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.


ERK phosphorylation is not increased in NF1 mutants.WT (DdB) and axeB knockout (HM1591) cells were harvested from bacterial growth plates then washed and incubated in HL5 medium and shaken for the indicated times before ERK activity was assessed using an antibody raised against a phosphorylated TEY motif. A band of the expected size of D. discoideum ErkB reproducibly increased in intensity over time in both strains, but more intensely in WT cells than NF1 mutants, perhaps reflecting starvation-induced development. A band of the approximate expected size of ErkA varied in its pattern of intensity in different experiments, but showed no tendency to be more intense in mutants. A single representative experiment is shown.DOI:http://dx.doi.org/10.7554/eLife.04940.023
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: ERK phosphorylation is not increased in NF1 mutants.WT (DdB) and axeB knockout (HM1591) cells were harvested from bacterial growth plates then washed and incubated in HL5 medium and shaken for the indicated times before ERK activity was assessed using an antibody raised against a phosphorylated TEY motif. A band of the expected size of D. discoideum ErkB reproducibly increased in intensity over time in both strains, but more intensely in WT cells than NF1 mutants, perhaps reflecting starvation-induced development. A band of the approximate expected size of ErkA varied in its pattern of intensity in different experiments, but showed no tendency to be more intense in mutants. A single representative experiment is shown.DOI:http://dx.doi.org/10.7554/eLife.04940.023

Mentions: Active Ras at the plasma membrane recruits class 1 phosphoinositide 3′-kinases (PI3Ks; Rodriguez-Viciana et al., 1997), allowing the spatially restricted formation of phosphatidylinositol trisphosphate (PIP3) and other inositol phospholipids that occurs during macropinocytosis (Araki et al., 1996; Buczynski et al., 1997; Hoeller et al., 2013). Confirming that the macropinosomes observed in NF1 mutants are mechanistically similar to those previously documented, we find that Ras activity at membrane ruffles in NF1 mutants is accompanied by recruitment of PH-domain reporters that bind the plasmanyl inositides produced by Dictyostelium class 1 PI3Ks (Figure 5A; Clark et al., 2014), as well as by actin polymerisation (Figure 5B). PH domains are also prominently recruited during macropinocytosis in wildtype cells; regions of recruitment tend to be larger in mutants reflecting the increased Ras signalling that results from the absence of NF1 (Figure 5C). This pattern of Ras activity and PIP3 formation is invariably observed in every instance of macropinocytosis in Dictyostelium. The contributions of other Ras effectors remain unclear; for example no increase in ERK activity is observed in NF1 mutants compared to wildtype cells (Figure 5—figure supplement 1).10.7554/eLife.04940.022Figure 5.Downstream signalling: connections between Ras and PI3K activity during macropinocytosis.


Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

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

ERK phosphorylation is not increased in NF1 mutants.WT (DdB) and axeB knockout (HM1591) cells were harvested from bacterial growth plates then washed and incubated in HL5 medium and shaken for the indicated times before ERK activity was assessed using an antibody raised against a phosphorylated TEY motif. A band of the expected size of D. discoideum ErkB reproducibly increased in intensity over time in both strains, but more intensely in WT cells than NF1 mutants, perhaps reflecting starvation-induced development. A band of the approximate expected size of ErkA varied in its pattern of intensity in different experiments, but showed no tendency to be more intense in mutants. A single representative experiment is shown.DOI:http://dx.doi.org/10.7554/eLife.04940.023
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: ERK phosphorylation is not increased in NF1 mutants.WT (DdB) and axeB knockout (HM1591) cells were harvested from bacterial growth plates then washed and incubated in HL5 medium and shaken for the indicated times before ERK activity was assessed using an antibody raised against a phosphorylated TEY motif. A band of the expected size of D. discoideum ErkB reproducibly increased in intensity over time in both strains, but more intensely in WT cells than NF1 mutants, perhaps reflecting starvation-induced development. A band of the approximate expected size of ErkA varied in its pattern of intensity in different experiments, but showed no tendency to be more intense in mutants. A single representative experiment is shown.DOI:http://dx.doi.org/10.7554/eLife.04940.023
Mentions: Active Ras at the plasma membrane recruits class 1 phosphoinositide 3′-kinases (PI3Ks; Rodriguez-Viciana et al., 1997), allowing the spatially restricted formation of phosphatidylinositol trisphosphate (PIP3) and other inositol phospholipids that occurs during macropinocytosis (Araki et al., 1996; Buczynski et al., 1997; Hoeller et al., 2013). Confirming that the macropinosomes observed in NF1 mutants are mechanistically similar to those previously documented, we find that Ras activity at membrane ruffles in NF1 mutants is accompanied by recruitment of PH-domain reporters that bind the plasmanyl inositides produced by Dictyostelium class 1 PI3Ks (Figure 5A; Clark et al., 2014), as well as by actin polymerisation (Figure 5B). PH domains are also prominently recruited during macropinocytosis in wildtype cells; regions of recruitment tend to be larger in mutants reflecting the increased Ras signalling that results from the absence of NF1 (Figure 5C). This pattern of Ras activity and PIP3 formation is invariably observed in every instance of macropinocytosis in Dictyostelium. The contributions of other Ras effectors remain unclear; for example no increase in ERK activity is observed in NF1 mutants compared to wildtype cells (Figure 5—figure supplement 1).10.7554/eLife.04940.022Figure 5.Downstream signalling: connections between Ras and PI3K activity during macropinocytosis.

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.