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Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

Bloomfield G, Traynor D, Sander SP, Veltman DM, Pachebat JA, Kay RR - Elife (2015)

Bottom Line: Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis.An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling.Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

No MeSH data available.


Related in: MedlinePlus

Phagocytosis is accompanied by Ras and PI3K activity in the same way as in macropinocytosis.NF1 mutants (HM1591) were transformed with an expression construct containing both mCherry-Raf1-RBD and PH(CRAC)-GFP and imaged in the presence of Klebsiella cells; the initial engulfment occurred out of the plane of acquisition, but Ras and PI3K activity remained visible as the nascent phagosome moved into view. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.030
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fig7s3: Phagocytosis is accompanied by Ras and PI3K activity in the same way as in macropinocytosis.NF1 mutants (HM1591) were transformed with an expression construct containing both mCherry-Raf1-RBD and PH(CRAC)-GFP and imaged in the presence of Klebsiella cells; the initial engulfment occurred out of the plane of acquisition, but Ras and PI3K activity remained visible as the nascent phagosome moved into view. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.030

Mentions: Finally, since macropinocytosis and phagocytosis are closely related processes we compared phagocytosis in NF1 mutants and wildtype cells. Mutant and wildtype strains grow well on bacteria (Figure 7—figure supplements 1, 2) and take up bacterium-sized polystyrene microspheres (1 μm and 1.8 μm diameter) at very similar rates (Figure 7A), although the standard strain Ax2 is marginally but consistently less effective at internalising smaller beads than the other strains (Figure 7B). Against expectation, we found that wildtype cells cannot efficiently ingest yeast or beads greater than 3 μm in diameter (Figure 7A,C), whereas NF1 mutant cells can ingest beads larger than 4 μm in diameter (Figure 7A) or yeast cells very readily (Figure 7C). In line with earlier findings in Ax2 cells (Clarke et al., 2010), RBD and PH domain reporters localised to phagosomes as they formed, essentially identically to their behaviour during macropinocytosis (Figure 7—figure supplement 3). We conclude that, as well as controlling macropinocytosis, NF1 limits the size of nascent phagosomes, supporting the idea that these large-scale endocytic processes share regulatory as well as structural features. The striking improvement in phagocytosis of larger cells after NF1 deletion also suggests that variation in or loss of this gene can have important ecological and evolutionary consequences by enabling predators to target additional prey species (Porter, 2011).10.7554/eLife.04940.027Figure 7.NF1 mutants can phagocytose larger particles than wildtypes.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

Bloomfield G, Traynor D, Sander SP, Veltman DM, Pachebat JA, Kay RR - Elife (2015)

Phagocytosis is accompanied by Ras and PI3K activity in the same way as in macropinocytosis.NF1 mutants (HM1591) were transformed with an expression construct containing both mCherry-Raf1-RBD and PH(CRAC)-GFP and imaged in the presence of Klebsiella cells; the initial engulfment occurred out of the plane of acquisition, but Ras and PI3K activity remained visible as the nascent phagosome moved into view. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.030
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374526&req=5

fig7s3: Phagocytosis is accompanied by Ras and PI3K activity in the same way as in macropinocytosis.NF1 mutants (HM1591) were transformed with an expression construct containing both mCherry-Raf1-RBD and PH(CRAC)-GFP and imaged in the presence of Klebsiella cells; the initial engulfment occurred out of the plane of acquisition, but Ras and PI3K activity remained visible as the nascent phagosome moved into view. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.030
Mentions: Finally, since macropinocytosis and phagocytosis are closely related processes we compared phagocytosis in NF1 mutants and wildtype cells. Mutant and wildtype strains grow well on bacteria (Figure 7—figure supplements 1, 2) and take up bacterium-sized polystyrene microspheres (1 μm and 1.8 μm diameter) at very similar rates (Figure 7A), although the standard strain Ax2 is marginally but consistently less effective at internalising smaller beads than the other strains (Figure 7B). Against expectation, we found that wildtype cells cannot efficiently ingest yeast or beads greater than 3 μm in diameter (Figure 7A,C), whereas NF1 mutant cells can ingest beads larger than 4 μm in diameter (Figure 7A) or yeast cells very readily (Figure 7C). In line with earlier findings in Ax2 cells (Clarke et al., 2010), RBD and PH domain reporters localised to phagosomes as they formed, essentially identically to their behaviour during macropinocytosis (Figure 7—figure supplement 3). We conclude that, as well as controlling macropinocytosis, NF1 limits the size of nascent phagosomes, supporting the idea that these large-scale endocytic processes share regulatory as well as structural features. The striking improvement in phagocytosis of larger cells after NF1 deletion also suggests that variation in or loss of this gene can have important ecological and evolutionary consequences by enabling predators to target additional prey species (Porter, 2011).10.7554/eLife.04940.027Figure 7.NF1 mutants can phagocytose larger particles than wildtypes.

Bottom Line: Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis.An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling.Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

No MeSH data available.


Related in: MedlinePlus