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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.


Two established axenic mutants possess identical complex mutations affecting the axeB gene.Genomic DNA isolated from the Kuspa laboratory stock AX4 and the Kay laboratory stock Ax2 was amplified using primers spanning the deletion-insertion mutation identified in the AX4 reference sequence, and sequence using a primer within the upstream gene. A black vertical line shows the 5′ boundary of the mutation; the boundaries and inserted sequence are identical. The mutation's effects on the parental DdB sequence are annotated in the sequence file deposited in the ENA database as HF565448. Resequencing of these strains' genomes, to be described elsewhere, confirmed this result.DOI:http://dx.doi.org/10.7554/eLife.04940.006
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fig1s2: Two established axenic mutants possess identical complex mutations affecting the axeB gene.Genomic DNA isolated from the Kuspa laboratory stock AX4 and the Kay laboratory stock Ax2 was amplified using primers spanning the deletion-insertion mutation identified in the AX4 reference sequence, and sequence using a primer within the upstream gene. A black vertical line shows the 5′ boundary of the mutation; the boundaries and inserted sequence are identical. The mutation's effects on the parental DdB sequence are annotated in the sequence file deposited in the ENA database as HF565448. Resequencing of these strains' genomes, to be described elsewhere, confirmed this result.DOI:http://dx.doi.org/10.7554/eLife.04940.006

Mentions: At first, other than two large duplications that do not correlate with axenicity (Figure 1—figure supplement 1), we could only identify one mutation affecting coding sequence in any of these strains relative to their parent, a seven basepair deletion in strain HM559 (Table 1). We noted that the reference genome sequence (Eichinger et al., 2005), derived from the axenic mutant strain AX4, also differs from its parent DdB in the same gene model (annotated as DDB_G0279251). Further analysis demonstrated that AX4 has lost almost nine kilobases of this region on chromosome 3, resulting in the deletion of most of the coding sequence of a large gene encoding a homologue of the Ras GTPase-activating protein (RasGAP) Neurofibromin (NF1), as well as part of the upstream gene (Figure 1A), with a short segment of extraneous sequence inserted. The 7 bp deletion mutation in HM559 lies within the C-terminal region of this NF1 homologue, and we found that another established axenic mutant, Ax2, has exactly the same deletion-insertion mutation as AX4 (Figure 1—figure supplement 2; Table 1).10.7554/eLife.04940.003Table 1.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

Two established axenic mutants possess identical complex mutations affecting the axeB gene.Genomic DNA isolated from the Kuspa laboratory stock AX4 and the Kay laboratory stock Ax2 was amplified using primers spanning the deletion-insertion mutation identified in the AX4 reference sequence, and sequence using a primer within the upstream gene. A black vertical line shows the 5′ boundary of the mutation; the boundaries and inserted sequence are identical. The mutation's effects on the parental DdB sequence are annotated in the sequence file deposited in the ENA database as HF565448. Resequencing of these strains' genomes, to be described elsewhere, confirmed this result.DOI:http://dx.doi.org/10.7554/eLife.04940.006
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374526&req=5

fig1s2: Two established axenic mutants possess identical complex mutations affecting the axeB gene.Genomic DNA isolated from the Kuspa laboratory stock AX4 and the Kay laboratory stock Ax2 was amplified using primers spanning the deletion-insertion mutation identified in the AX4 reference sequence, and sequence using a primer within the upstream gene. A black vertical line shows the 5′ boundary of the mutation; the boundaries and inserted sequence are identical. The mutation's effects on the parental DdB sequence are annotated in the sequence file deposited in the ENA database as HF565448. Resequencing of these strains' genomes, to be described elsewhere, confirmed this result.DOI:http://dx.doi.org/10.7554/eLife.04940.006
Mentions: At first, other than two large duplications that do not correlate with axenicity (Figure 1—figure supplement 1), we could only identify one mutation affecting coding sequence in any of these strains relative to their parent, a seven basepair deletion in strain HM559 (Table 1). We noted that the reference genome sequence (Eichinger et al., 2005), derived from the axenic mutant strain AX4, also differs from its parent DdB in the same gene model (annotated as DDB_G0279251). Further analysis demonstrated that AX4 has lost almost nine kilobases of this region on chromosome 3, resulting in the deletion of most of the coding sequence of a large gene encoding a homologue of the Ras GTPase-activating protein (RasGAP) Neurofibromin (NF1), as well as part of the upstream gene (Figure 1A), with a short segment of extraneous sequence inserted. The 7 bp deletion mutation in HM559 lies within the C-terminal region of this NF1 homologue, and we found that another established axenic mutant, Ax2, has exactly the same deletion-insertion mutation as AX4 (Figure 1—figure supplement 2; Table 1).10.7554/eLife.04940.003Table 1.

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.