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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

NF1 mutants are able to grow in suspension in rich axenic media.The established axenic mutant strain Ax2 was selected for high rates of growth in HL5 medium in shaken suspension; deleting axeB in the wildtype background only partially recapitulated this pheontype, the cells only being able to grow well in HL5 when attached to a substratum, indicating that at least one other mutation is required to account for the full axenic phenotype. However the single mutant is able to grow in shaken suspension when HL5 is supplemented with 10% foetal bovie serum. Cells were inoculated at a starting density of 5 × 105 cells per ml in 50 ml of medium in 250 ml Erlenmeyer flasks at 22°C and counted at the stated intervals using a haemocytometer. The means and standard errors are given for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.026
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fig6s2: NF1 mutants are able to grow in suspension in rich axenic media.The established axenic mutant strain Ax2 was selected for high rates of growth in HL5 medium in shaken suspension; deleting axeB in the wildtype background only partially recapitulated this pheontype, the cells only being able to grow well in HL5 when attached to a substratum, indicating that at least one other mutation is required to account for the full axenic phenotype. However the single mutant is able to grow in shaken suspension when HL5 is supplemented with 10% foetal bovie serum. Cells were inoculated at a starting density of 5 × 105 cells per ml in 50 ml of medium in 250 ml Erlenmeyer flasks at 22°C and counted at the stated intervals using a haemocytometer. The means and standard errors are given for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.026

Mentions: The observations described above indicate that wildtype cells perform qualitatively similar macropinocytosis to NF1 mutants, but on a smaller scale. This results in a markedly different outcome when the cells are incubated in HL5 medium: mutants can grow but the wildtype cannot. One possible explanation is that nutrient-uptake below a certain threshold leads to a growth arrest. To test this idea, we asked whether wildtype cells can maintain growth in an enriched axenic medium, as suggested by earlier work (Sussman and Sussman, 1967). Wildtype cells incubated in stationary cultures in HL5 supplemented with foetal bovine serum (or bovine serum albumin, data not shown) were able to grow, albeit still much more slowly than NF1 mutants cultured in the same medium (Figure 6A,B). The morphology of wildtype cells was not appreciably altered after several days of axenic growth, while NF1 mutants remained consistently more flattened and extensively ruffled than wildtype cells in the same conditions (Figure 6C). Wildtype cells were also found to degrade DQ-BSA efficiently after axenic growth in the presence of serum (Figure 6—figure supplement 1). Serum addition also stimulated the growth of NF1 mutants in shaking suspension (Figure 6—figure supplement 2). These findings suggest that the additional nutrients in the richer broth allow these cells to avoid the starvation-triggered growth arrest that can occur in axenic media.10.7554/eLife.04940.024Figure 6.Wildtype amoebae can grow axenically in medium supplemented with bovine serum.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

NF1 mutants are able to grow in suspension in rich axenic media.The established axenic mutant strain Ax2 was selected for high rates of growth in HL5 medium in shaken suspension; deleting axeB in the wildtype background only partially recapitulated this pheontype, the cells only being able to grow well in HL5 when attached to a substratum, indicating that at least one other mutation is required to account for the full axenic phenotype. However the single mutant is able to grow in shaken suspension when HL5 is supplemented with 10% foetal bovie serum. Cells were inoculated at a starting density of 5 × 105 cells per ml in 50 ml of medium in 250 ml Erlenmeyer flasks at 22°C and counted at the stated intervals using a haemocytometer. The means and standard errors are given for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.026
© Copyright Policy
Related In: Results  -  Collection

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

fig6s2: NF1 mutants are able to grow in suspension in rich axenic media.The established axenic mutant strain Ax2 was selected for high rates of growth in HL5 medium in shaken suspension; deleting axeB in the wildtype background only partially recapitulated this pheontype, the cells only being able to grow well in HL5 when attached to a substratum, indicating that at least one other mutation is required to account for the full axenic phenotype. However the single mutant is able to grow in shaken suspension when HL5 is supplemented with 10% foetal bovie serum. Cells were inoculated at a starting density of 5 × 105 cells per ml in 50 ml of medium in 250 ml Erlenmeyer flasks at 22°C and counted at the stated intervals using a haemocytometer. The means and standard errors are given for three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.04940.026
Mentions: The observations described above indicate that wildtype cells perform qualitatively similar macropinocytosis to NF1 mutants, but on a smaller scale. This results in a markedly different outcome when the cells are incubated in HL5 medium: mutants can grow but the wildtype cannot. One possible explanation is that nutrient-uptake below a certain threshold leads to a growth arrest. To test this idea, we asked whether wildtype cells can maintain growth in an enriched axenic medium, as suggested by earlier work (Sussman and Sussman, 1967). Wildtype cells incubated in stationary cultures in HL5 supplemented with foetal bovine serum (or bovine serum albumin, data not shown) were able to grow, albeit still much more slowly than NF1 mutants cultured in the same medium (Figure 6A,B). The morphology of wildtype cells was not appreciably altered after several days of axenic growth, while NF1 mutants remained consistently more flattened and extensively ruffled than wildtype cells in the same conditions (Figure 6C). Wildtype cells were also found to degrade DQ-BSA efficiently after axenic growth in the presence of serum (Figure 6—figure supplement 1). Serum addition also stimulated the growth of NF1 mutants in shaking suspension (Figure 6—figure supplement 2). These findings suggest that the additional nutrients in the richer broth allow these cells to avoid the starvation-triggered growth arrest that can occur in axenic media.10.7554/eLife.04940.024Figure 6.Wildtype amoebae can grow axenically in medium supplemented with bovine serum.

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