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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.


Localisation of GFP-Ras fusion proteins.DdB cells (WT) or HM1591 (axeB) cells expressing GFP or GFP-tagged RasG (‘G’), RasGG12T (‘G12’), RasS (‘S’), RasSG12V (‘S12’), RasSQ61R (‘S61’), or RasBG15T (‘B15’) were imaged by confocal microscopy to confirm proper localisation. All show some degree of enrichment on the plasma membrane except the dominant negative S17N mutants, which are very weakly fluorescent and do not show membrane localisation, presumably because they are deleterious and only cells with restricted expression grow through the selection procedure. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.020
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fig4s4: Localisation of GFP-Ras fusion proteins.DdB cells (WT) or HM1591 (axeB) cells expressing GFP or GFP-tagged RasG (‘G’), RasGG12T (‘G12’), RasS (‘S’), RasSG12V (‘S12’), RasSQ61R (‘S61’), or RasBG15T (‘B15’) were imaged by confocal microscopy to confirm proper localisation. All show some degree of enrichment on the plasma membrane except the dominant negative S17N mutants, which are very weakly fluorescent and do not show membrane localisation, presumably because they are deleterious and only cells with restricted expression grow through the selection procedure. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.020

Mentions: Two Dictyostelium Ras proteins have been linked to endocytic functions (Chubb et al., 2000; Hoeller et al., 2013); to examine their involvement in NF1-controlled events we expressed GFP-tagged versions of each in NF1 mutant and wildtype cells. All tested GFP-tagged Ras constructs localised to the plasma membrane, except for dominant negative (S17N mutant) RasG and RasS, expression of which was apparently poorly tolerated in these strains (Figure 4—figure supplement 4). None of the Dictyostelium Ras expression constructs phenocopied the loss of NF1; constitutively active RasG was deleterious to growth, as was expression of wildtype or constitutively active RasS (Figure 4—figure supplement 5), suggesting that improper activation of these isoforms interferes with endocytosis or other Ras-influenced processes leading to detrimental effects on cell growth.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

Localisation of GFP-Ras fusion proteins.DdB cells (WT) or HM1591 (axeB) cells expressing GFP or GFP-tagged RasG (‘G’), RasGG12T (‘G12’), RasS (‘S’), RasSG12V (‘S12’), RasSQ61R (‘S61’), or RasBG15T (‘B15’) were imaged by confocal microscopy to confirm proper localisation. All show some degree of enrichment on the plasma membrane except the dominant negative S17N mutants, which are very weakly fluorescent and do not show membrane localisation, presumably because they are deleterious and only cells with restricted expression grow through the selection procedure. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.020
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374526&req=5

fig4s4: Localisation of GFP-Ras fusion proteins.DdB cells (WT) or HM1591 (axeB) cells expressing GFP or GFP-tagged RasG (‘G’), RasGG12T (‘G12’), RasS (‘S’), RasSG12V (‘S12’), RasSQ61R (‘S61’), or RasBG15T (‘B15’) were imaged by confocal microscopy to confirm proper localisation. All show some degree of enrichment on the plasma membrane except the dominant negative S17N mutants, which are very weakly fluorescent and do not show membrane localisation, presumably because they are deleterious and only cells with restricted expression grow through the selection procedure. Scale = 5 μm.DOI:http://dx.doi.org/10.7554/eLife.04940.020
Mentions: Two Dictyostelium Ras proteins have been linked to endocytic functions (Chubb et al., 2000; Hoeller et al., 2013); to examine their involvement in NF1-controlled events we expressed GFP-tagged versions of each in NF1 mutant and wildtype cells. All tested GFP-tagged Ras constructs localised to the plasma membrane, except for dominant negative (S17N mutant) RasG and RasS, expression of which was apparently poorly tolerated in these strains (Figure 4—figure supplement 4). None of the Dictyostelium Ras expression constructs phenocopied the loss of NF1; constitutively active RasG was deleterious to growth, as was expression of wildtype or constitutively active RasS (Figure 4—figure supplement 5), suggesting that improper activation of these isoforms interferes with endocytosis or other Ras-influenced processes leading to detrimental effects on cell growth.

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.