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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.


Intracellular degradation of proteins occurs normally in NF1 mutants.To image degradation of internalized protein, cells of strains Ax2, DdB, and HM1591 were incubated in Loflo medium plus 50 µg/ml DQ Green BSA, either (A) for 60 min for cells taken directly from bacterial growth, or (B) for 15 min for cells incubated for 24 hr in loflo medium before DQ Green BSA was added; cells were imaged for green fluorescence of degraded peptides using the same laser power and gain in each case. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.015
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fig3s3: Intracellular degradation of proteins occurs normally in NF1 mutants.To image degradation of internalized protein, cells of strains Ax2, DdB, and HM1591 were incubated in Loflo medium plus 50 µg/ml DQ Green BSA, either (A) for 60 min for cells taken directly from bacterial growth, or (B) for 15 min for cells incubated for 24 hr in loflo medium before DQ Green BSA was added; cells were imaged for green fluorescence of degraded peptides using the same laser power and gain in each case. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.015

Mentions: Despite taking in a similar amount of fluid, the NF1 knockout mutant grows more slowly than Ax2 (see above). We examined whether the mutant processes ingested medium effectively by incubating cells with BODIPY-labelled bovine serum albumin (DQ-BSA), which becomes fluorescent only after lysosomal degradation releases fluorophores that previously quenched each other. Ax2 and the NF1 strain rapidly and comparably degrade protein after internalization by macropinocytosis and maturation of endosomes, (Figure 3—figure supplement 3). Wildtype DdB cells effectively degrade DQ-BSA when taken freshly from bacterial growth but not after overnight incubation in axenic conditions (Figure 3—figure supplement 3), again suggesting that they shut down endocytic feeding as part of a starvation response.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

Intracellular degradation of proteins occurs normally in NF1 mutants.To image degradation of internalized protein, cells of strains Ax2, DdB, and HM1591 were incubated in Loflo medium plus 50 µg/ml DQ Green BSA, either (A) for 60 min for cells taken directly from bacterial growth, or (B) for 15 min for cells incubated for 24 hr in loflo medium before DQ Green BSA was added; cells were imaged for green fluorescence of degraded peptides using the same laser power and gain in each case. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.015
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374526&req=5

fig3s3: Intracellular degradation of proteins occurs normally in NF1 mutants.To image degradation of internalized protein, cells of strains Ax2, DdB, and HM1591 were incubated in Loflo medium plus 50 µg/ml DQ Green BSA, either (A) for 60 min for cells taken directly from bacterial growth, or (B) for 15 min for cells incubated for 24 hr in loflo medium before DQ Green BSA was added; cells were imaged for green fluorescence of degraded peptides using the same laser power and gain in each case. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.015
Mentions: Despite taking in a similar amount of fluid, the NF1 knockout mutant grows more slowly than Ax2 (see above). We examined whether the mutant processes ingested medium effectively by incubating cells with BODIPY-labelled bovine serum albumin (DQ-BSA), which becomes fluorescent only after lysosomal degradation releases fluorophores that previously quenched each other. Ax2 and the NF1 strain rapidly and comparably degrade protein after internalization by macropinocytosis and maturation of endosomes, (Figure 3—figure supplement 3). Wildtype DdB cells effectively degrade DQ-BSA when taken freshly from bacterial growth but not after overnight incubation in axenic conditions (Figure 3—figure supplement 3), again suggesting that they shut down endocytic feeding as part of a starvation response.

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.