surface. (B) Confocal series of an axenically grown AX2 cell expressing PH-CRAC-RFP and the SCAR complex marker HSPC300-GFP. Arrowheads show regions marked by HSPC300-GFP. Asterisk indicates a PIP3 patch that does not form a macropinosome. The boxed regions are shown in detail (2.2× magnification) in the bottom row. This corresponds with Video 2. (C) TIRF image of an axenically cultivated AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP. (D) Profile plot of the line indicated in C. (E) Confocal image series of an axenic AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP during macropinocytosis. Colored dots indicate the edges (red, purple) and center (green) of the PIP3 patch. (F) Confocal image of an axenic AX2 cell expressing Lifeact-mRFP and PH-CRAC-GFP with a pseudopod (top left) and a macropinosome (top right). (G) Profile plot of a line drawn along the perimeter of the developing macropinosome in F. All indicated times are in seconds. Bars: (A, B, C, and F) 10 µm; (E) 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: SCAR, actin, and PIP3 in pseudopods and macropinosomes. (A) TIRF and DIC image of HSPC300-GFP during progression of a normal pseudopod of axenic AX3 cells. Cells were overlayed with 0.4% agarose to ensure contact with the glass surface. (B) Confocal series of an axenically grown AX2 cell expressing PH-CRAC-RFP and the SCAR complex marker HSPC300-GFP. Arrowheads show regions marked by HSPC300-GFP. Asterisk indicates a PIP3 patch that does not form a macropinosome. The boxed regions are shown in detail (2.2× magnification) in the bottom row. This corresponds with Video 2. (C) TIRF image of an axenically cultivated AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP. (D) Profile plot of the line indicated in C. (E) Confocal image series of an axenic AX2 cell expressing PH-CRAC-RFP and HSPC300-GFP during macropinocytosis. Colored dots indicate the edges (red, purple) and center (green) of the PIP3 patch. (F) Confocal image of an axenic AX2 cell expressing Lifeact-mRFP and PH-CRAC-GFP with a pseudopod (top left) and a macropinosome (top right). (G) Profile plot of a line drawn along the perimeter of the developing macropinosome in F. All indicated times are in seconds. Bars: (A, B, C, and F) 10 µm; (E) 2 µm.
Mentions: The ambiguous character of these actin structures in axenic cells complicates analysis. To more accurately distinguish the potential role of PIP3 in pseudopods and macropinosomes, we examined the behavior of the SCAR/WASP and verprolin homologue (WAVE) complex, which is the definitive marker for pseudopods. It remains associated with the leading edge throughout the progression of a pseudopod and disappears from the leading edge when the pseudopod stops advancing (Fig. 4 A; Hahne et al., 2001; Veltman et al., 2012). Interestingly, high concentrations of PIP3 do not colocalize with the SCAR complex, which is recruited to a small margin surrounding each patch (Fig. 4, B–D; and Video 2). This distribution is not only visible in the PIP3 patches that develop to form macropinosomes, but also in ones that remain planar and that cannot readily be classified as either a pseudopod or a nascent macropinosome (Video 1 and Fig. 4 B, asterisk). We were unable to find any event where a PIP3 patch was followed by recruitment of SCAR to the leading edge in the fashion that is typical for 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