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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.


NF1 mutants do not have an increase in overall Ras activity as assessed by confocal microscopy.Using GFP-Raf1-RBD reporter constructs, no increase in plasma-membrane associated active Ras was observed in the axeB : this was quantified from tilescans of cells from three independent experiments. In all cases, to ensure that the cells were in comparable state, they were used within 30 min of harvesting from bacterial growth.DOI:http://dx.doi.org/10.7554/eLife.04940.019
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fig4s3: NF1 mutants do not have an increase in overall Ras activity as assessed by confocal microscopy.Using GFP-Raf1-RBD reporter constructs, no increase in plasma-membrane associated active Ras was observed in the axeB : this was quantified from tilescans of cells from three independent experiments. In all cases, to ensure that the cells were in comparable state, they were used within 30 min of harvesting from bacterial growth.DOI:http://dx.doi.org/10.7554/eLife.04940.019

Mentions: We then asked whether inactivation of NF1 results in a global increase in Ras activity. Pulldowns of Ras-GTP using the Raf1 Ras-binding domain (RBD) from growing cells indicated no increase in Ras activity in mutants compared to wildtype cells (Figure 4—figure supplement 2). Similarly, confocal microscopy of cells expressing the GFP-RBD reporter revealed no difference between mutant and wildtypes in overall Ras activity estimated by determining the proportion the cell periphery labelled with the RBD (Figure 4—figure supplement 3). This is not surprising, since NF1 is only one of twelve putative RasGAPs encoded in the D. discoideum genome (not including IQ-GAPs, which generally act as small G protein effectors and scaffolds and do not stimulate Ras GTPase activity [Shannon, 2012]).


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

NF1 mutants do not have an increase in overall Ras activity as assessed by confocal microscopy.Using GFP-Raf1-RBD reporter constructs, no increase in plasma-membrane associated active Ras was observed in the axeB : this was quantified from tilescans of cells from three independent experiments. In all cases, to ensure that the cells were in comparable state, they were used within 30 min of harvesting from bacterial growth.DOI:http://dx.doi.org/10.7554/eLife.04940.019
© Copyright Policy
Related In: Results  -  Collection

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

fig4s3: NF1 mutants do not have an increase in overall Ras activity as assessed by confocal microscopy.Using GFP-Raf1-RBD reporter constructs, no increase in plasma-membrane associated active Ras was observed in the axeB : this was quantified from tilescans of cells from three independent experiments. In all cases, to ensure that the cells were in comparable state, they were used within 30 min of harvesting from bacterial growth.DOI:http://dx.doi.org/10.7554/eLife.04940.019
Mentions: We then asked whether inactivation of NF1 results in a global increase in Ras activity. Pulldowns of Ras-GTP using the Raf1 Ras-binding domain (RBD) from growing cells indicated no increase in Ras activity in mutants compared to wildtype cells (Figure 4—figure supplement 2). Similarly, confocal microscopy of cells expressing the GFP-RBD reporter revealed no difference between mutant and wildtypes in overall Ras activity estimated by determining the proportion the cell periphery labelled with the RBD (Figure 4—figure supplement 3). This is not surprising, since NF1 is only one of twelve putative RasGAPs encoded in the D. discoideum genome (not including IQ-GAPs, which generally act as small G protein effectors and scaffolds and do not stimulate Ras GTPase activity [Shannon, 2012]).

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.