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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 presence of NF1 homologues and other RasGAPs in the three main eukaryotic supergroups.While the corticates evidently ancestrally possessed RasGAPs (and Ras signalling), no NF1 homologues are detectable in the genomes of any presently available in the public databases. A previously discussed putative homologue in Stramenopiles has a START domain next to its RasGAP domain, not the unrelated CRAL/TRIO domain found in NF1 (van Dam et al., 2011). The other two supergroups, podiates and excavates, both possess NF1 (and MNF) homologues; if the root of the eukaryotic tree lies between the podiates and either Naegleria or Trichomonas this implies that NF1 was present in the LECA. Examples of RasGAPs and NF1 orthologues in the lineages shown here are given in Figure 2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.04940.011
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fig2s2: The presence of NF1 homologues and other RasGAPs in the three main eukaryotic supergroups.While the corticates evidently ancestrally possessed RasGAPs (and Ras signalling), no NF1 homologues are detectable in the genomes of any presently available in the public databases. A previously discussed putative homologue in Stramenopiles has a START domain next to its RasGAP domain, not the unrelated CRAL/TRIO domain found in NF1 (van Dam et al., 2011). The other two supergroups, podiates and excavates, both possess NF1 (and MNF) homologues; if the root of the eukaryotic tree lies between the podiates and either Naegleria or Trichomonas this implies that NF1 was present in the LECA. Examples of RasGAPs and NF1 orthologues in the lineages shown here are given in Figure 2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.04940.011

Mentions: The Dictyostelium NF1 gene encodes a protein with the same domain organisation as the human version, with CRAL/TRIO and PH-like domains at the C-terminal side of the catalytic RasGAP domain (Figure 2A). It is also of a similar size, with homology extending across most of the two proteins' lengths (Figure 2B). The D. discoideum NF1 orthologue is about as similar to the human protein as are those from the basal metazoa and choanoflagellates (Figure 2C). NF1 is an ancient protein, conserved considerably beyond the metazoan and fungal lineages in which it has been studied to date, with homologues in a variety of unicellular eukaryotes including the excavates Naegleria and Trichomonas as well as other amoebae (Figure 2C,D and Figure 2—figure supplement 1; Carlton et al., 2007; Fritz-Laylin et al., 2010; Clarke et al., 2013). RasGAPs are more broadly distributed than NF1, being present in further excavates as well as in certain ciliates, oomycetes, and the foraminiferan Reticulomyxa (Figure 2—figure supplement 2 and Figure 2—source data 1; van Dam et al., 2011; Glöckner et al., 2014). The dictyostelids, Entamoeba, Thecamonas, and Naegleria all possess separate smaller homologues with a similar domain organisation to NF1 but lacking homology outside of the central region; we term these proteins ‘MNF’ (for ‘miniature neurofibromin’). The D. discoideum NfaA protein (Zhang et al., 2008) falls into this class (Figure 2A,D and Figure 2—figure supplement 1), and is discussed further below.10.7554/eLife.04940.008Figure 2.NF1 is broadly conserved in a range of amoeboid species as well as animals and fungi.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

The presence of NF1 homologues and other RasGAPs in the three main eukaryotic supergroups.While the corticates evidently ancestrally possessed RasGAPs (and Ras signalling), no NF1 homologues are detectable in the genomes of any presently available in the public databases. A previously discussed putative homologue in Stramenopiles has a START domain next to its RasGAP domain, not the unrelated CRAL/TRIO domain found in NF1 (van Dam et al., 2011). The other two supergroups, podiates and excavates, both possess NF1 (and MNF) homologues; if the root of the eukaryotic tree lies between the podiates and either Naegleria or Trichomonas this implies that NF1 was present in the LECA. Examples of RasGAPs and NF1 orthologues in the lineages shown here are given in Figure 2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.04940.011
© Copyright Policy
Related In: Results  -  Collection

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

fig2s2: The presence of NF1 homologues and other RasGAPs in the three main eukaryotic supergroups.While the corticates evidently ancestrally possessed RasGAPs (and Ras signalling), no NF1 homologues are detectable in the genomes of any presently available in the public databases. A previously discussed putative homologue in Stramenopiles has a START domain next to its RasGAP domain, not the unrelated CRAL/TRIO domain found in NF1 (van Dam et al., 2011). The other two supergroups, podiates and excavates, both possess NF1 (and MNF) homologues; if the root of the eukaryotic tree lies between the podiates and either Naegleria or Trichomonas this implies that NF1 was present in the LECA. Examples of RasGAPs and NF1 orthologues in the lineages shown here are given in Figure 2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.04940.011
Mentions: The Dictyostelium NF1 gene encodes a protein with the same domain organisation as the human version, with CRAL/TRIO and PH-like domains at the C-terminal side of the catalytic RasGAP domain (Figure 2A). It is also of a similar size, with homology extending across most of the two proteins' lengths (Figure 2B). The D. discoideum NF1 orthologue is about as similar to the human protein as are those from the basal metazoa and choanoflagellates (Figure 2C). NF1 is an ancient protein, conserved considerably beyond the metazoan and fungal lineages in which it has been studied to date, with homologues in a variety of unicellular eukaryotes including the excavates Naegleria and Trichomonas as well as other amoebae (Figure 2C,D and Figure 2—figure supplement 1; Carlton et al., 2007; Fritz-Laylin et al., 2010; Clarke et al., 2013). RasGAPs are more broadly distributed than NF1, being present in further excavates as well as in certain ciliates, oomycetes, and the foraminiferan Reticulomyxa (Figure 2—figure supplement 2 and Figure 2—source data 1; van Dam et al., 2011; Glöckner et al., 2014). The dictyostelids, Entamoeba, Thecamonas, and Naegleria all possess separate smaller homologues with a similar domain organisation to NF1 but lacking homology outside of the central region; we term these proteins ‘MNF’ (for ‘miniature neurofibromin’). The D. discoideum NfaA protein (Zhang et al., 2008) falls into this class (Figure 2A,D and Figure 2—figure supplement 1), and is discussed further below.10.7554/eLife.04940.008Figure 2.NF1 is broadly conserved in a range of amoeboid species as well as animals and fungi.

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