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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

The axenic growth phenotype is specific to loss of the NF1 RasGAP protein.All axenic mutants we have examined so far possess mutations in axeB, the gene encoding NF1. To test whether other RasGAPs might also have related functions (but, for instance, a lower rate of spontaneous mutation), we also deleted the related RasGAP gene nfaA in both the wildtype and axeB  background. The nfaA single mutant (HM1709) does not grow axenically, as assessed by the crystal violet binding assay, while the axeB nfaA double mutant (HM1710) has slightly potentiated the axeB phenotype, suggesting that in the absence of NF1, the NfaA protein can substitute for it to some extent. Data are the means plus and minus the standard error for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.017
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fig4s1: The axenic growth phenotype is specific to loss of the NF1 RasGAP protein.All axenic mutants we have examined so far possess mutations in axeB, the gene encoding NF1. To test whether other RasGAPs might also have related functions (but, for instance, a lower rate of spontaneous mutation), we also deleted the related RasGAP gene nfaA in both the wildtype and axeB background. The nfaA single mutant (HM1709) does not grow axenically, as assessed by the crystal violet binding assay, while the axeB nfaA double mutant (HM1710) has slightly potentiated the axeB phenotype, suggesting that in the absence of NF1, the NfaA protein can substitute for it to some extent. Data are the means plus and minus the standard error for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.017

Mentions: Given the known function of NF1 in regulating Ras, and the conserved mutation affecting the RasGAP domain in one of our mutants, we examined the involvement of Ras signalling in macropinocytosis, and the specificity of individual RasGAPs in controlling it. First, we deleted the closely related MNF RasGAP, nfaA, from wildtype cells, and found that this does not confer the ability to grow axenically (Figure 4—figure supplement 1), suggesting that NF1 has specific functions not shared by other GAPs. This corroborates earlier findings that NfaA has a distinct function regulating pseudopodium formation during chemotaxis (Zhang et al., 2008).


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

The axenic growth phenotype is specific to loss of the NF1 RasGAP protein.All axenic mutants we have examined so far possess mutations in axeB, the gene encoding NF1. To test whether other RasGAPs might also have related functions (but, for instance, a lower rate of spontaneous mutation), we also deleted the related RasGAP gene nfaA in both the wildtype and axeB  background. The nfaA single mutant (HM1709) does not grow axenically, as assessed by the crystal violet binding assay, while the axeB nfaA double mutant (HM1710) has slightly potentiated the axeB phenotype, suggesting that in the absence of NF1, the NfaA protein can substitute for it to some extent. Data are the means plus and minus the standard error for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.017
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: The axenic growth phenotype is specific to loss of the NF1 RasGAP protein.All axenic mutants we have examined so far possess mutations in axeB, the gene encoding NF1. To test whether other RasGAPs might also have related functions (but, for instance, a lower rate of spontaneous mutation), we also deleted the related RasGAP gene nfaA in both the wildtype and axeB background. The nfaA single mutant (HM1709) does not grow axenically, as assessed by the crystal violet binding assay, while the axeB nfaA double mutant (HM1710) has slightly potentiated the axeB phenotype, suggesting that in the absence of NF1, the NfaA protein can substitute for it to some extent. Data are the means plus and minus the standard error for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.017
Mentions: Given the known function of NF1 in regulating Ras, and the conserved mutation affecting the RasGAP domain in one of our mutants, we examined the involvement of Ras signalling in macropinocytosis, and the specificity of individual RasGAPs in controlling it. First, we deleted the closely related MNF RasGAP, nfaA, from wildtype cells, and found that this does not confer the ability to grow axenically (Figure 4—figure supplement 1), suggesting that NF1 has specific functions not shared by other GAPs. This corroborates earlier findings that NfaA has a distinct function regulating pseudopodium formation during chemotaxis (Zhang et al., 2008).

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