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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

Schematic model of NF1 function in Dictyostelium.(A) While wildtype NF1+ amoebae ingest bacteria most readily, NF1− cells are also able to ingest larger particles such as yeast cells, and accumulate more fluid in macropinosomes. (B) The large concave membrane ruffles formed during phagocytosis and macropinocytosis both are marked by intense Ras signalling (green); NF1 localises dynamically to these regions, stimulating the GTPase activity of Ras proteins there, inactivating them and thereby limiting the expansion and spread of the ‘activated’ membrane domain.DOI:http://dx.doi.org/10.7554/eLife.04940.031
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fig8: Schematic model of NF1 function in Dictyostelium.(A) While wildtype NF1+ amoebae ingest bacteria most readily, NF1− cells are also able to ingest larger particles such as yeast cells, and accumulate more fluid in macropinosomes. (B) The large concave membrane ruffles formed during phagocytosis and macropinocytosis both are marked by intense Ras signalling (green); NF1 localises dynamically to these regions, stimulating the GTPase activity of Ras proteins there, inactivating them and thereby limiting the expansion and spread of the ‘activated’ membrane domain.DOI:http://dx.doi.org/10.7554/eLife.04940.031

Mentions: Both macropinosomes and phagosomes are significantly larger in NF1 mutants than in wildtypes, suggesting that NF1 stimulates Ras GTPase activity as endocytic ruffles form and spread, thereby limiting their size (Figure 8). The control of NF1 function is not well understood, but our results suggest that it might be locally inactivated during macropinocytosis and phagocytosis. In an intriguing parallel, growth factor treatment of mammalian cells leads to rapid degradation of NF1 by the proteasome (Cichowski et al., 2003) and also triggers membrane ruffling and macropinocytosis with similar kinetics (Brunk et al., 1976; Mellström et al., 1983). However, we could find no obvious fluid-uptake phenotype in NF1 mouse embryonic fibroblasts, suggesting that NF1 inactivation is not sufficient for stimulation of macropinocytosis in these cells (unpublished data). Given the known involvement of Ras signalling in promoting ruffling and macropinocytosis the possibility remains that NF1 function is conserved in metazoa, but in a context in which Ras activity is more heavily regulated, with additional layers of control not present in amoebae (Casci et al., 1999; Johnson et al., 2005).10.7554/eLife.04940.031Figure 8.Schematic model of NF1 function in Dictyostelium.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

Schematic model of NF1 function in Dictyostelium.(A) While wildtype NF1+ amoebae ingest bacteria most readily, NF1− cells are also able to ingest larger particles such as yeast cells, and accumulate more fluid in macropinosomes. (B) The large concave membrane ruffles formed during phagocytosis and macropinocytosis both are marked by intense Ras signalling (green); NF1 localises dynamically to these regions, stimulating the GTPase activity of Ras proteins there, inactivating them and thereby limiting the expansion and spread of the ‘activated’ membrane domain.DOI:http://dx.doi.org/10.7554/eLife.04940.031
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Schematic model of NF1 function in Dictyostelium.(A) While wildtype NF1+ amoebae ingest bacteria most readily, NF1− cells are also able to ingest larger particles such as yeast cells, and accumulate more fluid in macropinosomes. (B) The large concave membrane ruffles formed during phagocytosis and macropinocytosis both are marked by intense Ras signalling (green); NF1 localises dynamically to these regions, stimulating the GTPase activity of Ras proteins there, inactivating them and thereby limiting the expansion and spread of the ‘activated’ membrane domain.DOI:http://dx.doi.org/10.7554/eLife.04940.031
Mentions: Both macropinosomes and phagosomes are significantly larger in NF1 mutants than in wildtypes, suggesting that NF1 stimulates Ras GTPase activity as endocytic ruffles form and spread, thereby limiting their size (Figure 8). The control of NF1 function is not well understood, but our results suggest that it might be locally inactivated during macropinocytosis and phagocytosis. In an intriguing parallel, growth factor treatment of mammalian cells leads to rapid degradation of NF1 by the proteasome (Cichowski et al., 2003) and also triggers membrane ruffling and macropinocytosis with similar kinetics (Brunk et al., 1976; Mellström et al., 1983). However, we could find no obvious fluid-uptake phenotype in NF1 mouse embryonic fibroblasts, suggesting that NF1 inactivation is not sufficient for stimulation of macropinocytosis in these cells (unpublished data). Given the known involvement of Ras signalling in promoting ruffling and macropinocytosis the possibility remains that NF1 function is conserved in metazoa, but in a context in which Ras activity is more heavily regulated, with additional layers of control not present in amoebae (Casci et al., 1999; Johnson et al., 2005).10.7554/eLife.04940.031Figure 8.Schematic model of NF1 function in Dictyostelium.

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