Limits...
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

Phylogram of NF1 and MNF homologues.This represents the same tree as Figure 2D, displayed rectilinearly instead of radially. Selected NF1 homologues from Metazoa and Fungi are included; outside of these taxa all identified homologues are included. The code used, aligned sequences and tree files have been deposited in FigShare (with DOIs 10.6084/m9.figshare.1057805–808). For NfaA-related proteins we suggest the name MNF for ‘miniature Neurofibromin’ to avoid confusion with the unrelated Naegleria Nfa1 protein (Shin et al., 2001).DOI:http://dx.doi.org/10.7554/eLife.04940.010
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4374526&req=5

fig2s1: Phylogram of NF1 and MNF homologues.This represents the same tree as Figure 2D, displayed rectilinearly instead of radially. Selected NF1 homologues from Metazoa and Fungi are included; outside of these taxa all identified homologues are included. The code used, aligned sequences and tree files have been deposited in FigShare (with DOIs 10.6084/m9.figshare.1057805–808). For NfaA-related proteins we suggest the name MNF for ‘miniature Neurofibromin’ to avoid confusion with the unrelated Naegleria Nfa1 protein (Shin et al., 2001).DOI:http://dx.doi.org/10.7554/eLife.04940.010

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)

Phylogram of NF1 and MNF homologues.This represents the same tree as Figure 2D, displayed rectilinearly instead of radially. Selected NF1 homologues from Metazoa and Fungi are included; outside of these taxa all identified homologues are included. The code used, aligned sequences and tree files have been deposited in FigShare (with DOIs 10.6084/m9.figshare.1057805–808). For NfaA-related proteins we suggest the name MNF for ‘miniature Neurofibromin’ to avoid confusion with the unrelated Naegleria Nfa1 protein (Shin et al., 2001).DOI:http://dx.doi.org/10.7554/eLife.04940.010
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: Phylogram of NF1 and MNF homologues.This represents the same tree as Figure 2D, displayed rectilinearly instead of radially. Selected NF1 homologues from Metazoa and Fungi are included; outside of these taxa all identified homologues are included. The code used, aligned sequences and tree files have been deposited in FigShare (with DOIs 10.6084/m9.figshare.1057805–808). For NfaA-related proteins we suggest the name MNF for ‘miniature Neurofibromin’ to avoid confusion with the unrelated Naegleria Nfa1 protein (Shin et al., 2001).DOI:http://dx.doi.org/10.7554/eLife.04940.010
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