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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 new axenic mutant strains possess overlapping duplications on the same chromosome.The samtools ‘depth’ command was used to calculate the depth of coverage at each position along the chromosomes. A rolling median was obtained (window size 999) to remove outliers, then each chromosome examined by sampling every 1000th position and plotting them sequentially using R (www.r-project.org); the ‘index’ in the plots refers to these 1000 nucleotide divisions. Contiguous segments with approximately double the average depth reflect likely duplication events. Only two such segments could be identified, on overlapping regions at one end of chromosome four in strains HM557 and HM558: shown here are the plots for this chromosome in all four strains resequenced. These duplications are large, spanning hundreds of kilobases and many genes, and it is possible that they contribute to these strains' growth phenotypes; this hypothesis remains to be tested.DOI:http://dx.doi.org/10.7554/eLife.04940.005
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fig1s1: Two new axenic mutant strains possess overlapping duplications on the same chromosome.The samtools ‘depth’ command was used to calculate the depth of coverage at each position along the chromosomes. A rolling median was obtained (window size 999) to remove outliers, then each chromosome examined by sampling every 1000th position and plotting them sequentially using R (www.r-project.org); the ‘index’ in the plots refers to these 1000 nucleotide divisions. Contiguous segments with approximately double the average depth reflect likely duplication events. Only two such segments could be identified, on overlapping regions at one end of chromosome four in strains HM557 and HM558: shown here are the plots for this chromosome in all four strains resequenced. These duplications are large, spanning hundreds of kilobases and many genes, and it is possible that they contribute to these strains' growth phenotypes; this hypothesis remains to be tested.DOI:http://dx.doi.org/10.7554/eLife.04940.005

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 new axenic mutant strains possess overlapping duplications on the same chromosome.The samtools ‘depth’ command was used to calculate the depth of coverage at each position along the chromosomes. A rolling median was obtained (window size 999) to remove outliers, then each chromosome examined by sampling every 1000th position and plotting them sequentially using R (www.r-project.org); the ‘index’ in the plots refers to these 1000 nucleotide divisions. Contiguous segments with approximately double the average depth reflect likely duplication events. Only two such segments could be identified, on overlapping regions at one end of chromosome four in strains HM557 and HM558: shown here are the plots for this chromosome in all four strains resequenced. These duplications are large, spanning hundreds of kilobases and many genes, and it is possible that they contribute to these strains' growth phenotypes; this hypothesis remains to be tested.DOI:http://dx.doi.org/10.7554/eLife.04940.005
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: Two new axenic mutant strains possess overlapping duplications on the same chromosome.The samtools ‘depth’ command was used to calculate the depth of coverage at each position along the chromosomes. A rolling median was obtained (window size 999) to remove outliers, then each chromosome examined by sampling every 1000th position and plotting them sequentially using R (www.r-project.org); the ‘index’ in the plots refers to these 1000 nucleotide divisions. Contiguous segments with approximately double the average depth reflect likely duplication events. Only two such segments could be identified, on overlapping regions at one end of chromosome four in strains HM557 and HM558: shown here are the plots for this chromosome in all four strains resequenced. These duplications are large, spanning hundreds of kilobases and many genes, and it is possible that they contribute to these strains' growth phenotypes; this hypothesis remains to be tested.DOI:http://dx.doi.org/10.7554/eLife.04940.005
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.