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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

In contrast to fluid uptake, membrane uptake is not increased in NF1 mutants.DdB (WT) and the NF1  mutant HM1591 (axeB) were harvested from bacterial growth plates, washed, and resuspended in KK2 buffer and shaken at room temperature for 15 min before being added a stirred fluorimeter cuvette containing FM1-43 dye. When cells are added, the fluorescence of the sample increases rapidly as dye enters the plasma membrane; as membrane is internalised, compensating fresh membrane is exposed to the surface enabling more dye to bind and fluoresce. While fluid uptake is several-fold higher in mutants, we found no evidence of increased membrane uptake rates, indicating that most membrane is taken up as small vesicles or narrow tubules with large surface-area to volume ratios.DOI:http://dx.doi.org/10.7554/eLife.04940.014
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fig3s2: In contrast to fluid uptake, membrane uptake is not increased in NF1 mutants.DdB (WT) and the NF1 mutant HM1591 (axeB) were harvested from bacterial growth plates, washed, and resuspended in KK2 buffer and shaken at room temperature for 15 min before being added a stirred fluorimeter cuvette containing FM1-43 dye. When cells are added, the fluorescence of the sample increases rapidly as dye enters the plasma membrane; as membrane is internalised, compensating fresh membrane is exposed to the surface enabling more dye to bind and fluoresce. While fluid uptake is several-fold higher in mutants, we found no evidence of increased membrane uptake rates, indicating that most membrane is taken up as small vesicles or narrow tubules with large surface-area to volume ratios.DOI:http://dx.doi.org/10.7554/eLife.04940.014

Mentions: Established axenic mutants have very high rates of macropinocytosis (Hacker et al., 1997). To examine fluid uptake in our NF1 knockout mutant, we incubated amoebae with fluorescent dextran and compared them with both the wildtype DdB strain and the established axenic mutant Ax2. When they are harvested directly from bacterial growth plates, NF1 mutants ingest fluid at about the same rate as an established axenic strain, Ax2, and more than four times more rapidly than wildtype cells (Figure 3A). After prolonged incubation in HL5 medium without bacteria, Ax2 cells and NF1 mutants increase their uptake further while wildtype cells decrease it, such that after 24 hr mutants take in fluid at a rate more than twenty times higher than the wildtype (Figure 3A). Fluid uptake is linear at the earliest times measured with no evidence for rapid recycling of fluid in any of these strains (Figure 3—figure supplement 1), in agreement with earlier studies of axenic mutants (Aubry et al., 1993; Padh et al., 1993). Membrane uptake measured using the accumulation of FM1-43 dye was not increased in NF1 mutants (Figure 3—figure supplement 2), consistent with an earlier comparison of Ax2 with wildtype cells (Aguado-Velasco and Bretscher, 1999). Since this assay predominantly measures uptake into small vesicles or tubules with a high surface to volume ratio, we conclude that clathrin-dependent and -independent micropinocytotic processes in these cells (Neuhaus et al., 2002; Hirst et al., 2014), are unaffected by NF1 loss.10.7554/eLife.04940.012Figure 3.NF1 mutants grow axenically in HL5 medium and have increased fluid uptake.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

In contrast to fluid uptake, membrane uptake is not increased in NF1 mutants.DdB (WT) and the NF1  mutant HM1591 (axeB) were harvested from bacterial growth plates, washed, and resuspended in KK2 buffer and shaken at room temperature for 15 min before being added a stirred fluorimeter cuvette containing FM1-43 dye. When cells are added, the fluorescence of the sample increases rapidly as dye enters the plasma membrane; as membrane is internalised, compensating fresh membrane is exposed to the surface enabling more dye to bind and fluoresce. While fluid uptake is several-fold higher in mutants, we found no evidence of increased membrane uptake rates, indicating that most membrane is taken up as small vesicles or narrow tubules with large surface-area to volume ratios.DOI:http://dx.doi.org/10.7554/eLife.04940.014
© Copyright Policy
Related In: Results  -  Collection

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

fig3s2: In contrast to fluid uptake, membrane uptake is not increased in NF1 mutants.DdB (WT) and the NF1 mutant HM1591 (axeB) were harvested from bacterial growth plates, washed, and resuspended in KK2 buffer and shaken at room temperature for 15 min before being added a stirred fluorimeter cuvette containing FM1-43 dye. When cells are added, the fluorescence of the sample increases rapidly as dye enters the plasma membrane; as membrane is internalised, compensating fresh membrane is exposed to the surface enabling more dye to bind and fluoresce. While fluid uptake is several-fold higher in mutants, we found no evidence of increased membrane uptake rates, indicating that most membrane is taken up as small vesicles or narrow tubules with large surface-area to volume ratios.DOI:http://dx.doi.org/10.7554/eLife.04940.014
Mentions: Established axenic mutants have very high rates of macropinocytosis (Hacker et al., 1997). To examine fluid uptake in our NF1 knockout mutant, we incubated amoebae with fluorescent dextran and compared them with both the wildtype DdB strain and the established axenic mutant Ax2. When they are harvested directly from bacterial growth plates, NF1 mutants ingest fluid at about the same rate as an established axenic strain, Ax2, and more than four times more rapidly than wildtype cells (Figure 3A). After prolonged incubation in HL5 medium without bacteria, Ax2 cells and NF1 mutants increase their uptake further while wildtype cells decrease it, such that after 24 hr mutants take in fluid at a rate more than twenty times higher than the wildtype (Figure 3A). Fluid uptake is linear at the earliest times measured with no evidence for rapid recycling of fluid in any of these strains (Figure 3—figure supplement 1), in agreement with earlier studies of axenic mutants (Aubry et al., 1993; Padh et al., 1993). Membrane uptake measured using the accumulation of FM1-43 dye was not increased in NF1 mutants (Figure 3—figure supplement 2), consistent with an earlier comparison of Ax2 with wildtype cells (Aguado-Velasco and Bretscher, 1999). Since this assay predominantly measures uptake into small vesicles or tubules with a high surface to volume ratio, we conclude that clathrin-dependent and -independent micropinocytotic processes in these cells (Neuhaus et al., 2002; Hirst et al., 2014), are unaffected by NF1 loss.10.7554/eLife.04940.012Figure 3.NF1 mutants grow axenically in HL5 medium and have increased fluid uptake.

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